Artificial insemination of mammalians with low quantity of sex-separated spermatozoa

FIELD: animal science.

SUBSTANCE: for the purpose to obtain farm animals of preset sex it is necessary to apply male spermatozoa to detect sex sign in certain multitude of spermatozoa and sort spermatozoa according to the detected sex sign and collect them into the collector supplied with a shock absorbing element. Then one should obtain a sorted combination of spermatozoa to introduce it for female of the given mammalian type to fertilize, at least, one female ovicell at resultativeness level being, at least, 50% against that of pregnancy achieved at applying conventional combination of spermatozoa for artificial insemination. For separating spermatozoa one should create the source of cells that supplies spermatozoa for sorting procedure. Chemically one should coordinate capsular liquid for creating the medium out of capsular liquid for spermatozoa being coordinated with liquid medium of spermatozoa both before and after sorting technique. Then it is important to identify one of spermatozoa's properties. Spermatozoa should be differentiated in accordance to their sex and separated at the rate of, at least, 1200 operations/sec. Then spermatozoa with desired sex sign should be collected into the collector supplied with shock-absorbing element. To obtain multiple embryos it is necessary to induce polyovulation in a female of the given mammalian type to obtain, at least, two ovicells. Moreover, a polyovulation-favoring pharmaceutical preparation should be introduced at 12-h-long interval at any days between the 2nd and the 18th d of estrus cycle. It is necessary to detect sex in certain multitude of spermatozoa in a male of the given mammalian type to separate these spermatozoa according to sex sign and introduce separated spermatozoa into female uterus of the given mammalian type after estrus onset to fertilize multitude of ovicells in the uterus. The innovation enables to efficiently separate male spermatozoa in farm animals and obtain from them the offspring of desired sex and, also, obtain multiple embryos of the preset sex.

EFFECT: higher efficiency of artificial insemination.

70 cl, 4 dwg, 4 ex

 

I. technical FIELD

This invention relates primarily to the field of gender selection in the progeny of mammals. Especially it concerns aspects of artificial insemination with low doses of semen and increased production of eggs to ensure the desired gender of the offspring. In particular, the invention concerns the development of artificial insemination with low doses of sexed semen, regardless of how sperm separation by sex for sperm separation by sex by flow cytometry for artificial insemination with low doses of sexed semen, as well as ways of achieving increased ovulation, etc.

II. The LEVEL of TECHNOLOGY

For centuries people tried to learn how to choose the gender specific derived progeny. Besides the obvious psychological aspects of sex selection for offspring mammals has significant economic consequences when applied to animals used for producing food products, such as cattle, as well as for the prize animals, such as horses, etc. It's a huge commitment has led to various attempts to produce offspring with the selected floor. Apparently, among them the most close to getting the desired results, the method consisting in the separation and selection of X and Y sperm before insemination.

the bottom of difficulties, faced when trying to separate X and Y sperm, is that it requires a large amount of semen. In natural fertilization, the number of produced sperm cells in some animal species are billions; artificial insemination this figure is smaller, but nevertheless use significant amounts of sperm. For example, when artificial insemination is usually used from ten million to five hundred million sperm (depending on species). Thus, even in conditions of artificial insemination requires a huge amount of sperm.

To achieve separation of sperm bearing the X - and Y-chromosomes, tried a variety of ways. These methods range from a magnetic methods, such as described in U.S. patent No. 4276139, to methods using columns such as described in U.S. patent No. 5514537, and to gravimetric methods, such as discussed in U.S. patent No. 3894529, in the replacement patent No. 32350, in U.S. patent No. 4092229, 4067965 and 4155831. Attempted to use the electrical properties, as shown in U.S. patent No. 4083957, as well as a combination of electric and gravimetric properties, as discussed in U.S. patent No. 4225405, 4698142 and 4749458. Efforts were also attempts to use different designs for the Yu sperm motility, as shown in U.S. patent No. 4009260 and 4339434. Developed chemical methods, such as described in U.S. patent No. 4511661 and 4999283 (including monoclonal antibodies), in U.S. patent No. 5021244, 5346990, 5439362 and 5660997 (including membrane proteins) and in U.S. patent No. 3687803, 4191749, 4448767 and 4680258 (including antibodies), as well as methods, including the addition of serum components, such as shown in U.S. patent No. 4085205. Although each of these methods presents as a highly effective, but in fact at the present time none of them give the desired level of pre-sex selection. Regardless of the actual use of the methods of separation by gender, competing combination of high amounts of sperm naturally present in the semen, and approach, consisting in the separation of sperm bearing the X - and Y-chromosomes, made desirable the development of ways to achieve insemination using lower quantities of sperm.

Currently, the only quantitative method used to separate sperm carrying the X - and Y-chromosome, is a method based on individual differences and on the separation of sperm using methods flow cytometry. This method has become possible as a result of women and discoveries, including differential absorption of the dye by a sperm carrying the X - and Y-chromosomes. Previously described in U.S. patent No. 4362246 and was significantly expanded by the methods described by Lawrence Johnson Lawrence Johnson) in U.S. patent No. 5135759. Proposed by Johnson method using flow cytometry to separate sperm carrying the X - and Y-chromosome so has taken forward this study that for the first time it became possible commercial application of the method of separation by sex such sperm. Although the separation of sperm on the floor still remains experimental, but it was significantly improved by the use of high-speed flow cytometers, such as flow cytometer MoFlo®manufactured by the company Cytomation Inc., and described in a number of other patents, including U.S. patent No. 5150313, 5602039, 5602349 and 5643796, as well as in international patent publication PCT WO 96/12171. Although the use of cytometric MoFlo® company Cytomation significantly increase the speed of separation of sperm on the floor and while this increase of speed is especially important when large quantities of sperm, which are often used, but there are still some unresolved issues. Despite an almost tenfold increase in the rate of separation of sperm on the floor using a MoFlo cytometer®, what about some reasons still require a much shorter length of time separation of sperm on the floor. Firstly, it was found that almost the efficiency of sperm is very dependent on the time of their storage. The longer they remain unused, the more they lose their effectiveness. Secondly, the time required for the processes of semen collection, separation of sperm on the floor and insemination, does speed matter of great commercial value. Thus, the nature of sperm associated with the dependence of their performance on the retention time and the time required for the processes associated with insemination, makes speed an important element to achieve high efficiency and high performance insemination.

There are also other difficulties, both practical and theoretical. From a practical point of view, it is desirable to obtain sets of sexed spermatozoa with the use of inexpensive consumable components and substances. From the point of view of reducing costs it is also desirable, in order to allow the separation of sperm on the floor (and taking sperm and insemination), with the greatest possible productivity. Thus, for commercial production and success in this area, improvements that can provide increased efficiency, can still be significant. Along with the practical aspect, the related costs, there is also the practical aspect associated with the subtlety and sensitivity of the whole process. In this regard, it is desirable to simplify the method and make it more understandable, so that operator error or level of skill played as a lesser role. The methods can also be combined, which makes insemination in small doses even more desirable.

In addition to the details of the process, has always been known that spermatozoa are extremely sensitive to external influences. Although at first glance this factor seems easy to understand, but in fact all the degree of sensitivity of these cells are not yet fully investigated. In the context of flow cytometry, as a rule, most of the partial cells or particles often have a spherical shape and therefore physiologically capable of withstanding a number of harmful effects. But this does not apply to sperm. In fact, as described by the present invention, conventional methods of flow cytometry essentially can be unsuitable for cytometrical separation of spermatozoa in some applications. Sensitivity is associated with different aspects, ranging from the problems associated with dilution and full flow cytometer the need for isolation and identification of each cell individually, and with the pressure of other with whom resume, when conventional flow cytometry to the present invention influenced the partial cells or substances. Sensitivity can be a particularly important factor for sperm, because, apparently, despite the fact that sperm can pass through the flow cytometer and to be separated by gender for no apparent harmful side effects, but the cells themselves may be to such an extent affected by stress that their behavior in the process of insemination is less than optimal. Thus, it is obvious that there is interaction between the factors, which cause unusual problems connected with prospects for the separation of sperm on the floor and use them for artificial insemination.

Another difficulty lies in the fact that despite the great achievements resulting from use of the patent Johnson and related technologies, in fact, until the present invention was extremely difficult to achieve insemination lower doses of sexed semen, regardless of the method used cell division. Although the history of this issue has some achievements in the insemination with low doses, but it turned out that these achievements rather exist in theory or in laboratory practice, and not in those conditions, which are usually p and the application of the method of production. In this regard, it is important not just to carry out the insemination with low doses, but to carry out the insemination with low doses of such indicators, defined by the percentage of the onset of pregnancy, which is comparable to the existing conventional methods of artificial insemination high doses of semen, without separation of sperm on the floor. Thus, achievements, obtained by the authors of the present invention in artificial insemination as sexed sperm, and low doses of semen, represent significant progress for the first time can make possible the application of this method in production.

The next problem faced by professionals in practice and also in spite of the great progress made by the patent Johnson and related technology - is that the problem itself is artificial insemination with high efficiency insemination has a statistical nature, in which, apparently, interact with many factors. Thus, the proposed solution can, to some extent involve a combination of factors, which, after a careful statistical study will be defined as necessary, either individually or in combination with other factors. This definition is even more slojnee is the, actual results vary depending on the type of animal, and it may be difficult to confirm their authenticity due to the fact that testing and statistical sampling in large databases in the initial stages of research requires a lot of efforts. For these reasons, the present invention may also include the combination of factors that may, individually or in combination, represent a solution corresponding to the applications. The present description, therefore, should be considered as quite broad, covering various combinations and permutations of the described methods. May be not open to the present time the synergistic interaction with other factors. Here are those factors that are in the process of separation by sex, or perhaps in flow cytometry, and in stages, such as stage semen collection, as well as the stage insemination. To date, the first studies conducted in cattle, but this does not mean that these methods are limited to only one kind of animal or only sperm. It is obvious that the used methods can be applied not only sperm but also in other areas where you want to share sensitive elements or simply to minimize the stress, call aimie the influence of flow cytometry to sort the elements.

Interestingly, while the present invention uses an approach aimed at minimizing the harmful effects of the process of sperm separation or stress on the sperm cells, other methods actually used approaches, which moves in the opposite direction from this direction, increasing the pressure, increasing requirements for speed and others in this spirit. Essentially, the shift towards insemination with low doses and high-speed processing, separately or interconnected way, apparently, raised the problem of limiting each other. Thus, although there has long been an unmet need for high insemination with low doses of sexed semen, and though it has long had the means and elements for its implementation, but to the present invention, the specialists in this field of technology, it seems, has seen achievements or maybe a combination of achievements, allowing it to perform. Perhaps they, to some extent, failed to appreciate the fact that this problem involves the interaction of factors, as well as special requirements for certain types of cells (sperm or perhaps species-specific sperm)used in this technical field. Interestingly, as shown by the list of earlier attempts made in the present about what Isani, significant efforts have been made, but they are not explicitly given opportunities to understand the problem inherent in such areas as artificial insemination with low doses of sexed semen, and apparently, it was concluded that since natural fertilization requires billions of sperm, then, apparently, there are physical limitations to the implementation of artificial insemination quantities of sperm that are as much as four orders of magnitude less. Thus, it is not surprising that existed to some extent the actual teaching, which stood just to the side of the direction in which were the authors of the present invention. It is possible that their results can even be considered to some extent unexpected, because they showed that it is possible to achieve artificial insemination with low doses of sexed semen with high efficiency, comparable to that obtained with conventional artificial insemination high doses not sexed semen. Some may also seem surprising that the methods and achievements of the present invention are effectively combined to obtain the presented results. Although the result of each individual of the way some of them can be considered as insignificant, in fact, small changes lead ultimately to the floor is the value of significant progress in the final result - to consider whether individually or in combination with other minor changes.

Thus, until the present invention it was impossible to achieve high performance in artificial insemination with low doses of sexed semen, with the required levels of performance or simplified procedures required for use in a production environment. In addition to the achieved production level of artificial insemination with low doses of sexed semen, the present invention also describes ways to achieve superior performance, and thereby improve the desired end result, namely artificial insemination with low doses of sexed semen on a commercial basis.

III. Description of the INVENTION

Accordingly, the present invention claims the achievement at the commercial level, insemination with low doses of semen and the results of its application to a given floor in mammals. It also applies to the superior capsule and collector system for the separation of sperm cells to determine their sex by using method flow-cytometrical separation. In this method of separation is usually used in the capsule flow cytometer fluid (hereinafter briefly called "capsular fluid") Zam is both fluid which minimizes the stress on spermatozoa during their separation. In addition, the system of collection of sexed sperm is improved in such a way as to minimize both physical and chemical stresses, which are sperm. Presents various methods and substances, but experts in the art can easily understand that it is possible to use various combinations and permutations, thus, to optimize the implementation of the method for different types of animals, methods of separation, goals, work, and other parameters associated with a specific application of the method.

