MXPA00006526A

MXPA00006526A - Sex-specific insemination of mammals with low number of sperm cells

Info

Publication number: MXPA00006526A
Application number: MXPA/A/2000/006526A
Authority: MX
Inventors: George E Seidel; Lisa Herickhoff; John Schenk
Original assignee: Xy Inc
Priority date: 1997-12-31
Filing date: 2000-06-30
Publication date: 2002-02-26

Abstract

Artificial Insemination is achieved for sexed mammalian offspring in a commercially practical manner and with dosages of insemination sperm which were not previously thought to be practical for commercial implementation. Improved insemination systems particularly adapted to use for sex-selected sperm sorting include systems which achieve superovulation and then multiple embryo production with sexed embryos. These systems combine with other techniques, including techniques for enhanced sheath fluid (3) and other strategies which minimize stress on the sperm cells (18), and potentially, a 2.9 percent sodium citrate sheath solution for bovine species and a hepes bovine gamete media for equine species. Improved collection systems (14) and techniques for the process are describeb so that commercial application of sperm samples as well as the resulting animals may now be achieved in the field.

Description

INSEMINATION WITH SPECIFIC SEX TO MAMMALS THROUGH LOW NUMBER OF SPERM CELLS I. TECHNICAL FIELD This invention relates generally to the field of sex selection in the progeny of mammals. This is especially relevant for the aspects of low dose artificial insemination and increased egg production to create the desired progeny sex. Particularly, the invention relates to the obtaining of artificial insemination sexed with low doses with respect to classification techniques, to systems for the classification of sperm through flow cytometry for specific sex and low dose products in artificial insemination techniques and results of increased ovulation or the like. II. BACKGROUND For years it has been desired to select the sex of the specific progeny. Beyond the obvious psychological aspects, the current sex selection of mammalian progeny has significant economic consequences when considering its application to animals that produce food such as cattle as well as famous trophy animals-such as horses and the like. This great desire has resulted in a significant variety of efforts to achieve the progeny of selected sex. Probably the effort that seems most similar to achieve the desired results has been the efforts in the classification and selection between the sperm of X and Y before insemination. One of the challenges that the efforts in the selection of sperm of X and Y have faced is the large number of sperm involved. In natural insemination the sperm is produced in some species by billions; in artificial insemination it is less, but a large significant number of sperm is still used. For example, artificial insemination techniques commonly use ten million to 500 million sperm (depending on the species). In this way, a significant number of sperm is necessary even in an artificial insemination environment. Many methods have been tried to achieve separation of the X and Y chromosomes that carry the sperm. These methods have varied from magnetic techniques such as that described in U.S. Patent No. 4276139 to column techniques such as that described in U.S. Patent No. 5514537 to gravimetric techniques as discussed in U.S. Patent No. 3894529, reissued patent No. 32350, US Patents No. 4092229; 4067965 and 4155831. Electrical properties have also been attempted as shown in EU 4083957 as well as a combination of electrical and gravimetric properties as discussed in US Patent Nos. 4225405, 4698142 and 4749458. Efforts have also been made to motility as shown in U.S. Patent Nos. 4009260 and 4339434. Chemical techniques such as those shown in U.S. Patent Nos. 4511661 and 4999283 (including monoclonal antibodies) and U.S. Patent Nos. 5021244, 5346990 , 5439362 and 5660997 (including membrane proteins) and U.S. Patent Nos. 3687803, 4191749, 4448767 and 4680258 (including antibodies) as well as the addition of serum components as shown in U.S. Patent No. 4085205. Although each of these techniques has been presented as being highly efficient, none of these techniques currently produces the desired sex pre-selection levels. However, despite the fact that the separation technique is used eventually, the competitive combinations of high sperm numbers naturally present and the separation approaches of the X and Y chromosomes that carry the sperm, have made it desirable to develop a ability to achieve insemination with a number of spermatozoa. Currently, the only quantitative technique used to achieve separation of the X and Y chromosomes that carry sperm, has been the one that includes the discrimination and individual separation of the spermatozoa through flow cytometry techniques. This technique seems possible as a result of advances and discoveries that include the differential absorption of dye from the X and Y chromosomes that sperm carry. This was discussed above in U.S. Patent No. 4362246 and was diffused significantly through the techniques described by La rence Johnson in U.S. Patent No. 5,135,759. Johnson's technique of using flow cytometry to separate X and Y chromosomes that carry the sperm, has been such a significant breakthrough that for the first time has made possible the commercial separation of such sperm. Although still experimental, separation has been significantly improved through the use of high-speed flow cytometers such as the MoFlo® flow cytometer produced by Cytomation, Inc. and described in a variety of other patents including US Patent Nos. .5150313, 5602039, 5602349 and 5643796 as well as in the International PCT Patent publication WO 96/12171. Although the use of the Cytomation MoFlo® cytometers have allowed a great increase in speed and although these speed increases are particularly relevant given the large number of sperm frequently used, certain problems have still remained. In spite of the advances of almost ten times in the possible speed by means of the flow cytometer MoFlo®, it has been desired to shorten and shorten the classification times for several reasons. First, it has been discovered that as a practical matter, sperm cells are time critical. They lose their effectiveness while the longer they remain unused. Second, the timing of collection, classification and insemination has made speed an element of high commercial importance. In this way, the critical nature of the tier of the sperm cells and the process, have made speed an essential element to achieve high efficiency and successful rates. There are also other problems that vary from practice to theory. On the practical side, it has been desired to obtain sperm samples of classified sex using components and economic disposable substances.