The purpose of the invention, therefore, is simply to achieve the implementation of artificial insemination lower doses of sexed semen by the way, working in a real production environment. The goal also is to achieve improved separation for items such as sperm. A related objective is to minimize the harmful impact of the operation of the separation of cells or other sensitive components, which can be subjected to separation. For the method flow-cytometrical division specific objective is to minimize the harmful impact of caps is through fluid in cells, and to offer capsule liquid, which has a positive effect on the cells, improving conditions of work with them in various conditions associated with flow cytometry stress. A parallel goal is to offer substances and methods that are particularly suitable for sperm in General, and also for sperm of bulls, stallions sperm, and to separate such sperm on components carrying the X - and Y-chromosomes. Similarly, the objective is to minimize the harmful effects that has on cells in the phase of collection (for example, after separation), as well as to minimize both physical and chemical harmful impacts to these sexed cells. Thus, the aim is to achieve this separation, when the cells as little as possible would be invalid.

Another object of the invention is to achieve the implementation of artificial insemination lower doses of sexed semen at levels that are comparable to the levels obtained for conventional artificial insemination high doses not sexed semen. To this end the present invention proposes a General system of artificial insemination, which can be used to achieve this goal in practice, in the production environment. T is thus, the above goal of minimizing stress or possible damaging effects on sperm, are important. The separation in a way that allows you to have both high speed and separation at low stresses, and which is particularly adapted for the separation of spermatozoa in the context of low doses of their application, is also an important aim of the invention. An important goal is to offer such a capsule or other fluid for flow cytometry, which does not have a negative impact on fertility, sperm and which is suitable for artificial insemination.

Other objectives of the invention are described in other sections of the present description and claims.

IV. BRIEF DESCRIPTION of DRAWINGS

Figure 1 is a schematic illustration of sorcerey system in accordance with the method flow-cytometrical separation according to the present invention.

Figure 2 is a schematic representation of the captured cells in the zone of free fall a conventional flow cytometer.

Figure 3 is a conceptual diagram that shows the differences resulting from the present invention.

Figure 4 is a schematic illustration of the flow of the divided cells collected in the zone of subsidence.

V. BEST RE IS THEM carrying out the INVENTION

As will be seen from the following, the basic concept of the present invention can be combined and implemented in different ways. The invention includes simply feasible in the production practice of artificial insemination with low doses of sexed semen and its results. For methods flow-cytometrical split the invention also includes an improved system of flow cytometry and authoring system aggregates of sperm specified floor that can be used in artificial insemination, as well as the animals obtained in this manner. The invention includes a combination of ways in which you can achieve high performance insemination in a production environment. In addition, the methods described in General terms so that they can be used for specific systems and applications, based on General principles. What is the description of the improved device, it should be borne in mind that these improvements are not only certain ways, but they also can be varied and combined in many different ways. It is important to bear in mind, and this applies to all the above that each of these aspects is covered by the present description.

As mentioned, the main goal is the Department of the research Institute of sperm, bearing X chromosome from the sperm that carry the Y chromosome. This exercise method by which distinguish these two types of sperm, so that each type can be packaged separately and separately to work with him. Currently the selection is preferably carried out using flow cytometry. Flow cytometry in General is a well-known method. For example, its main aspects are presented and discussed in a number of patents firm Cytomation, Inc., such as the U.S. patents and other listed publications. All of these patents or references cited in the present description, is included as a reference; therefore, specialists in the art can easily understand the basic principles used.

Essentially, flow cytometry involves the separation of elements, such as cells that enter the device running cytometer from any type of source cells. The conceptual apparatus is shown in figure 1. The device is flowing cytometer includes a source (1) cells, feed the cells or any other type of elements to be analyzed in a flow cytometer. Cells are located inside the nozzle (2) in such a way that these cells are surrounded by a capsule of liquid (3). Capsular fluid (3) usually takes any source (4) capsule liquid so that when the source (1) cells delivers the yrs, capsular fluid (3) is simultaneously supplied through the nozzle (2). Thus, it is easy to understand how the capsular fluid (3) forms the environment for the cells, consisting of a capsule of liquid. After running cytometr received several liquids under some pressure, they flow from the nozzle (2) and out through the hole (5) of the nozzle. By including in the device device type oscillator (6), which can be very accurately controlled using the device (19) control oscillator inside the nozzle (2) you can create pressure waves and transmit them to fluid flowing from the nozzle (2) through hole (5) of the nozzle. Since the oscillator (6) thus affects the capsular fluid (3), jet (7)arising out of the hole (5) of the nozzle, from time to time and regularly forms drops (8). As the cells are surrounded by an environment that represents capsule liquid, drops (8) can contain individually selected cells or other elements.

Because drops (8), usually contain isolated cells, flow cytometer can distinguish and to separate droplets, based on the fact, contained or not contained certain cell or cells within a given drop. This is done by using the system (9) cell recognition. Recognition of cells includes at least any type of sensor (10), d is giraudeau cells, contained within each droplet (8), as described in detail in the work of Larry Johnson, namely, in U.S. patent No. 5135759. As described by the patent Johnson for sperm, the system (9) recognition of cells may be affected, depending on the relative presence or relative absence of a specific dye that can be excited by any stimulant such as laser exciter (11). Although the dye stains all types of sperm, but different length of the X-chromosome and Y-chromosome creates different levels of staining. Thus, by detecting the degree of staining present in the sperm, it is possible to distinguish cells that carry the X chromosome, from cells carrying the Y chromosome, on the basis of their different levels of emissions.

In order to achieve complete separation and highlight the corresponding cell in the method flow-cytometrical separation, the signals received by the sensor (10), served in any type sorcerous the recognition system (12), which quickly makes the decision and can charge every drop of (8), depending on whether contained or not contained in the drop (8) desired cell. Thus, santerna sensing system (12) allows electrostatic reflectors (13) reject drops (8) on the basis of whether they contain or do not contain with testwuide cell or other element. As a result of this flow cytometr parts of cells, causing them to fall into one or more collectors (14). Thus, by the recognition of certain properties of cells or other elements flow cytometer can distinguish cells based on their specific characteristics and place them in the appropriate collector (14). In the system currently used for the separation of sperm drops with sperm containing the X chromosome, is charged positively and thus deviate in one direction, and drops the sperm containing the Y chromosome, become negatively charged and deflected in the other direction, and the flow of defective cells (i.e. those that could not be divided by sex) are not charged and therefore going in netcontrol jet, acting in the suction tube or similar device.

Looking at figure 2, you can more clearly imagine this process. As shown in this figure, the nozzle (2) is a stream (7), which is due to the oscillator (6) (figure 2 not shown) to form droplets (8). Because the source (1) cells (figure 2 not shown) may submit spermatozoa (15), colored according to the method of Johnson, the sensor (10) differential determines the light stimulation, which arose under the influence of the laser exciter (11), so that the flow cytometer can the t to control the presence or absence of charge in every drop, when it is separated from the jet (7). Such management results in positively charged, negatively charged and uncharged drops (8) on the basis of their content. In figure 2 some drops shown as rejected droplets (16). These rejected droplets (16) represent the drops containing spermatozoa (15) of one or other sex. They are then placed in the appropriate collector (14) for later use.

One of the aspects of flow cytometry, particularly important for its application for separation of sperm, is a high-speed operation of the flow cytometer. Achievements in this direction were made, in particular, the company Cytomation, which produces a flow cytometry under the trademark MoFlo®. These flow cytometry have a very increased rate of separation that makes flow cytometry method that allows to subdivide the sperm on the floor in a production environment (among other commercial applications of this method). These flow cytometry operate at a very high speed of separation, that is, at a speed, which is much higher devices used another brand. Specifically, a flow cytometry brand MoFlo® company Cytomation operate at the frequency of oscillator components higher than about five kilohertz, and more specifically, they can work item and the frequency in the range from 10 to 30 and even 50 kHz. Thus, the droplets are formed at very high frequencies, and cells that are in the environment of the capsule liquid can quickly fly out of the nozzle (2). As a result, each of the components, namely the nozzle (2), the oscillator (6), etc. that are part of the system of the flow cytometer, it is possible to configure or to choose so that the result is a high-speed cell sorters. In this application a high-speed cell sorting device for separating spermatozoa reach speeds of separation, which comprises more than 500 transactions per minute. In fact, high-speed cellular sorcery reach speeds of separation in the range from 1000 to 1200 transactions per minute. It is very important, and this should be borne in mind that the term "high speed" is relative, so if will be developed other improvements on the flow cytometer or specific applications, the aspect indicated by the term "high", can either be changed, or its value may be absolute. Under any definition, the main principle is that the separation can occur at speeds under which the parameters and physical characteristics of the flow cytometer are essential for the cells in the separation of specific types of cells such as sperm cells.

One aspect vysokoskorostnogo the separation, which, apparently, plays a role in the separation of spermatozoa using flow-cytometrical way, is that being in a flow cytometer, the cells are subjected to pressure and other stress factors. For example, when operating at high speeds (or, in the alternative definition, "high-speed") flow cytometry can work under pressure in 344,80 PA and even 413,86 PA. These pressure indicators can be considered high, because they can lead to exposure to shared cells. The key point, as indicated in the present description, for this aspect of the invention is that the threshold stresses for a particular cell are the determining factor. In addition, as it turned out later, the threshold effects of stress can be a function of the combined effects, such as a particular kind of animal or specific operations carried out with cells before or after stresses. The key here is that the stress on the cells can change their viability and their ability to give desired results. In the case of pressure, it may happen that only effects on sperm increased pressure as a result of work under this pressure, the flow cytometer can lead to pony the structure of red blood cells. The present invention in one of its aspects is directed to minimizing these stresses, and thus, results in increased efficiency, as well as the possibility of using lower doses of semen, as described below.

As for the aspect associated with stress effects on cells, the present invention operates in such a way that it reduces these stresses to a minimum. These stresses can be minimized at any point in the cycle of collection and separation of sperm and even insemination of animals. It is important that the stress caused by processing cells in the flow cytometer, is essential for this application. In one of the embodiments of the invention specially selected capsular fluid so that it was coordinated with the liquid environment in which there are cells to divide and/or liquid environment in which there are cells after division. Although, of course, can be adjusted with either a liquid medium in which the cells are to separation or liquid medium which contains cells after division, but in one of the embodiments of the present invention regulate the composition of the capsule liquid (3) in such a way that it had significantly less stress on the cells than was the case prior to the present invention. the present invention is remarkable in that in particular, it focuses not on the technical aspects of the operation of the flow cytometer, the processing of the cells and removing the effects of stress on them. For example, although it is known the use of liquids having a specific pH or osmotic pressure, the present invention sets that can be defined chemical composition, to which cells can be hypersensitivity. Such sverhreaktsiya chemical compositions, of course, can vary depending on the cells or even depending on previous processing of the cells. Currently, it is important that sperm there are certain metabolic chemical composition, such as citrates, which seems to prevent unusually high levels of stress effects on cells. Thus, sverhreaktsiya chemical composition can be defined as such compositions, to which cells show specific reactivity in the context of their functional activity and ways of processing cells. As for the sperm, it was found that the metabolic composition, in particular based on the citrate composition for sperm of bulls and based on hepes-buffer compositions for sperm stallions can be very important. Thus, the us is Aasee invention acts as that by certain types of operations or selection of substances it minimizes changes to the cells.

With regard to capsular fluid, in accordance with one embodiments of the invention chosen substance, chemically coordinated in such a way that it caused minimal changes. Thus, by selecting the appropriate capsular fluid not only in the context of parameter flow cytometry, but rather in the context of the parameters of the cells, it is possible to improve the situation changes with the cells, and the end result of cell division. This is conceptually presented in figure 3. The figure 3 shows some type of chemical factors (such as citrate or other factors), how can it exist within the different phases of this process. For example, the four shown in the figure, the phase can be represented as the following for the method flow-cytometrical separation, but not be limited to: phase I can be a stay of cells in source (1) cells, phase II may show the presence of cells in the environment of the capsule liquid during their separation, phase III may represent cells, collected after separation, and phase IV may represent the recovered cells in the storage medium after separation. These che who ever got phase, as they are shown for the prototype can be widely varied with respect to chemical factors of the environment. However, as conceptually shown in the figure, in the conditions of the present invention, the cells can undergo very little change, and it is particularly notable that the difference between phases I and II can be practically absent. This is the result of selecting the appropriate capsule environment, as mentioned above. Thus, due to the fact that cells exposed to a suitable capsule environment, the cells of the present invention can have significantly lower levels of stress.