Also on the cost side, it has been desired to be able to achieve classification (as well as collection and insemination) in a labor event as efficient as possible. Thus, for commercial production and success in the field, improvements that can only represent an increase in efficiency may still be significant. In relation to the practical aspect of the costs, it is the practical aspect of the delicacy and sensitivity of the whole process. In this regard, it has been desired to simplify the process and make it as procedurally strong as possible so that the error of the operator or expert can play an ever decreasing role. They have also combined to make insemination with even more desirable low doses. In addition to the delicacy of the process, it has always been known that sperm by themselves are cells that are extremely delicate. Although this factor at first glance seems to be easily understandable, in fact, the total degree of sensitivity of the cells has not yet been fully explored. In the context of flow cytometry in general, most of the cells or particles classified frequently have been spherical or otherwise physically capable of resisting a variety of maltreatment. This is not the case with sperm cells. In fact, as described in the present invention, the procedure through normal flow cytometry techniques may, in fact, be unacceptable for the cytometric classification of sperm cells in certain applications. The range of sensitivities from the dilution problems and the inherent needs of flow cytometry to isolate and distinguish each cell individually as well as the pressure and other stresses that typical flow cytometry has, prior to the present invention, are imposed on the cells or other substances that were classified. This may also represent a unique factor for sperm cells because it seems that even though the sperm cells may appear to pass through the flow cytometer and are classified with non-visually discernible side effects, in fact, the cells themselves they may have been stressed to the point that their performance is less than optimal in the insemination process. In this way an interaction of factors seems to be involved and they have reached unusual problems from the perspective of classifying sperm cells and their final use for artificial insemination. Another problem that has remained - despite the great advances achieved through the Johnson Patent and the related art - is the fact that prior to the present invention it has been extremely difficult to achieve insemination of lower doses with sexed sperm, regardless of the separation technology used. Although historically, some low-dose insemination achievements have occurred, they appear to be more in a theoretical or laboratory environment than in environments that are similar to be experienced or applicable to a commercial application. In this regard, the desire has not only been to achieve low dose insemination but rather to achieve low dose insemination with successful fertility rates that are comparable to the existing non-sexed high dose artificial insemination efforts. Thus, the advances achieved by the present inventors in artificial insemination both sexed and low dose, represent significant advances that can for the first time, make commercial application possible. Another problem that has been faced by those in the industry - again, in spite of the great advances of the Johnson Patent and related technology - is the fact that the problem by itself, namely artificial insemination with a high success rate, it is statistical in nature in which a multitude of factors seem to interact. In this way, the best solutions can to some degree involve a combination of factors that, when studied statistically in detail, will prove to be necessary, either in isolation or in combination with other factors. Such a determination is further compounded by the fact that the results themselves vary by species and may be difficult to investigate due to the fact that it is probably not worth the effort in the initial stages of statistical and test sampling on a sufficiently large data. For these reasons the invention can also include a combination of factors that can, individually or in combination, represent the appropriate solutions for a given application. This description is therefore considered sufficiently broad so that the various combinations and permeations of the techniques described can be achieved. There may be undiscovered synergies with other factors. Such factors may vary from classification factors or perhaps, from the flow cytometer, from the stages to those in the collection as well as in the insemination stages. However, at present, studies have been mainly conducted on bovine species, it is not considered that these techniques will be limited to such species or, for that matter, only for sperm cells. It seems that the techniques used may have application beyond the sperm cells in areas that involve either sensitive elements to be stored or only the minimization of the stress impacts of flow cytometry when classifying the element. Interestingly, although the present invention takes an approach to minimize the impacts of classification or stress on sperm cells, others seem to have actually removed stages of this direction by increasing the pressures and demands for speed and others such as Performance. Essentially, driving for low-dose insemination and high-speed processing can, in an individual or perhaps interrelated manner, have had problems that are limited to each other. Thus although there has been a long-felt but not satisfied need for low-dose, high-speed sexed insemination and although implementation techniques and elements have been available prior to the present invention, advances or perhaps combinations of advances have been apparently overlooked by those experts in the field. Perhaps to some degree they failed to appreciate that the problem involved an interaction of factors as well as peculiar needs for the type of cells (sperm cells or perhaps specific species sperm cells) involved in this field. Interestingly, as the list of previous efforts in this exhibit shows, substantial but apparently unsuccessful attempts have been made to understand the problem inherent in such an area as low dose sex insemination, and perhaps they have assumed that due to the service event Naturally involving perhaps billions of sperm, there may have been physical limitations to the achievement of artificial insemination with numbers that are as many as four orders of magnitude smaller in number. Thus it may not be surprising that there existed to some degree a real teaching away from the technical direction that the present inventors followed. Maybe the results can. still considered unexpected to a degree, because they have shown that artificial low-dose insemination can be achieved, sexed with successful rates comparable to those of artificial insemination of high doses not sexed. It may still be surprising to some that the techniques and advances of the present invention are in fact combined to achieve the great results shown. Although each technique can be isolated, seen by some as not extraordinary, in fact, the subtle changes seem to provide significant advances in the final result - whether they are considered alone or in combination with other subtle changes. In this way, the achievements - from successful rates of low-dose, sexed artificial insemination, to the present invention, with required levels of performance or simplified procedures similar to those needed to achieve commercial implementation, had not been possible. However, beyond the sexed insemination of low dose at a commercially achieved level, the present invention also describes techniques that allow the achievement of improved performances and thus facilitates the desired final result, ie, artificial insemination sexed dose low on commercial bases. III DESCRIPTION OF THE INVENTION According to the foregoing, the present invention claims the achievement at a commercial level of the low-dose insemination and the results as applied to predetermine the sex of the mammal. Improved coating and collection systems are also provided for the classification of sperm cells in order to determine their sex through a flow cytometer separation technique. In this separation technique, the coating fluid as typically used in a flow cytometer is replaced with a fluid that minimizes stress on the sperm cells as they are sorted. In addition, the collection system has been improved to minimize both the physical and chemical stress to which the sperm cells are subjected. Various techniques and substances are represented but as will be readily understood by those skilled in the art, various combinations and permutations can be used in a manner that can be optimized for their realization based on the species, separation techniques, objectives and other parameters involved in a specific processing application. Thus, it is an object of the invention to achieve only sexed insemination with low doses in a manner that works under real commercial circumstances. It is also a goal to achieve the best classification for substances such as sperm cells. A related purpose is to minimize the impact of the classification function that the cells or other sensitive elements that can be classified have themselves. For a technique of classification by flow cytometry, a particular objective is to minimize the impact that the coating fluid imposes on the cells and potentially provide a coating fluid that acts affirmatively to help the cells in handling the various stresses involved. A parallel objective is to provide substances and techniques that are especially suitable for sperm cells in general, for bovine sperm cells, for de-equine sperm cells and for the separation of such sperm cells in X and X chromosomes. And that carry the components. Similarly, one goal is to minimize the impacts that the collection phase (for example after the classification has on the cells and minimize the physical impact as well as the chemical impacts on such sperm cells of classified sex). achieve a classified result as unaffected as possible.Another objective of the invention is to achieve classified insemination of low dose levels and with success rates that are comparable to those of typical high dose, non-sexed artificial insemination. In the objective, a purpose in the total system for artificial insemination is present that can achieve this objective in a commercially practical way, so the above purposes of minimizing stress or potential damage to sperm cells is important. a way that provides both high-speed and low-stress classification and that is dapte especially for the classification of sperm cells in a low dose context, is also an important goal. The purposes of providing coating and other fluids that do not adversely affect sperm fertility and are compatible with artificial insemination, they are also important. Naturally, further objects of the invention are described through other areas of the specification and claims.