One of the potential uncertainties that may exist for this phenomenon is that some chemical compositions can be more sverhreaktsiya than other chemical compositions. Although, of course, this may vary depending on the species of sperm, its processing, and even the type of cells used, but it was found that the cell viability for their intended use (in this case for artificial insemination) greatly varies, either by natural causes or as a result of the separation, or because of both these reasons, and therefore, cells exhibit hypersensitivity to the Oprah is elenai chemical composition. By selecting certain metabolic chemical compositions, most preferably citrates or chemicals from the citric acid cycle, you may receive the greatest benefits. So, for bull semen capsule liquid (3) select and coordinate so that it was a composition with a content of about 2.9% sodium citrate. Specifically, 2,9%solution of sodium citrate can be obtained as follows:

1. Place 29,0 grams of sodium citrate (Na3With6H5O7·2H2O) in a volumetric flask of 1000 ml

A. Dissolve the sodium citrate in 3/4 servings of water, and then add water to volume.

2. Add deionized or purified using the device Nanopure water, bringing to a final volume of 1000 ml.

3. Transfer the resulting solution into bottles and prostokrasivaya when the pressure 1,021 ATM (118,3°C) for at least 30 minutes.

A. Autoclavicle solution under conditions, which reduces evaporation to a minimum (loosely covered with a lid).

b. Make sure that the water has not boiled away.

4. Slowly cool at room temperature.

5. Store unopened in a cool room where the temperature is 5°C.

Further, for use as a capsule liquid solution of sodium citrate can be filtered.

6. Filled profile is route through a filter with holes 0.22 micron.

It is interesting to note that sperm stallions such a composition is not so good, as for the sperm of bulls. It has been found that sperm stallions good medium with hepes-buffer, such as hepes-environment for gametes bulls, in particular the environment HBGM3 previously developed J.J.Parrish for sperm of bulls. This environment is discussed in the article "Capacitation of Bovine Sperm by Heparin", 38 Biology of Reproduction 1171 (1988), which is incorporated into this description by reference. This is surprising not only because it is not the same type of substance that is used for sperm of bulls, but in fact the buffer, which was originally developed for application to sperm of bulls. Thus, to apply for sperm stallions choose this capsule liquid, which contains hepes buffer. This solution may have a pH value at room temperature of about 7,54 (pH at 39°C = 7,4) and the following composition:

ChemicalDry weight (g/500 ml)
CaCl20,145
KCl20,115
MgCl2·6N2About0,004
NaH2·PO4·H2O0,018
NaCl 2,525
The sodium pyruvate0,011
Lactic acid (60%)of 1.84 ml
HEPES4,765
NaHCO30,420
BSA (bovine serum albumin) (fraction V)3,0

One other aspect that may play a role in the present invention is that it uses cells can exhibit unusual sensitivity. On the one hand, this may be due to the fact that the sperm belong to the class neustanovivshiesya cells. This means that they do not possess the property of self-healing and as a result, they require a much more careful treatment than that which is typical for flow cytometers and other equipment designed to work with cells. Thus, we can assume that by the invention the improvement is particularly applicable when the flow cytometer or other separating device generates a source of sperm. Other potentially related to this aspect of the invention, which can have a unique value for this class of cells, like sperm, is the fact that their DNA is preparamos, rereplacenocase and retranscribing. Any of these factors may come into effect, and the poet is mu they can be considered either individually or together. Thus, the provisions of the present invention can be applicable to all gametes or even for viruses and similar objects, which are preparerow, retranslateui and retranscribing cells.

Another aspect of flow cytometry, which can also be important, is the correct way to treat cells as in chemical and physical sense, after their separation. As shown in figure 4, when inside droplets (8) cells sink to the bottom of the collector (14), can be very important that the container represents part of a collector, had the right size, so it worked as a means of preventing mutual cell damage each other and the container itself. Although it is known the location of source collector fluid (17) at the bottom of the container, to collect cells so that they do not hit the bottom of the container, but it turned out that for better result you can simply expand the container to fit the different variations in the characteristics of the jet, and also took into account the inevitable splashing occurring due to the shock of the cells of the container. On the one hand, it can act as a cushioning element that facilitates the careful handling of the cells, which can be mechanically fragile, meaning that they can split the change or be damaged under the impact of the blow. Thus, when the source cytometer do physically fragile cells to be separated, can be very important to create some type of shock-absorbing element, such as a wide collector tube, the width (18) holes which serves for positioning the walls of the container thereby to prevent contact with the cells. Thus, this tube is arranged so that its side walls are not so close to each other that there is any significant contact between shared by the cells and the walls of the tubes. Thus, in addition to the use of the collector fluid (17), may also be desirable to have a wide collector tube. Perhaps it will be enough just to make a wide opening of the container, which is part of the collector (14). For applications using high speed separation of sperm found that it is enough to have a container with an inner diameter of the hole constituting at least 15 mm, Specifically, found that when used for such applications 14-ml test tubes Falcon is minimum damage to the cells in the reservoir (14).

It should be noted that even 14-millilitrovoj test tube Falcon may not be optimal. Specifically, suppose that the design number is cornago container, the corresponding geometry of the jet (i.e. the container that corresponds to the stream")may be optimal. This corresponds to the spray container may have any or all of the following characteristics: a relatively wide hole, elliptical hole, the ratio of height to width that is less than currently used, the location of the parties at an angle or otherwise coordinated way, for example, the side walls may be parallel to the incident jets, etc. May also be desirable to have a mounting element, such as a movable element or the environment, such as ball bearings or the like that allows you to change the orientation of the impinging jet, you want to collect. In addition, the physical characteristics of this class of containers, as the existing tube (described as a test tube-type "Falcon"), may include not only the width of the tubes, but also the material (such as polystyrene, to which cells do not adhere), which made the container, and the like (These materials are well known for 14-mm Falcon tubes). Thus, the container and its collector fluid can also serve as shock absorbing elements to minimize physical damage to the cells. It may also be due to its size, to facilitate the collection is relevant to the respective amounts of semen without substantial dilution.

Another aspect of the collector fluid (17) can be that it can also serve to minimize the chemical effects of stress on the cells. On the one hand, because it can be important to ensure the supply of nutrients to the cells both before and after separation, the collector fluid (17) can be selected so that it provides a coordinated level of nutrients, so that these levels were balanced both before and after separation. For sperm of a bull, for which nutrients are used citrate egg yolk, when the content of egg yolk of two percent, found that use of the content citrate egg yolk of six percent (i.e. six percent content of egg yolk in a solution of citrate) provides good results. This is a result of the influence of volumes, existing before and after the split operation. The initial volume of the collector fluid (17) may be (before the split) from about 2 ml of the Operation of division can add approximately two times larger volume (the result that by the end of the separation fluid volume is three times the original volume), with very little content citrate egg yolk in solution (due to the fact that he was going to clap what I and other reasons, associated with flow cytometer). Thus, the end result in regard to the content of citrate egg yolk may be equivalent to the initial value, namely, two percent content of egg yolk in a solution of citrate, thanks to the used volumes. Thus, the collector fluid (17) can be chosen such that it provided for the creation of a final collector liquid environment, balanced with the initial nutrient content or other liquid medium. Due to this, it can serve to minimize the time and extent of changes in the content of the song, which are cells. Naturally, such a liquid medium can be contained in a flow cytometer or can exist for some time before entering into it, and the important point is to minimize stress, which are the cells at any time in their life cycle. In addition, because the original content of chemical substances can be varied (for example, the content of egg yolk in the citrate can be changed in the direction of increasing or decreasing), then the conditions of the original collector of the liquid or its various volumes also can be varied so that the end result was the same. Thus, before beginning the process divided the I manifold liquid has a content of egg yolk in the six percent solution of citrate, and after the separation of the collector fluid with sexed semen may have a content of egg yolk in a solution of citrate of two percent is the same as the initial content of nutrients.

Please note that continued use of these sperm can be in other ways treated with a solution of citrate with 20% egg yolk, however, these changes do not have to stress on the cells, because it is just part of the process of artificial insemination. Although, of course, the levels can vary, which is understandable to experts in the field of technology, nitrate buffer with 20% egg yolk may have the following composition:

I. FINAL COMPOSITION:

80% solution of sodium citrate (72 mm)

20% (by volume) of egg yolk

II. COOKING FOR 1 LITER:

A. a Solution of citrate

1. Place 29,0 grams of sodium citrate (Na3With6H5O7·2H2O) in a volumetric flask of 1000 ml

2. Add deionized or purified using the device Nanopure water, bringing to a final volume of 1000 ml.

3. Transfer the resulting solution into bottles and prostokrasivaya when the pressure 1,021 ATM (118,3°C) for at least 30 minutes.

A. Autoclavicle solution under conditions, reducing their evaporation to a minimum (loosely covered with a lid).

b. Make sure that the water has not boiled away.

4. Slowly cool at room temperature.

5. Store unopened in a cool room where the temperature is 5°C.

C. PREPARATION of EGGS

1. Buy fresh chicken eggs in a reliable commercial enterprise.

2. Wash the eggs to keep them clean of dirt (do not use too much detergent) and rinse them.

3. Immerse the eggs in 70% ethanol for 2-5 minutes.

4. Remove eggs from ethanol and let them dry (or dry) and keep it on a clean towel.

C. GETTING THINNER

1. Take a sterile clean glass dish.

2. A-faction (faction without glycerol)

A. Place 800 ml of 2.9% solution of sodium citrate in a graduated cylinder 1000 ml

b. The levels of antibiotics for a fraction of diluent that does not contain glycerin (fraction A) can be the following:

i. Tylosin = 100 mg/ml

ii. Gentamicin = 500 mg/ml

iii. Linko-spectin = 300/600 mg/ml

C. Add 200 ml of fresh egg yolk, as described below (section D).

i. Very carefully stir.

d. The result will be a fraction of the diluent on the basis of 2.9%solution of sodium citrate, 20% egg yolk and antibiotics at concentrations known as non-toxic for bull semen.

that is, the diluent can be stored until the next day at 5°C.

g. Heat to 37°With, before you can use the next day.

D. to add egg yolk to the buffer, use the following procedure:

1. Wash and dry the eggs (see above).

2. Break the egg and separate the yolk from whites with yolk separator. Alternatively, 2-3 times pour the yolk from one half shell to the other. Do not break the shell around the egg yolk.

3. Place the yolk on sterile filter paper of size 15 cm

4. Keep the filter paper over the graduated cylinder containing the buffer, squeeze the yolk (breaking its shell) and let the yolk run down the filter paper into a cylinder. Usually one egg, you can get about 12-15 ml of yolk.

Another aspect that can interact with different factors of the present invention is to use low doses of semen for artificial insemination and the like. Additional rationale for this aspect of artificial insemination of sexed semen can be found in the publication "Prospects for Sorting Mammalian Sperm" by Rupert P. Amman and George E. Seidel, Jr., Colorado Associated University Press (1982), which is incorporated into this description by reference. As mentioned, in the natural fertilization spent the greater the number of sperm, making about a billion sperm. Conventional artificial insemination are currently engaged in the use of millions of sperm for cattle and hundreds of millions of sperm for horses. The term "low dose" is meant that the amount used for one insemination sperm is less than half, or preferably less than 10% of the amount of semen released during normal artificial insemination. Thus, the term "low dose" should be considered in the context of the doses used in conventional artificial insemination or as an absolute number. For bull semen, which currently varies from 1 to 10 million sperm, by way of low-dose considered such a way that the absolute quantity of sperm is about 500000 and maybe even 300,000 sperm. In fact, when using the methods of the present invention showing the possibility of achieving good performance of artificial insemination using quantities of sperm components 100000 and 250000 sperm (percent occurrence of pregnancy 41% and 50%, respectively), as described in "Uterine Horn Insemination of Heifers With Very Low Numbers of Non-frozen and Sexed Spermatozoa", published in Theriogenology 48 1255 (1997), which included the present description by reference. Because the sperm, obviously, show sensitivity to dilution, these results may have significant impact on the use of sperm samples with low doses of sperm, in the context of various methods of the present invention. The absolute number of sperm may depend on species. For horses low can be considered a dose that make up less than twenty-five, ten, five or even one million sperm.