IV BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of a classification system according to a flow cytometer separation technique for the present invention. Figure 2 is a diagram of the cells introduced in the free fall area of a typical flow cytometer. Figure 3 is a conceptual diagram showing the differences as they appear roughly as a result of the present invention. Figure number 4 is a diagram of the stream of cells sorted as they are collected in the area of the deposit area. V BEST MODE FOR CARRYING OUT THE INVENTION As will be appreciated, the basic concepts of the present invention can be combined and incorporated in a variety of ways. The invention includes only sexed insemination of low dose for commercial practice and the results. For the techniques of separation by flow cytometry, the invention also includes both improved flow cytometry systems as well as systems for the creation of sex-specific sperm samples that can be used in artificial insemination and the animals produced by such techniques. The invention includes, above all, processes through which highly successful rates are possible even in commercial environments. In addition, the techniques are described in a general manner so that they can be applied to specific systems and applications once the general principles have been understood. Although the improvements to the device are described, it will be understood that these improvements not only carry out certain methods, but may also vary and be combined in different ways. Importantly, regarding all of the above, each of these facets must be understood in order to be covered by this description. As mentioned, the basic objective is the separation of the sperm carrying the X from the sperm carrying the Y. This is done so that the two types of sperm are isolated so that each can be packaged and treated separately. . Currently the isolation is preferably done through the use of flow cytometry. Flow cytometry is generally a technique that is well understood. For example, the basic aspects of this are shown and described in a variety of Cytomation, Inc. patents such as US Patents. and other publications listed above. Each of these patents and the references cited therein, are incorporated for reference; thus those skilled in the art can easily understand the basic principles included. Essentially, flow cytometry includes classification elements, such as cells that are provided to the flow cytometer instrument through some types of cell sources. Figure 1 shows a conceptual instrument. The flow cytometer instrument includes a source of cells (1) that acts to establish or supply cells or some other type of element to be analyzed by the flow cytometer. The cells are deposited inside a nozzle (2) in such a way that the cells are surrounded by a coating fluid (3). The coating fluid (3) is normally supplied by means of some source of coating fluid (4) so that the source of cells (1) supplies its cells, the coating fluid (3) is fed concurrently through the nozzle (2). In this way it can be easily understood how the coating fluid (3) forms a fluid coating environment - for the cells. Since various fluids are provided to the flow cytometer at some pressure, they flow out of the nozzle (2) and exit through the hole (5) of the nozzle. By providing some type of oscillator (6) that can be controlled very precisely through an oscillator control (19), pressure waves can be established within the nozzle (2) and transmitted to the fluids leaving the nozzle (2) through the hole (5) of the nozzle. Since the oscillator (6) thus acts on the coating fluid (3), the current (7) coming out of the holes (5) of the eventual nozzle and regularly forms drops (8).