Another aspect that may be important, is that in the system of artificial insemination using sperm sexed by the methods of the present invention or by other means. Thus, in the case when the method of flow cytometry collector (14) is used to obtain sperm for artificial insemination, the methods of the present invention is particularly applicable. In addition, it is also possible that the combination use of for artificial insemination and the use of low doses can work together to create such interaction, making the various methods of the present invention is particularly suitable. Of course, sexed semen can be used not only for artificial insemination, but in other ways, such as in vitro fertilization, etc.

The method of collection, separation sperm is optional insemination of animals using flow cytometry or other methods of separation of sperm, includes a number of stages. In the context of insemination of cows, first take the bull sperm through the use of an artificial vagina. This occurs with the release of approximately 1.5 billion sperm/ml of This bull semen can be checked using a spectrophotometer to determine its concentration, and can also be examined microscopically to ensure that it meets the standards in terms of motility and viability of spermatozoa. You can then add antibiotics. The original sample may have from about 60 to 70 percent of sperm with increasing mobility in the ejaculate. For sperm treatment can be used to dilute any type TALP (tirumalaisamudram), in order to obtain a working concentration of sperm (for flow-cytometrical analysis), approximately 100 million sperm per ml TALP not only nourishes the sperm, but also can make them sverhkachestvennye for stage of painting. Before staining for sperm of some species of animals such as horses, you can spend centrifugation. Staining can be performed in accordance with the method of multiple or single staining, the latter method is the subject of a patent Johnson and related technologies. Staining can in order to carry out while adjusting the content of a diluent, to obtain an appropriate nutrient medium. In applying for bull semen you can add about 20% egg yolk in a solution of citrate immediately after staining. Further, when the staining of spermatozoa was found that the best results can be obtained when using higher amounts of dye than you would expect. The high concentration of the dye may include the use of quantities of dye components dozens of micromol, as described in the following examples, which used a 38 micromol dye Hoechst 33342.

After adding the dye, you can use the incubation period, such as incubation for 1 hour at 34°to speed up the absorption of the dye at concentrations of about 100 million sperm per ml can Then be filtered to remove lumps of sperm, and then diluted to the desired concentration for the separation of sperm on the floor, about 100 million sperm per ml After that you can make the separation of sperm on the floor in accordance with the various methods discussed above, after which the sperm enter the unloading phase (collect). As mentioned above, after collecting sperm samples may be available, containing about 2% citrate egg yolk (of semen). Then it clicks the set of technical documents can be concentrated to about 3-5 million sperm per ml, by centrifugation, after which the capsule liquid and the protective liquid can be removed. You can then have a final dilution by adding either 20% citrate egg yolk, or Cornell universal diluent or the like. Cornell universal diluent may have the following composition per 1000 ml:

14.5 g of sodium citrate

2.1 g NaHCO3

0.4 g KCl

3.0 g glucose

9,37 g glycine

0.87 g of citric acid

For the composition with 20% egg yolk, you can use 800 ml of the above composition and about 200 ml of egg yolk.

After this last dilution can be obtained from 3 to 5 million sperm per ml (semen). Then, the sample (a set of spermatozoa) can be cooled to slow the metabolism of the sperm that will allow you to use it over a longer period of time. For horses the sample can then be used to alcaravaneras or other methods of insemination, well known to specialists in this field of technology. For bull semen samples can again be diluted until the desired level of dose. It is established that the dilution may affect the viability of sperm, and it is therefore desirable to avoid too much dilution through the use of a smaller amount is and samples. Regardless of the methods of separation of sperm on the floor, it is now possible to obtain approximately 300,000 sperm at 0,184 Jr. in Addition, it may be desirable to maintain the content of seminal plasma by about five percent, although the results of this requirement currently controversial. Samples of sperm can then be placed in straws for future use in artificial insemination and transported to subject insemination for cows and heifers.

In order to achieve the convenient timing of artificial insemination, it is possible to synchronize the onset of oestrus in cows and heifers using known methods, such as the use of prostaglandin F2αin accordance with methods well known in the art. The latter substance may be especially valuable because recently there have been reports that it may increase fertility in heifers, which are discussed in the article "Prostoglandin F2α- A Fertility Drug in Dairy Cattle?", 18 Theriogenology 245 (1982), which is incorporated into this description by reference. Although we have recently received the results did not confirm this assumption, perhaps, the present invention demonstrates that it is particularly legitimate in situations of artificial insemination low to the AMI sexed semen. For cows artificial insemination can be performed through the use of equipment for transplanting embryos, placing sperm deep into the uterine horn. This can be done not at the peak moment, as is usually done by artificial insemination, and a little later, about 12 hours after that time, because there is some possibility that fertility artificial insemination of sexed semen may come later. Equipment for transferring embryos is recommended because of the uterus can be very sensitive to this artificial insemination with low doses of sexed semen.

In addition, the methods can be combined and also in order to achieve higher productivity. In particular, the methods of the present invention, enabling high-speed separation and insemination with low doses of sexed sperm, it is also possible for animals with multiple ovulation (paleovalley). Preovulation can be achieved through the use of Pro-preovulation pharmaceutical preparations or in any other way. Pro preovulation pharmaceutical drugs can act directly or not directly, for example, through a sequence of reactions, reaching zoom is authorized in comparison with the normal production of eggs. Combination with paleovalley is unexpected, as previously suggested that polyvalence prevents the implementation of such a combination. The transport of sperm in animals with paleovalley difficult; therefore, animals are often artificially seminal several times and/or multiple doses of semen. In addition, before splitting procedure, the sperm on the floor were relatively slow; therefore, it was interesting to determine the effectiveness of fertilization after a single insemination in cattle with paleovalley-driven pharmaceutical agent, such as FSH (a hormone that stimulates follicles), using only 600000 total number of thawed spermatozoa, with these new combinations of methods.

For example, the twelve crossbred Angus heifers caused preovulation using standard methods: 6, 6, 4, 4, 2, 2, 2 and 2 mg of FSH was injected intramuscularly at intervals of twelve hours, starting between 9 s and 12 s-day estrus cycle; 25 and 12.5 mg of prostaglandin F-2 alpha was injected intramuscularly with the 6th and 7th injections of FSH. Semen from bulls of known fertility were stained with Hoechst dye 33342, and then divided by sex by using a MoFlo flow cytometer®/cell sorting device, receiving at 700-800 live sperm of each sex per second. The average purity of the separation was 89% desired by the and. Sexed semen was concentrated until 3,36×106spermatozoa/ml by centrifugation at 650 g for 10 minutes, cooled to 5°and kept for 4 hours. Then 184 μl were loaded into plastic straws 0.25 ml; half the dose was injected into each uterine horn after 20-24 hours after the onset of estrus, with automatic capsules with side holes for transplanting embryos. Embryos were removed using standard surgical techniques 7 or 16 days after estrus. The results were similar for embryos, taken on 7-th and 16-th day, and the same for X - and Y-sperm. The embryos were taken at 9 heifers. Just was 52 embryo (average of 4.3+/-5,3 on the donor) in the normal stages of development, 13 embryos with developmental delay and 31 unfertilized egg. At forty-six embryos identified gender by PCR using primers for DNA sequence specific for the Y chromosome; 43 (93%) of them were expected sex. Although this study involved a small number of animals, it was found that the insemination of heifers with paleovalley using only 600000 (live) unfrozen sexed sperm gave results similar to the results of conventional methods. You can also make variations of the method described above, including, but not limited to, split the sperm else than flow-cytometrics, by the way, the achievement of preovulation other ways increase fertility in other ways, etc.

Further, the consistent application of methods of separation of sperm by sex, based on DNA content, high-speed flow cytometers/cell sartarov, as well as methods of insemination of heifers number of sperm that make up less than 500000 total number of spermatozoa, without reducing fertility, allowed only a few years to achieve commercial production viable sexed spermatozoa of cattle. There is a huge number of applications for sperm sexed with an accuracy of >85%. Apparently, the most obvious of these is the artificial insemination of one of the subpopulations of cattle (both dairy and beef) for repair consisting of females of the herd, and create the opposite subpopulations (both dairy and beef cattle), consisting of a completely different type of bulls, for rearing calves for meat. A very important point above is the insemination of heifers with sperm carrying X-chromosomes, for heifers, at birth, which happens to be fewer cases of obstructed labor than at birth, calves, apparently, due to the smaller size of Chicks. In addition, the child young producers dairy cattle is much more effective when the prevalence among calves heifers. Among the calves more than 85% of heifers also makes feasible such breeding dairy cows, in which they on average earn less than two surviving calves during the period of their life that is attractive because it reduces the problems associated with pregnancy and childbirth. Be also feasible to same-sex system of breeding beef cattle, in which each female produces replacement yourself, and at the age of between 2 and 3 years its score, which accounts for the much higher percentage of feed used for animal growth, and less interest in maintaining the grown animals. Sexed sperm is also useful for in vitro fertilization and insemination of cows with paleovalley for transplant embryos. Often one floor calves more valuable than another floor, and although there are accurate methods of separation of embryos on the floor, but they require a large amount of time, and half of the resulting embryos will be less valuable floor. It is suggested that accurately sexed semen will be well suited for artificial insemination of livestock, if the load when the separation of sperm on the floor were low, and fertility disturbed only minimally. Probably will increase significantly the percentage of beef cattle artificially inseminated split is on the floor with sperm.

Unexpectedly it was found that the best results can be obtained when the insemination is not by placing sperm inside the body of the uterus, as is usually done, and by placing it deep in the uterine horn. You may also seem surprising that in the investigated samples revealed no differences between the ipsilateral and contralateral insemination, if it was carried out deep in the uterine horn. The word "deep" is to be understood that the tool for insemination reliably enter into the horns of the uterus tool for transplanting embryos. The fact that the results of the ipsilateral and contralateral insemination vary insignificantly, led the authors of the present invention to the proposed use of insemination both of these methods, so that the process of identification of relevant horns of the uterus may be no longer needed.

In the insemination, of course, desirable to obtain the animal desired sex. This animal can be obtained by using the previously discussed systems, through the use of sets of sexed sperm. It should also be borne in mind that the methods of the present invention can find application in other methods, such as laproscopically insemination, acepromazine insemination, etc.

As examples conducted the following experiments. X is although not all of them used all the aspects of the invention described here, but they convincingly demonstrate the advantages obtained with the aid of various aspects of the invention. In addition, a summary of some of the experiments contained in the article "Uterine Horn Insemination of Heifers With Very Low Numbers of Non-frozen and Sexed Spermatozoa", cited earlier. This article summarizes the results of some studies showing the effectiveness of the present invention. As for experiments, one conducted with the use of sexed frozen semen, and have achieved high performance, as described below:

EXAMPLE I

Angus heifers at the age of 13-14 months, moderate fatness, was sinhronezirovala by injecting 25 mg prostaglandin F-2 alpha in 12-day intervals and were seminal through 6-26 hours after they noted the steady estrus. Fresh semen obtained from three bulls at the age of 14-26 months, incubated at 38 μm Hoechst 33342 at a concentration of 75×106spermatozoa/ml in TALP medium for 1 hour at 34°C. Sperm were separated according to sex chromosomes on the basis of epifluorescence excited by a laser at 351 and 364 nm, 150 mW, using a MoFlo flow cytometer®/cell sorting device operating at a pressure 344,74 kPa, and the use of 2.9%sodium citrate as a capsule liquid. The sperm that carry the X chromosome (with a purity of ≈ 90%, which is conducted by the Yali by re-dividing the processed ultrasound aliquot of sperm) were collected with speed ≈ 500 live sperm/sec in test tubes Eppendorf 2 ml containing 100 ál of Cornell universal diluent (CUE) with 20% egg yolk. The collected sperm was centrifuged at 600 g for 10 minutes and resuspendable to 1.63×106live sperm/ml in the CUE. To get the control liquid undivided by sex sperm stained with Hoechst dye 33342 semen was diluted capsular fluid to 9×106spermatozoa/ml, centrifuged, and then resuspendable to 1.63×106sperm with increasing mobility per ml, in the CUE. Separated by sex and the fluid control semen was cooled to 5°C for 75 minutes and was loaded into a 0.25-ml straws (184 μl on a straw). Straws transported at a temperature of from 3 to 5°With a device for cooling drinks with controlled temperature, at a distance of 240 km/ to insemination, which was carried out later, from 5 to 9 hours after sperm separation. Insemination of sexed and liquid control sperm was performed using blue capsules with side holes (IMV), using half of each of the straws in each uterine horn (3×105live sperm to the calf). As a standard control, semen from the same bull were frozen in straws 0.5 cm3using STD is bound methods (average of 15.6× 106motile sperm per dose, after thawing), thawed at 35°C for 30 sec, and then introduced into the body of the uterus. Options were distributed among the 3 bulls and 2 operators of artificial insemination in the ratio of 3:2:2 inseminations for option sexed semen and two controls. Pregnancy was determined by ultrasound way through 31-34 days after insemination and confirmed through 64-67 days after insemination, when the fruit was possible to determine the sex (blind). The data presented in the table.