Because the cells are surrounded by a fluid coating environment, the drops (8) may contain individually isolated cells or other elements within them. Since the drops (8) generally contain isolated cells, the flow cytometer can distinguish and separate the drops based on whether or not it is the appropriate cell (s) and whether it is / are contained in the drop. This is carried out through a cell detection system (9). The cell detection system includes at least one type of detector (10) that responds to the cells contained within each drop (8) as discussed in detail in the seminal work by Larry Johnson (not to be repetitive), ie in the U.S. Patent Do not. - 5135759. As explained in the Johnson patent for sperm cells, the cell detection system (9) can make an action depending on the relative presence or relative absence of a particular dye, which can be excited by some stimulant such as laser exciter (11). Although each type of sperm cell is stained by the dye, the length in which the X chromosome differs and the Y chromosome makes the different levels of staining. Thus, by detecting the degree of ink present in the sperm cells, it is possible to discriminate between the sperm carrying the X and the sperm that carry the Y through their different levels of emission. In order to achieve the separation and final isolation of the appropriate cells in a separation technique by flow cytometer, the signals received by the detector (10) they are fed to some kind of classifying discrimination system (12) which makes the decision very quickly and can differentially load each drop. (8) based on whether you have decided that the desired cell exists or does not exist within the drop (8). In this way the classifying discriminator system (12) acts to allow the electrostatic deflection plates (13) to deflect the drops (8) based on whether or not they contain the appropriate cell or other element. As a result, the flow cytometer acts to classify the cells by having them deposited in one or more collectors (14). Thus, when detecting some property of the cells or other elements, the flow cytometer can discriminate between the cells based on a particular characteristic and place them in the appropriate collector (14). In the system currently used to classify sperm, the drops of sperm that carry the X are positively charged and thus deflect in one direction, the drops of sperm that carry the Y are negatively charged and thus deflect in another way, and the current waste (which are unclassifiable cells) is discharged and thus collected in an unclassified stream in a suction tube or the like. With reference to Figure 2, the process can still be understood further. As shown in that figure, the nozzle (2) emits a current (7) which due to the oscillator (6) (not shown in Figure 2) forms drops (8). Since the source of cells (1) (not shown in Figure 2) can supply sperm cells (15) which have been stained according to the Johnson technique, it is differentially determined by the detector (10), the lightly stimulation by the laser exciter (11) so that the existence or non-existence of a charge in each drop (8) can be controlled as they are separated from the stream (7) by the flow cytometer. This control results in drops (8) - positively charged, negatively charged and uncharged based on their content. As shown in Figure 2, certain drops are shown as deflected drops (16). These deflected drops (16) are those that contain sperm cells (15) of one or the other sex. These are deposited in an appropriate collector (14) for later use. One of the aspects of flow cytometry that is particularly important for its application in the classification of sperm, is the high-speed operation of a flow cytometer. Advances have been made particularly for the flow cytometers available from Cytomation, Inc. under the trademark MoFlo®. These flow cytometers have extraordinarily increased the classification rates, and thus have made flow cytometry a technique that is ideal to make viable the commercial application of sperm classification (among other commercial applications). They act to achieve high-speed classification, which is a speed that is noticeably greater than those used in other circumstances. Especially, the Cytomation MoFlo® flow cytometers act with oscillatory frequencies greater than approximately five kilohertz and more specifically can operate in ranges of 10 to 30 or even 50 kilohertz. Thus, the drops are formed at very high frequencies and the cells contained within the fluid coating environment can be emitted very rapidly from the nozzle (2). As a result, each of the components such as the nozzle (2) oscillator (6) and the like that make up and are part of a flow cytometer system can be configured or selected to result in a high-speed cell sorter. . In the application of a high-speed cell sorter for the classification of sperm cells, classification is achieved at speeds greater than 500 classifications per second. In fact, the classification speeds in the thousand and one thousand two hundred ranges have already been achieved through a high-speed cell sorter. Importantly, it should be understood that the term "high speed" is a relative term in such a way that as further advances in flow cytometry and specific applications are achieved, the aspect that is considered "high" may vary or it can remain absolute. In any definition, the. The general principle is that classification can occur at speeds at which the parameters and physical characteristics of the flow cytometer are significant for the cells themselves when particular cells are classified as sperm cells. Another aspect of the high-speed classification