OptionThe number of heifers are inseminatedThe number of pregnant heifers on 31-34 dayThe number of pregnant heifers at 64-67 dayThe number of female fetuses
Sexed semen4520 (44%)IS (42%)18 (95%)a
Liquid control2815 (54%)15 (54%)8 (53)b
Frozen control2916 (55%)15 (52%)12 (80%)c
a, bSex ratio of indicators with different letter indexes differ (P<0,02).

Although the percentage of pregnant the minute when the insemination of sexed semen was only 80% of the controls, but this difference was not statistically significant (>0,1). One pregnancy in each group, i.e. the variant with insemination of sexed semen and controls with liquid and frozen semen, 64-67 days were lost; 18 of 19 fruits (95%) for the variant with insemination of sexed sperm were female, and in the control groups from 20 to 30 fruits (67%) were female. In the control insemination with liquid semen results by the percentage of pregnancy were almost identical to those obtained in the control insemination of frozen semen, containing more than 50 times higher number of motile sperm (more than 120 times greater total number of spermatozoa)that demonstrates the effectiveness of insemination with low doses of semen entered into the horns of the uterus. The inventors were able to significantly alter the sex ratio through the use of flow-cytometrical technology and artificial insemination.

Likewise conducted an experiment with insemination is not unfrozen sexed sperm, the results of which are summarized below:

EXAMPLE 2

The aim of the experiment was to determine the effectiveness of insemination (percentage of pregnancy) when heifers were seminal extremely low quantities of frozen semen in the ideal production of the built environment. Sperm obtained from three Holstein bulls with above average fertility, was diluted in gomogenizirovannom milk plus 7% glycerol (CSS) plus 5% homologous seminal plasma to concentrations of 2×105, 5×105and 10×106(control) total number of sperm in French straws volume of 0.25 ml and frozen in a moving vapor of liquid nitrogen. Sperm were subjected to thawing in water at 37°C for 20 sec. Holstein heifers at the age of 13-15 months weighing 350-450 kg by injection was administered at 25 mg prostaglandin F-2 alpha (Lutalyse®), twice with an interval of 12 days and seminal them using the catheter for transferring embryos and capsule with a side opening, entering by half the number of sperm deep into each uterine horn after 12 or 24 hours after detection of estrus. The experiment was performed in five replications, 5 months, with a balanced use of two operators artificial insemination. Outside temperature when breeding animals often ranged from -10 to -20°therefore, measures were taken to keep the equipment for insemination warm. Pregnancy was determined by detection of visible fetal ultrasound at 40-44 days after estrus, and confirmed it on 55-62 days; 4 202 of fruit for between these TLDs is I research were lost. The percent occurrence of pregnancy on 55-62 days was 55/103 (53%), 71/101 (70%) and 72/102 (71%), for options, respectively, with 2×105, 5×105and 10×106(control) total sperm count (P<0,1). The percent of pregnancy differed (P<0.05) in the use of sperm from different bulls (59, 62 and 74%), but did not differ when using different operators (64 and 65%) and different timing of insemination after the onset of estrus (65% for the variant with 12 HR and 64% for the variant with 24 hour, N=153 in each case). For the described methods interest springing heifers were almost identical at doses of 5×105and 10×106the total number of sperm per insemination.

Also conducted the experiment with insemination with sexed frozen semen, the results of which are summarized below:

EXAMPLE 3

The sperm was taken from bulls in the cooperative Atlantic Breedres, was diluted in the ratio 1:4 with diluent based on HEPES-buffer + 0.1% of BSA (bovine serum albumin) and transported to a distance of 160 km (about 2 hours) in Besville, Maryland, where it was divided by gender at ambient temperature by the method of flow cytometry in 20%thinner "TEST", using previously described methods (Biol. Reprod. 41:199). Reached speeds of separation of components up to 2×106sperm of each sex for 5-6 hours, with a purity of ≈ 90%. Sperm concentrate is was demonstrated by centrifugation (300 g for 4 minutes) to 2× 106spermatozoa/ml Some sperm were separated in a diluent containing homologous seminal plasma (final concentration 5%). Sexed semen were sent by air in Colorado on distance ≈ 2,600 km) and stored either at ambient temperature or at 5° (chilled during transportation for more than 6 hours in the heat-insulated device model Equitainer having a compartment for ice). Heifers or dry cows with 11-36 hours before it was determined estrus, seminal in the range from 9 to 29 hours after the end of the split operation of the sperm. The sperm (from 1 to 2×105spermatozoa in 1 ml) was injected deep into the uterine horn, located ipsilaterally in relation to the ovary, which was the largest follicle that was determined by ultrasound at the time of insemination.

None of the 10 females did not become pregnant after insemination with the sperm, which is transported and stored at ambient temperature. Of the 29 females are inseminated with sperm that during transport was cooled to 5°, 4 weeks after insemination pregnant were 14 females and 12 (41%) were pregnant 8 weeks after insemination. Eleven of the 22 females are inseminated with sperm taken not later than 10 hours after its division by sex, were pregnant and during examination after 8 weeks after insemination, and only 1 of 7 females are inseminated with sperm taken through 17-24 hours after its division by sex, was pregnant. The addition of seminal plasma had no significant effect. One of the 12 fruits were not having sex, which was expected based on the separation of sperm, the sex of one of the fruit was outstanding, and 10 fruits were expected floor, which was determined by ultrasound at the time of 60-70 days of pregnancy.

Then another 33 heifers were osumenyi using 0.05 ml of semen (diluted as described above), with the introduction of it into each of the horn under the control of ultrasound; only 3 of them were pregnant during the examination 4 weeks after insemination, and only 1 remained pregnant after 8 weeks after insemination. However, in this group did not use those bulls that were used for the previous group, and all of insemination was performed after 18-29 hours after sperm separation by gender. Have similarly osumenyi 38 heifers (approximately 22 hours after sperm separation) 200 km from the laboratory of the authors of the present invention, sexed semen taken from another bull; none of these heifers was not pregnant during the examination 8 weeks after insemination.

Summarizing, we can say that in cattle, it is possible to achieve pregnancy by artificial insemination Spa is my divided according to sex chromosomes using flow cytometry, and the ratio of the sexes receive the fruits of roughly corresponds predicted by the results of the second analysis sexed sperm for DNA content (90%). However, the percent of pregnancy in these preliminary experiments, requiring the transportation of sperm for long distances, varied a lot. Fertility decreased sharply when using semen collected after 17 hours of separation by sex, however, the purity of the experiment was slightly disturbed by the fact that at different times of capture of the sperm used sperm from different bulls. Further research is needed to determine whether the observed variation in the percentage of pregnancy due to the origin of sperm from different bulls, or they were the result of the methods of insemination, the interval between the separation of sperm on the floor and insemination, or other factors.

And finally, also conducted an experiment with insemination undivided on the floor unfrozen sperm, the results of which are summarized below:

EXAMPLE 4

The purpose was to determine the percent of pregnancy when heifers were seminal very small amounts of sperm in ideal experimental conditions. Sperm taken from three Holstein bulls, was diluted in orderscom universal diluent plus 5% homologous seminal plasma, concentrations up to 1×105or 2.5×106sperm cells in 0.1 ml; 2,5×106the total number of sperm cells in 0.25 ml was used as control. Fully diluted semen was packaged in a plastic French straws volume of 0.25 ml, which prevented doses of semen for insemination, comprised of 0.1 or 0.25 ml of Semen was cooled to 5°and used through 26-57 hours after its collection. Holstein heifers at the age of 13-15 months weighing 350-450 kg by injection was administered at 25 mg prostaglandin F-2 alpha (Lutalyse®), with intervals of 12 days, and was seminal them using the catheter for transferring embryos and capsule side hole, inserting the sperm into the uterine horn after 12 or 24 hours after detection of estrus. Insemination was carried out ipsilateral toward the side on which ultrasound 12 hours after estrus found the largest follicle; side ovulation specified by detecting yellow body with ultrasound in 7-9 days after estrus. Pregnancy was determined by detection of the fetus by ultrasound through 42-45 days after estrus. The experiment was performed in four replicates, with balanced use of the three operators of artificial insemination. Side ovulation accurately determined at 205 of 225 heifers (91%); however, unexpectedly it was found that the% is you pregnancy were nearly identical for the ipsilateral and contralateral insemination. The percent occurrence of pregnancy was 38/93 (41%), 45/87 (52%) 25/45 (56%), for options, respectively, with 1×105, 2,5×105and 2.5×106sperm insemination (P>0,1). Rates of pregnancy were significantly different (P<0.05) in the use of different operators, but did not differ when using sperm from different bulls. With these methods you can reduce the number of sperm required for one insemination, enough to make use of sexed using the flow cytometer sperm had commercial value.

As mentioned and as can be seen from different experiments, the observed differences are statistically significant and therefore it is possible to conduct a number of additional experiments to determine the appropriate combination and restrictive strategies. So, next will be determined by the relationship between the various influences, for example, it is possible to investigate the influence of the dye and the combined effects of the dye laser excitation.

The discussion included in this description serves as the base description. The reader should be aware that the specific description of the invention cannot exhaustively cover all possible variations in its implementation; refers to the availability of many alternatives. Description the ie cannot fully explain the General nature of the invention and may not fully show as each character and element can actually be representative of a broader function or of a great variety of alternative or equivalent elements. And it is also assumed in the present description. In cases where the invention is described in terms-oriented device, it is understood that each element of the device performs the function. The claims related to the staff, can apply not only to the described device, but also the claims relating to the method or process may relate to the functions performed by the invention and each element. Neither the description nor the terminology does not limit the scope of the claims, the items which can be subordinate. Keep in mind that you can perform a number of changes which do not go beyond the limits of the invention. These changes also enabled by default in the present invention. However, they do not go beyond the scope of the invention. An extensive description of the covering as fully disclosed therein variant(s) of the invention and a great variety of implicit alternative embodiments, and a wide range of methods and processes, and the like are also covered by the present description.

In addition, each of the various elements of this image is to be placed and its formula can be done in many different ways. The present description should be considered as covering each such variation, be it a variation of the manner of exercise of any option device, method or process, or even just a variation of any element. In particular, it should be borne in mind that since the present description refers to elements of the invention, the words describing each element can be expressed in equivalent terms related to the device or terms relating to the method - even if the same are only function and result. Such equivalent, extended or even generic terms should be considered as covering in the description of each item or activity. Such terms can be replaced by other, when it is desirable to make clearer mean them values for the present invention. As one example, it should be borne in mind that all actions can be expressed in the form of methods to perform these actions, or as elements that cause this action. Similarly, each described a physical item should be considered as encompassing description of the action that this physical element allows. As only one example of this aspect, the description of "the collector" (devices for collecting sperm) should pony the AMB as encompassing description of the action "collect" - no matter described or whether it is only implied and Vice versa, if there is only a description of the action "collect", this description should be understood as encompassing description "collector". Such changes and alternative terms should be considered as the default included in the description. Further, it should be borne in mind that in addition to the originally submitted the claims, the claims may be varied so that it more fully represented at least: (i) devices, as they are here presented and described, ii) the related methods are presented and described, iii) similar, equivalent or even implicit variations of these devices and methods, iv) alternative solutions that perform each of the described and represented functions, v) alternative solutions and ways of performing each of the functions shown as running by default that presented and described, vi) each feature, component and phase are shown as separate and independent inventions, vii) the various combinations and permutations of each of the above.

For understanding the present invention can be useful in a number of published reference materials. They are listed below and hereby incorporated by reference; however, with regard to claims, who can be considered inconsistent with the patenting of this/these invention(s), such statements certainly should not be considered as made by the applicant. Potentially useful references include: U.S. patents№: 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; RE32350. Useful links can also include the following publications: "Insemination of Holstein Heifers With Very Low Numbers Of Unfrozen Spermatozoa." G.E.Seidel, Jr., C.H.Alien, Z.Brink, J.K.Graham, and M.B.Cattell, Colorado State University, Fort Collins, Atlantic Breeders Cooperative, Lancaster, PA., DUO Dairy, Loveland, CO. July 1995; "Artificial Insemination With X-and Y-Bearing Bovine Sperm", G.E.Seidel, Jr., L.A.Johnson, C.A.Alien, G.R.Welch, M.D.Holland, Z.Brink and M.B.Cattell, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO; Germplasm and Gamete discrimination Lab, ARS, USDA, Beltsville, MD; Atlantic Breeders Coop, Lancaster, PA; DUO Diary, Loveland, CO, USA, January 1996; "Insemination of Heifers with Very Low Numbers of Frozen Spermatozoa." G.E.Seidel, Jr., C.H.Alien. Z.Brink, M.D.Holland, and M.B.Cattell, Colorado State University, Fort Collins, Atlantic Breeders Cooperative, Lancaster, PA, DUO Dairy, Loveland, CO, July 1996; "Production of Lambs by Low Dose Intrauterine Insemination With Flow Cytometrically Sorted and Unsorted Semen," D.G.Cran, W.A.C.McKelvey, M.E.King, D.F.Dolman, T.G.McEvoy, P.J.Broadbent and J.J.Robinson, Mastercalf, Craibstone, Bucksburn, Aberdeen, AB21 9TN, UK Scottish Agricultural College, Craibstone, Bucksburn, Aberdeen. AB21 UA, UK, Theriogenology, Page 267; "Uterine Horn Insemination of Heifers With Very Low Numbers of Nonfrozen and Sexed Spermatozoa," G.E.Seidel, Jr., C.H.Alien, L.A.Johnson, M.D.Holland, Z.Brink, G.R.Welch, J.K.Graham and M.B.Cattell, Animal Reproduction and Biotechnology Laboratory Colorado State University, Atlantic Breeders Cooperative, Lancaster, PA 17601, Germplasm and Gamete discrimination Laboratory, ARS, USDA, Beltsville, MD 20705, DUO Diary, Loveland, CO 80538, Theriogenology 48: 1255-1264, 1997; "Capcitation of Bovine Sperm by Heparin," J.J.Parrish, J.Susko-Parrish, M.A.Winer, and N.L.First, Department of Meat and Animal Science, University of Wisconsin, Madison, WI 53706, Biology Of Reproduction 38, 1171-1180 (1988); "Prostaglandin F2a - A Fertility Drug In Dairy Cattle?", K.L.Macmillan and A.M.Day, Ruakura Animal Research Station, Private Bag, Hamilton, New Zealand, Theriogenology, September 1982, VOL.18 No.3, pages 245-253; "Prospects For Sexing Mammalian Sperm," Colorado Associated University Press, Animal Reproduction Laboratory College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, 80523 Edited by Rupert P. Amann and George E. Seidel, Jr., 1982; "Effects of Egg Yolk-Citrate and Milk Cam sex online on Chromatin Structure and Viability of Cryopreserved Bull Sperm", J Dairy Sci 74:3836, D.S.Karabinus and D.P.Evenson and M.T.Kaproth; "Assessment of Ram and Boar Spermatozoa during Cell-sorting by Flow Cytomerty", Reprod. Dom Anim 32:251; "Superovulation of Goats with Purified pFSH Supplemented with Defined Amounts of pLH", Therio. 43:797, M.A.Nowshari, J.F.Beckers, and W.Holtz; "Gender Preselection in Mammals: An Overview", Dtsch. tierarztl. Wschr. 103:285, L.A.Johnson.

Everywhere in this description, in cases where the context otherwise requires, the word "comprise" or variations such as "comprises", "comprising" and the like, should be understood as denoting the inclusion of the specified element or component or group of elements or components, but not the exclusion of any other element or component or group of elements or components.

1. The method of obtaining cattle given the floor, including stage

A. taking the sperm of a bull;

b. determination of sex from a variety of sperm bull;

C. sorting the sperm of a bull according to the definition of their sex, priznaisa a speed of at least 1200 operations per second and collection of separated spermatozoa in the collector, equipped with shock-absorbing element, and the environment of the capsule fluid for the sperm of a bull contains about 2.9% sodium citrate;

d. obtain a sorted set of bull sperm for artificial insemination, containing not more than 100,000 to not more than 300,000 sorted sperm of a bull;

that is, the introduction of the totality of sperm for artificial insemination of the female of this species, namely cattle;

f. fertilization at least one egg in the female mammal, a representative of the cattle, the level of performance that comprise at least 50% compared with the level of achievement of pregnancy achieved using conventional aggregate of sperm for artificial insemination;

g. produce offspring of the desired sex of the mammal, the representative of cattle.

2. The method according to claim 1, in which stage of fertilization at least one egg in the female of this species mammals, representative of cattle, includes a step of fertilization at least one egg in the female mammal, a representative of the cattle, the level of performance that comprise at least 90%.

3. The method according to claim 1 or 2, in which the introduction phase grade is focused set of bull sperm for artificial insemination of the female of this species mammals, representative cattle, and fertilization at least one egg in the female of this species of mammals.

4. The method according to claim 1, in which the introduction phase of a sorted set of bull sperm for artificial insemination of the female of this species mammals, representative of cattle, and fertilization at least one egg in the female of this species mammals, representative of cattle, with the level of achievement at least 50%, in the field, include the stage of the successive introduction of a significant number of populations sorted bull sperm for artificial insemination significant number of females of this species mammals, representative of cattle, in the conditions of the farms or ranches.

5. The method according to any one of claims 1 to 3, in which the mammal, the representative of cattle, has the horns of the uterus, and in which stage the introduction of the sorted set of bull sperm for artificial insemination of the female of this species mammals, representative of cattle, includes a step of introducing a sorted set of bull sperm for artificial insemination as IPSI-and contralateral to the horns of the uterus of a mammal, representative of large agathosma.

6. The method according to any one of claims 1 to 3, in which the mammal, the representative of cattle, has at least one horn of the uterus, and in which stage the introduction of the sorted set of bull sperm for artificial insemination of the female of this species mammals, representative of cattle, includes a step of introducing a sorted set of bull sperm for artificial insemination deep in the uterine horn.

7. The method according to claim 4, in which stage the introduction of the sorted set of bull sperm for artificial insemination of the female of this species mammals, representative of cattle, includes a step of introducing a sorted set of bull sperm for artificial insemination deep in the uterine horn.

8. The method according to claim 5, in which stage the introduction of the sorted set of bull sperm for artificial insemination of the female of this species mammals, representative of cattle, additionally includes a step of introducing a sorted set of bull sperm for artificial insemination in the uterine horn with a tool for transplanting embryos.

9. The method according to claim 6, in which stage the introduction of the sorted set of bull sperm for artificial insemination of the female of this species mammals, pre is the representative of cattle, also includes the stage of introduction of the sorted set of bull sperm for artificial insemination in the uterine horn with a tool for transplanting embryos.

10. The method according to claim 4, in which stage the introduction of the sorted set of bull sperm for artificial insemination of the female of this species mammals, representative of cattle, includes a step of introducing a sorted set of bull sperm for artificial insemination within 12 hours after the time that is generally considered optimal for a single artificial insemination of cattle.

11. The method according to claim 8, in which the stage of obtaining the sorted set of bull sperm for artificial insemination involves the step of obtaining the unfrozen combination of bull sperm for artificial insemination, and the stage of sorting sperm of a bull according to the definition of their sex takes place during the sort and stage of introduction of the sorted set of bull sperm for artificial insemination of the female of this species mammals, representative of cattle, carry out not later than 17 hours from the time of sorting.

12. The method according to claim 8, in which the stage of obtaining the sorted set of spermatozoa DL is artificial insemination includes a step for unfrozen sorted population of sperm for artificial insemination, and stage sorting of spermatozoa according to the definition of their sex takes place during the sort and stage of introduction of the totality of sperm for artificial insemination of the female of this species mammals is carried out not later than 10 hours from the time of sorting.

13. The method according to item 12, in which the stage of determining sex by a certain set of bull sperm involves the step of staining the sperm of a bull when the content of the colorant is at least 38 mm.

14. The method according to claim 1, wherein the stage of defining sex with a variety of bull sperm and sorting the sperm of a bull according to the definition of their sex, and the environment of the capsule fluid for the sperm of a bull contains about 2.9% sodium citrate, include stage

A. create a cell source which supplies the sperm of a bull for sorting.

b. chemical coordination capsular fluid to create an environment of capsular fluid for the sperm of a bull, coordinated with the liquid environment of spermatozoa before and after sorting;

C. recognition of one of the properties of the sperm of a bull;

d. distinguish between sperm of a bull, with the indication of the desired gender; and

E. collecting the sperm of a bull, with the indication of the desired gender is.

15. The method according to claim 2, in which the stage of determining sex by a certain set of bull sperm and sorting the sperm of a bull according to the definition of their sex, and the environment of the capsule fluid for the sperm of a bull contains about 2.9% sodium citrate, include stage

A. create a cell source which supplies the sperm of a bull for sorting.

b. chemical coordination capsular fluid to create an environment of capsular fluid for the sperm of a bull, coordinated with the liquid environment of spermatozoa before and after sorting;

C. recognition of one of the properties of the sperm of a bull;

d. distinguish between sperm of a bull, with the indication of the desired gender; and

e. collecting the sperm of a bull, with the indication of the desired sex.

16. The method according to claim 1, wherein the stage of defining sex with a variety of bull sperm and sorting of spermatozoa according to the definition of their sex, and the environment of the capsule fluid for the sperm of a bull contains about 2.9% sodium citrate, include stage

A. create a cell source which supplies the sperm of a bull for sorting.

b. create an environment of capsular fluid for sperm bull;

C. recognition of one of its the TV sperm of a bull;

d. distinguish between sperm of a bull, with the indication of the desired gender; and

E. collecting the sperm of a bull, with the indication of the desired floor, thus protecting the sperm of a bull from a collision with the manifold so that the cushioning element includes a source of collector fluid at the bottom of the collector and the collector has a large configuration that does not allow collision sperm with header.

17. The method according to claim 1, wherein the stage of defining sex with a variety of bull sperm and sorting of spermatozoa according to the definition of their sex, and the environment of the capsule fluid for the sperm of a bull contains about 2.9% sodium citrate, include stage

A. create a cell source which supplies the sperm of a bull for sorting.

b. create an environment of capsular fluid for sperm bull;

C. recognition of one of the properties of the sperm of a bull;

d. distinguish between sperm of a bull, with the indication of the desired gender; and

e. collecting the sperm of a bull, with the indication of the desired sex, the collector fluid on the basis of citrate containing about 6% egg yolk before the stage of collection.

18. The method according to claim 1, further comprising a stage conducive to preovulation formats whitesage drug calling the production of multiple eggs by the female of this species mammals, representative of cattle, in which stage of fertilization at least one egg in the female of this species mammals, representative of cattle, carried out with the level of performance that comprise at least 50%, includes a step of fertilization of multiple eggs produced by the female of this species mammals, representative of cattle, to obtain multiple embryos given the floor.

19. The method according to p in which stage of the application Pro-preovulation pharmaceutical drug causing the production of multiple eggs by the female of this species mammals, representative of cattle, includes a step of multiple doses of follicle stimulating hormone several times a day, and contributing to preovulation pharmaceutical drug is administered with an interval of half a day, on any day between the 2nd to the 18th day of the estrus cycle.

20. The method according to claim 19, in which stage multiple-dose follicle stimulating hormone includes a step introduction follicle stimulating hormone with intervals that make up about half of the day, in doses 6, 6, 4, 4, 2, 2, 2, and 2 mg between 9 and 12 days of estrus cycle inclusive and involves the step of introducing 25 and 12.5 mg prostaglandin F-2-alpha together with the sixth and seventh doses accordingly, follicle-stimulating hormone.

21. The method according to claim 1, in which stage of sorting cells with a speed of at least 1200 operations per second includes the operation of the flow cytometer in the range from 5 to 50 kHz.

22. The method according to clause 16, in which stage fencing sperm of a bull from a collision with a container for collecting includes a collection container having physical characteristics that are appropriate for the stream.

23. The method according to clause 16, in which stage fencing sperm of a bull from a collision with a container for collecting includes the step of installing the collection container having a diameter of at least 15 mm

24. The method of obtaining horses given the floor, including stage

A. taking stallion sperm from a male mammal, a representative of the horse;

b. determination of sex from a variety of stallion spermatozoa;

C. sort of stallion spermatozoa according to their definition sex with a speed of at least 1200 operations per second and collection of separated spermatozoa in the manifold, equipped with a shock-absorbing element, and the environment from the capsule liquid for stallion sperm contains hepes buffer;

d. obtain a sorted set of stallion spermatozoa for artificial insemination, containing not more than 1 to not b is over 25 million sorted stallion spermatozoa;

that is, the introduction of a set of stallion spermatozoa for artificial insemination of the female of this species mammals, representative of the horse;

f. fertilization at least one egg in the female of this species mammals, representative of the horse, with the level of achievement of pregnancy, constituting at least 35%, at least 41%, at least 50% and at least 90% compared to the level of achievement of pregnancy achieved using conventional aggregate of sperm for artificial insemination;

g. produce offspring of the desired sex of the mammal, a representative of the horse.

25. The method according to paragraph 24, in which the introduction phase of a sorted set of stallion spermatozoa for artificial insemination of the female of this species mammals, representative of the horse, and fertilization at least one egg in the female of this species mammals, representative of the horse, with a performance level of at least 35%, are carried out in the field.

26. The method according to paragraph 24, in which the introduction phase of a sorted set of stallion spermatozoa for artificial insemination of the female of this species mammals, representative of the horse, and fertilization at least one egg in the female of this species mammals, pre is the representative of horse, with a performance level of at least 35%, in the field, include the stage of the successive introduction of a significant number of populations sorted stallion spermatozoa for artificial insemination significant number of females of this species mammals, representative of the horse, in the conditions of the farms or ranches.

27. The method according to paragraph 24, in which the horse has the horns of the uterus, and in which stage the introduction of the sorted set of stallion spermatozoa for artificial insemination of the female of this species mammals, representative of the horse, includes a step of introducing a sorted set of stallion spermatozoa for artificial insemination as IPSI-and contralateral to the horns of the uterus of a female mammal, a representative of the horse.

28. The method according to paragraph 24, in which the mammal, the representative of the horse has at least one horn of the uterus, and in which stage the introduction of the sorted set of stallion spermatozoa for artificial insemination of a female mammal, a representative of the horse, includes a step of introducing a sorted set of stallion spermatozoa for artificial insemination deep in the uterine horn.

29. The method according to item 27, in which stage the introduction of the sorted set of stallion spermatozoa for artificial insemination SAMC is this type of mammals, representative horse, also includes the stage of introduction of the sorted set of stallion spermatozoa for artificial insemination deep in the uterine horn.

30. The method according to p in which stage of introducing at least part of the sorted set of stallion spermatozoa for artificial insemination of the female of this species mammals, representative of the horse, additionally includes a step of introducing a sorted set of stallion spermatozoa for artificial insemination in the uterine horn with a tool for transplanting embryos.

31. The method according to clause 29, in which stage the introduction of the sorted set of stallion spermatozoa for artificial insemination of the female of this species mammals, representative of the horse, additionally includes a step of introducing a sorted set of stallion spermatozoa for artificial insemination in the uterine horn with a tool for transplanting embryos.

32. The method according to p, in which stage the introduction of the sorted population of sperm for artificial insemination of the female of this species mammals involves the step of introducing a sorted population of sperm for artificial insemination within 12 hours after the time that is generally considered optimal for the simultaneous artificial insemination.

33. JV the property on p, in which stage of obtaining a sorted set of stallion spermatozoa for artificial insemination includes a step for unfrozen sorted set of stallion spermatozoa for artificial insemination, and the stage sort of stallion spermatozoa according to the definition of their sex takes place during the sort and stage of introduction of the sorted set of stallion spermatozoa for artificial insemination of the female of this species mammals, representative of the horse, carry out not later than 17 hours from the time of sorting.

34. The method according to p in which stage of obtaining a sorted set of stallion spermatozoa for artificial insemination includes a step for unfrozen sorted set of stallion spermatozoa for artificial insemination, and the stage sort of stallion spermatozoa according to the definition of their sex takes place during the sort and stage of introduction of the sorted set of stallion spermatozoa for artificial insemination of the female of this species mammals, representative of the horse, carry out not later than 10 hours from the time of sorting.

35. The method according A.25, in which stage the definition of sex have a variety of stallion spermatozoa VK is uchet stage dyeing of stallion spermatozoa when the content of the colorant at least 38 km.

36. The method according to paragraph 24, in which the stage of defining sex with a variety of stallion spermatozoa and sort of stallion spermatozoa on the basis of their sex, and the environment from the capsule liquid for stallion sperm contains hepes buffer, include stage

A. create a cell source which supplies the sperm stallion for sorting.

b. chemical coordination capsular fluid to create an environment of capsular fluid for sperm stallion, coordinated with the liquid environment of spermatozoa before and after sorting;

C. recognition of one of the properties of stallion spermatozoa;

d. discernment of stallion sperm with the desired sex characteristic; and

that is, the gathering of stallion spermatozoa, with the indication of the desired sex.

37. The method according to paragraph 24, in which the stage of defining sex with a variety of stallion spermatozoa and sort of stallion spermatozoa according to the definition of their sex, and the environment from the capsule liquid for stallion sperm contains hepes buffer, include stage

A. create a cell source which supplies the sperm stallion for sorting.

b. receive capsular fluid for sperm stallion;

C. recognition of one of the properties of stallion spermatozoa;

d. discernment of stallion sperm with the desired sex characteristic; and

that is, the gathering of stallion spermatozoa, with the indication of the desired floor, thus protecting the sperm from a collision with the manifold so that the cushioning element includes a source of collector fluid at the bottom of the collector and the collector has a large configuration that does not allow collision sperm with header.

38. The method according to paragraph 24, in which the stage of defining sex with a variety of stallion spermatozoa and sort of stallion spermatozoa according to the definition of their sex include stage

A. create a cell source which supplies the sperm stallion for sorting.

b. obtain a capsule liquid for stallion spermatozoa;

C. recognition of one of the properties of stallion spermatozoa;

d. discernment of stallion sperm with the desired sex characteristic; and

e. collection of stallion sperm with the desired sex characteristic, the collector fluid on the basis of citrate containing about 6% egg yolk before the stage of collection.

39. The method according to paragraph 24, further comprising a stage conducive to preovulation pharmaceutical drug causing the production of multiple eggs what amcol this type of mammals, representative horse, which involves the step of introducing a dose of follicle-stimulating hormone several times a day, and contributing to preovulation pharmaceutical drug is administered with an interval of half a day, on any day between the 2nd and 18th days of the estrus cycle.

40. The method according to § 39, in which the dose of follicle-stimulating hormone several times a day includes an introduction follicle stimulating hormone with intervals that make up about half of the day, in doses 6, 6, 4, 4, 2, 2, 2 and 2 mg between 9 and 12 days of estrus cycle inclusive, and includes a step of introducing 25 and 12.5 mg prostaglandin F-2-alpha together with the sixth and seventh doses, respectively follicle-stimulating hormone.

41. The method according to p, in which stage the sort of stallion spermatozoa with a speed higher than or equal to 1200 operations per second includes the operation of the flow cytometer in the range from 5 to 50 kHz.

42. The method according to paragraph 41, in which the stage of collection of stallion sperm with the desired characteristic includes a step of enclosing stallion spermatozoa from a collision with a container for collecting and stage depreciation includes the original collector fluid at the bottom of the collector and the collector has a large configuration that does not allow collision sperm with header.

43. The method according to § 42, in which the article is Diya fencing of stallion spermatozoa from a collision with a container for collecting, has a large configuration that does not allow collision of stallion spermatozoa, includes the step of installing the collection container having a diameter of at least 15 millimeters.

44. Method of separating sperm, which includes stages

A. create a cell source which supplies the sperm for sorting.

b. chemical coordination capsular fluid to create an environment of capsular fluid for sperm, coordinated with the liquid environment of spermatozoa before and after sorting;

C. recognition of one of the properties of sperm;

d. distinguish between sperm cells according to their gender;

e. separation of spermatozoa with a speed of at least 1200 operations per second; and

f. collection of sperm cells having the desired sex characteristic, the collector, equipped with a shock-absorbing element.

45. The method according to item 44, in which stage the chemical coordination of the collector fluid to create the ultimate environment of the collector fluid for sperm coordinated with liquid medium prior to sorting.

46. The method according to item 44, in which stage a source of cells includes a source of sperm of a bull.

47. The method according to item 44, in which stage a source of cells includes a step of creating a source of sperm is of totidem stallion.

48. The method according to item 44, in which stage the chemical coordinating capsular fluid to create an environment of capsular fluid for sperm, coordinated with the liquid environment of spermatozoa before and after sorting, includes a step of creating a cell source which supplies the sperm of a bull to sort and retrieve the capsule environment, containing about 2.9% sodium citrate.

49. The method according to p in which stage chemical coordination capsular fluid to create an environment of capsular fluid for sperm, coordinated with the liquid environment of spermatozoa before and after sorting, includes a step of creating a source of cells that supply stallion sperm sorting, and receiving capsule medium containing hepes buffer.

50. The method according to item 44, in which stage a source of cells includes a step of providing an initial nutrients to cells, and also includes a step of providing nutrients collector fluid cells, and the stage of gathering having a desired characteristic of cells in the collector fluid includes a step of balancing the number of original nutrients and nutrients collector fluid upon completion of the collection phase cells.

51. The method according to item 44, in which the stage of gathering having h is iny characteristic of cells in the collector fluid includes a step for the collector fluid on the basis of citrate, containing about 6% egg yolk before the stage of collection.

52. The method according to § 51, in which stage a source of cells includes a step of creating a source of sperm of a bull.

53. The method according to item 44, further comprising a stage insemination of a mammal using a sorted set of spermatozoa for insemination.

54. The method according to item 44, further comprising the stage of collecting sperm with the desired sex characteristic, which includes a cushion to protect sperm from a collision with the collector, and the protection of spermatozoa involves the collection of spermatozoa in the original collector fluid at the bottom of the collector and the collector has a large configuration that does not allow collision sperm with header.

55. The method according to item 54, in which the stage of collecting sperm in the original collector fluid at the bottom of the collector, and the collector has a large configuration that does not allow collision sperm with header includes the step of installing a container for collection.

56. The method according to § 55, at which stage the installation of the collection container includes the installation of a collection container having a diameter of at least 15 mm

57. The method according to item 44, in which the stage of sorting cells with a speed of at least 1200 operations per second VC is uchet stage of operation of the flow cytometer in the range from 5 to 50 kHz.

58. The method of sorting sperm, which includes stages

A. create a cell source which supplies the sperm for sorting.

b. obtain a capsule liquid to create the environment of the capsule fluid for sperm;

C. recognition of one of the properties of sperm;

d. distinguish between sperm cells according to their gender;

C. sorting of spermatozoa with a speed of at least 1200 operations per second; and

f. collecting sperm with the desired sex characteristic, including the state of depreciation to protect sperm from a collision with a collector, and a shock-absorbing element includes the original collector fluid at the bottom of the collector and the collector has a large configuration that does not allow collision sperm with header.

59. The method according to § 58, in which stage of depreciation to protect sperm from a collision with header includes the step of installing a container for collection.

60. The method according to § 58, at which stage the installation of the collection container includes the installation of a collection container having a diameter of at least 15 mm

61. The method according to p, further comprising a step for assorted collection of sperm for artificial insemination.

62. The method of obtaining the centre of the development of the embryos of a given sex from females of one species of mammals, including

A. the achievement of preovulation females of this species mammals to obtain at least two eggs, and contributing to preovulation pharmaceutical drug is administered at intervals of 12 hours in any day between the 2nd and 18th days of estrus cycle;

b. sex determination in a variety of sperm of the male of this species mammals;

C. separation of sperm by sex according to the method according to item 44 or 58;

d. the introduction of separated spermatozoa into the uterus of the female of this species mammal after the onset of estrus; and

E. fertilization of multiple eggs in the uterus to obtain multiple embryos given the floor.

63. The method according to item 62, in which the introduction of pharmaceutical product, contributing to preovulation, carried out with the help of at least seven injections conducted at intervals of 12 hours, of which at least the sixth and seventh injection synchronized estrus.

64. The method according to p, in which the introduction of sorted spermatozoa in the uterus includes the introduction of sperm into both uterine horns.

65. The method according to p, in which the introduction of sperm into both uterine horns includes the introduction of the sperm approximately between 20 and 24 h after onset of estrus.

66. The method according to item 62, in which the introduction of pharmaceutical product, the JV is to account for preovulation and causing the formation of multiple eggs includes an introduction follicle-stimulating hormone several times a day.

67. The method according to p, in which the achievement of preovulation the mammal with the aim of obtaining at least two eggs additionally includes co-administration of follicle-stimulating hormone and prostaglandin F-2 alpha, and specified the introduction synchronize with extrusion.

68. The method according to p in which follicle-stimulating hormone is administered at intervals of about 12 h in doses 6, 6, 4, 4, 2, 2, 2 and 2 mg between 9 and 12 days cycle hormone in the sixth and seventh injections of follicle stimulating hormone in doses of 25 and 12.5 mg

69. The method according to item 62, further comprising a stage

A. staining of sperm a male mammal with 38 μm dye;

b. sorting of spermatozoa in accordance with the floor with a speed of at least 1200 operations per second; and

C. concentration of sorted spermatozoa.

70. The method according to item 62, in which the introduction of sorted spermatozoa involves the use of a sorted set of sperm for artificial insemination.

Priority items:

31.12.1997 according to claims 1-17, 21-38, 41-57,

29.01.1998 on p-20, 39-40, 58-70.



 

Same patents:

FIELD: veterinary.

SUBSTANCE: naturally occurring minerals suitable for use in enterosorption process, especially in sorption of nickel and lead, are proposed. Zeolite originated from Kamyshlovsk deposit situated in Sverdlovsk region is added to main dairy cow diet in amount 0.15-0.20 g per 1 kg animal weight a day over a 25-30 day period.

EFFECT: achieved environmentally safe produce.

4 tbl

FIELD: animal science.

SUBSTANCE: it is necessary to introduce biologically active additives "Gorasel" or "Tykvorsel" being selenopyrane solutions in the mixture of vegetable oils. These biologically active additives should be injected subcutaneously per 10 ml/cow 4 times: the first time - immediately afterlaunching, and then 30, 20 and 10 d before calving. The innovation suggested enables to increase milk yield and milk quality.

EFFECT: higher efficiency.

1 tbl

FIELD: animal science.

SUBSTANCE: it is necessary to introduce perorally an alcohol-honey extract of walnuts of milk-wax maturity at adding glycine and flower pollen. The preparation should be introduced 5 d before and after the impact of stress factors 30 min before feeding per 10-15 ml/animal daily. As a result of applying the suggested preparation the body weight losses in animals are decreased considerably, the body weight gain is increased along with the quantity and quality of animal production.

EFFECT: higher efficiency of prophylaxis and correction.

1 ex, 2 tbl

FIELD: veterinary science.

SUBSTANCE: the innovation deals with supplementing the diet with iodinated common salt and a medicinal preparation perorally as metronidasol and a preparation of sulfanyl amide group. Additionally, one should introduce a diuretic, and as medicinal preparation one should additionally use penicillin sodium salt intramuscularly and 0.5%-metronidasol solution intravenously. Moreover, for pregnant cows of dairy herd the preparations should be introduced once annually for the whole productive period in the following sequence: a 6-d-long course up to calving dealing with introducing metronidasol at the dosage of 1.5 g/animal and norsulfasol or sulfadimezine at the dosage of 1 g/animal. At calving day it is necessary to inject intravenously 0.5%-metronidasol solution at the dosage of 200 ml/animal thrice in 8 h. Since the 2nd to the 5th d inclusively it is important to introduce metronidasol perorally at the dosage of 2.5 g/animal and norsulfasol or sulfadimezine at the dosage of 1 g/animal. Since the 6th to the 15th d inclusively one should inject intramuscularly penicillin sodium salt at the dosage of 2 mln U/animal - twice daily. On the 16th d one should inject intravenously 0.5%-metronidasol solution at the dosage per 200 ml/animal thrice in 8 h. With prophylactic purpose for stud bulls it is necessary to introduce preparations twice annually at 6-mo-long intervals according to the following scheme: during the 1st and the 2nd d - intravenously 0.5%-metronidasol solution at the dosage of 300 ml/animal thrice at 8-10-h-long interval. Since the 3d to the 5th d inclusively - perorally metronidasol at the dosage of 2.5 g/animal and norsulfasol or streptocid at the dosage of 2 g/animal. Since the 6th to the 15th d - intramuscularly penicillin sodium salt at the dosage per 3 mln U/animal twice daily. On the 16th d - intravenously 0.5%-metronidasol solution at the dosage of 300 ml/animal thrice in 8 h. With prophylactic purpose for cattle youngsters the preparations should be introduced starting since 1-mo age according to the following scheme: during the 1st d - intravenously 0.5%-metronidasol solution at the dosage of 10 ml/animal thrice at 8-10-h-long interval. During ten days - perorally metronidasol at the dosage of 0.125 g/animal and norsulfasol at the dosage of 0.5 g/animal once daily. On the 13th and the 14th d - intravenously 0.5%-metronidasol solution at the dosage of 10 ml/animal thrice at 8-10-h-long interval, for 4-mo-aged calves during the first 2 days - intravenously 0.5%-metronidasol solution at the dosage of 20 ml/animal thrice at 8-10-h-long interval. During 18 days - perorally metronidasol at the dosage of 0.5g/animal and norsulfasol at the dosage of 0.5g/animal once daily, for 6-mo-aged calves during 10 days running - intramuscularly penicillin sodium salt at the dosage of 500 thousand U/animal twice daily. For 12-mo-aged calves during the first 2 days - intravenously 0.5%-metronidasol solution at the dosage of 100 ml/animal thrice at 8-10-h-long interval, since the 3d to the 5th d inclusively - perorally metronidasol at the dosage of 1 g/animal and norsulfasol or streptocid at the dosage of 1 g/animal once daily, since the 6th to the 12th d inclusively - intramuscularly penicillin sodium salt at the dosage of 1 mln U/animal twice daily. For 17-mo-aged calves during the first 2 days - intravenously 0.5%-metronidasol solution at the dosage of 150 ml/animal thrice at 8-10-h-long interval; since the 3d to the 5th d - perorally metronidasol at the dosage of 1.5 g/animal and norsulfasol at the dosage of 1 g/animal; for 18-mo-aged calves before pairing for 10 d - intramuscularly penicillin sodium salt at the dosage of 1 mln U/animal twice daily. The innovation is of high efficiency and enables to shorten the terms of therapy along with widened groups of animals under treatment and increased action of medicinal preparations.

EFFECT: higher efficiency.

3 cl

FIELD: animal farming.

SUBSTANCE: method involves using colamine as means for reducing losses of young cattle products during transportation and pre-slaughtering keeping of young cattle; feeding colamine to animals 7 days before transportation in an amount of 20-50 mg/kg of live weight.

EFFECT: reduced losses of animals live weight and increased resistance to stress effects.

5 tbl, 1 ex

FIELD: animal science.

SUBSTANCE: it is necessary to apply mitesel and glycosel additives as antidepressants at feeding animals 5 d before their transportation at the rate of 30 mg/kg body weight. The innovation enables to decrease negative impact of transportation stress and reduce the losses of body weight in cattle youngsters.

EFFECT: higher efficiency of prophylaxis.

1 tbl

FIELD: veterinary medicine.

SUBSTANCE: method involves taking animal blood samples, producing erythrocyte suspension by mixing blood and physiologic salt solution and centrifuging it. Reaction is carried out on 96-cell plastic flat-bottom tablet. Erythrocyte suspension and immunospecific sera are introduced into the cells, shaken, placed into thermostat and reading data. The tablets are shaken with tablet-type photometer. Visible light absorption capacity in bandwidth of 630-650 nm is determined in samples by means of the tablet-type photometer before placing the tablet into thermostat. The samples are incubated at 37°C in thermostat for 40 min. Then, the samples are additionally shaken and incubated for more 1.5-2.0 h, and visible light absorption capacity in bandwidth of 630-650 nm is determined once more. The reaction readings are taken by subtracting visible light absorption indices of the samples after incubation from those before incubation. The received values are transformed into serologic test points.

EFFECT: high estimation accuracy; facilitated animal testing process.

6 tbl

FIELD: poultry farming.

SUBSTANCE: method involves addition of lithium citrate in the dose 30-35 mg/kg of live mass to water used for watering poultry 24 h before the stress effect. Method provides enhancing safety and productivity of poultry.

EFFECT: improved method of correction.

11 tbl

FIELD: animal science.

SUBSTANCE: one should supplement the feedstuff with Phosprenyl, at the dosage of 0.05 ml/kg by the weight of offspring at whelping - once; in case of lactation and before weaning the whelps - twice or thrice. The innovation increases the offspring output, the safety of offspring, increases body weight and body size and quality of skins.

EFFECT: higher efficiency.

6 tbl

FIELD: agriculture.

SUBSTANCE: method involves introducing copepods (Acanthocyclops viridis, Eucycllops serrulatus, Cyclops strennus) and Cladocera (Daphnia magna, Daphnia pulex) crayfish at copepods and Cladocera ratio of 1:1 in amounts providing population density of 30-50 spc/l of water of biotope of mollusks-alternate host of common fasciola.

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1 tbl, 9 ex

FIELD: animal science.

SUBSTANCE: the present innovation deals with dynamic loading onto cardio-vascular system in animals. Selection should be carried out by the following parameters: , ΔT3 and Δn, where ΔT1 - the time for pulse increase at running, ΔT2 - the time for pulse stabilization after running, ΔT3 - the time for pulse increase after running, Δn - the increase of pulse frequency after running. One should select animals into milking herd at the following values; ΔT3 ≤ 10 sec, Δn ≤ 10 beats/min. The method enables to present perspective evaluation of lactation capacity in animals.

EFFECT: higher efficiency of selection.

1 dwg, 1 ex, 1 tbl

FIELD: veterinary.

SUBSTANCE: method comprises introducing glauconite into diet of milk cows in ecologically unfriendly zone in amount corresponding to 0.15-0.20 g per 1 kg cow's weight once a day over a 25-30 day period.

EFFECT: reduced level of lead both in body and in blood of cows.

3 tbl

FIELD: veterinary medicine.

SUBSTANCE: method involves interpreting dynamic omega potential behavior pattern relative to its initial level during 6-7 min after applying artificial pain irritation. The method is applied beginning from animal age of 1 month. Omega potential is measured before and after pain irritation test. Omega potential level growing down, the animal is considered to be of low stress stability. Omega potential level growing high, the animal is considered to be of high stress stability.

EFFECT: enhanced effectiveness of method.

2 dwg, 2 tbl

FIELD: veterinary medicine.

SUBSTANCE: poultry's diet during the first and second phases of egg yield should be supplemented with roxazym enzymatic complex at the ratio towards husk-bearing components being 0.007 - 0.009 : 20. The present innovation prevents inflammation of oviducal vagina in laying hens.

EFFECT: higher efficiency of prophylaxis.

3 ex

FIELD: poultry science, poultry selection.

SUBSTANCE: the method deals with evaluating egg shell pigmentation in 30-wk-aged hens of maternal stock followed by selecting the youngsters obtained from these hens. Two weeks before finishing the process hens should undergo stress impact to re-evaluate their egg shell pigmentation. Poultry offspring that changed the values of chromaticity by 2 points and higher should be rejected. The innovation enables to increase efficiency of selecting hens for higher safety and egg yield.

EFFECT: higher efficiency of selection.

1 ex

FIELD: veterinary science.

SUBSTANCE: the suggested preparation contains condensate obtained after distillation with urinary vapor of sows being in heat, acetic acid, butyric acid, caproic acid at the following ratio of components, weight%: acetic acid 0.5-2.0, butyric acid 5.0-20.0, caproic acid 0.5-5.0, condensate - the rest up to 100. The preparation is of high biological activity.

EFFECT: higher efficiency.

1 ex, 1 tbl

FIELD: poultry science.

SUBSTANCE: the present innovation deals with visual evaluation in chicken followed by testing them by the value of bioelectric potential. Chickens with bioelectric potential being significantly higher against average values are considered to be stress-resistant ones and those with bioelectric potential being significantly lower against average values in concrete population are concluded to be stress-sensitive ones. The method is very simple in its implementation and efficient for large-scale selection in poultry on stress-resistance.

EFFECT: higher efficiency.

1 cl, 2 dwg, 2 ex, 4 tbl

FIELD: animal science.

SUBSTANCE: the suggested biopreparation named "Lactobifadol" should be applied 1 mo before the impact of the mentioned stress factor at the dosage of 100-200 mg/kg body weight, daily. Application of "Lactobifadol" enables to normalize physiological body state and decrease the losses of meat production.

EFFECT: higher efficiency of prophylaxis.

1 ex, 2 tbl

FIELD: animals science.

SUBSTANCE: the present innovation deals with intramuscular injection of sodium salt preparation cloprostenol 30-45 min before placing at the dosage of 750 mcg/animal. The method provides increased reproductive function, enhances sexual reflex, increases the volume of ejaculate, concentration, activity and quality of spermatozoa.

EFFECT: higher efficiency of breeding.

2 ex, 3 tbl

FIELD: animal science.

SUBSTANCE: one should measure bioelectric potential in biologically active centers N 5, 7, 11, 41, 44 to establish its average value and at its value being above 48 mcA one should conclude upon high growth energy in calves. The application of the suggested method enables to predict potential growth energy in calves beforehand.

EFFECT: higher efficiency and accuracy of evaluation.

2 ex, 1 tbl

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