that appears to be at play when sperm cells are classified through a flow cytometer separation technique is that of pressure and other stresses to which sperm cells are subjected. inside the flow cytometer. For example, when operating at high speed (and an alternative definition of "high speed"), flow cytometers can be operated at a pressure of 3.51 kg / cm2 (50 pounds per square inch) and still 4.22 kg / cm2 (60 pounds) and more pounds per square inch. These pressures can be considered high because they can result in effects on the cells that are being classified. The key as described in the present invention for this facet, is the fact that the stress thresholds of the particular cells are the determining factor. Additionally, as additional knowledge is acquired it can be shown that the stress thresholds are a function of the combined effects such as the particular species or the previous or subsequent particular management of the cells. The key in this respect is that the stress imposed on the cells can, in fact, alter their viability and their ability to achieve the desired result. In the case of pressure, it may be that only the sperm cells are subjected to a higher pressure as a result of the operation of the flow cytometer in which that pressure can result in a decreased performance of the cells. The present invention to an aspect acts to minimize this stress and thus result in greater efficiency as well as doses as low as will be discussed later. When considering the stress aspect in the cells, the present invention acts, in a way that minimizes stress. This stress can be minimized at any point in the cycle or total collection process, classification or even in the insemination of the animal. Importantly, the stress imposed by the handling of the cells within the flow cytometer seems significant for this application. In one embodiment of the invention, the coating fluid is specifically selected such that it can serve in a coordinated manner with both (or any) of the fluid pre-sorting cell environment or the post-sorting fluid cell environment. Although it is naturally possible to adjust any of the pre-or post-classification fluids, in one embodiment the invention adjusts the coating fluid (3) in a manner that imposes significantly less stress on the cells than those previously performed. In one aspect the invention is remarkable in that it removes the total focus from that of the operation of the flow cytometer to a focus on stress, handling and removal of the cells by themselves. For example, although it is known to use fluids having an appropriate pH factor or osmolality, the present invention recognizes that certain chemical compositions may exist for which the cells may be hypersensitive. These hypersensitive chemical compositions can vary naturally based on the cells or as in the previous handling of the cells. Importantly, it currently appears that for certain sperm cells certain metabolic chemical compositions such as citrate seem to avoid unusually high stresses on cells. Thus, hypersensitive chemical compositions can be defined as those to which the cells are particularly sensitive in the context of their functionality and therefore to the existing management techniques. As for the sperm cells it seems that the metabolic compositions, specifically the stability of citrate for bovine sperm cells and the stability of the hepes regulator for equine sperm cells can be very important. Thus, the present invention acts to minimize changes through the type of operation or the selection of substances that can act as a means to minimize the changes experienced by cells. For the coating fluid, a substance is selected according to one embodiment of the invention so that it can be chemically coordinated to the minimum changes present. Thus, by selecting the appropriate coating fluid, not only in the context of the flow cytometry parameters, but preferably also in the context of the cell parameters themselves, the changes experienced by the cells and proteins can be improved. total results of the classification. This is shown conceptually in Figure 3. Figure 3 shows some type of chemical factor (such as citrate or other factors) as it may exist during the various phases of the process. For example, the four phases shown can represent the following shown by a flow cytometer separation technique, but not to be limited in that way: phase I can represent the existence of cells within the source of cells (1), Phase II can show the existence of the cells as they are classified in the fluid coating environment, phase III can show the cells as they are collected after sorting, and phase IV can show the reconstituted cells in a Storage medium after sorting. These four phases as shown by the prior art can widely experience environments of different chemical factors. However, as shown conceptually, in the present invention the cells may undergo very small changes, most notably, the immersion or fall experienced between phase I and phase II may be virtually absent. This is as a result of the selection of the appropriate coating fluid as mentioned above. A) Yes, as a result of undergoing an appropriate coating fluid, the cells of the present invention may experience a much lower level of stress. One of the potential generalities that may exist with respect to this phenomenon is the fact that certain chemical compositions may represent more hypersensitive chemical compositions than others. Although this may vary naturally based on sperm species, management, or even the type of cells involved, it seems that the viability of the cells for their intended purpose (artificial insemination, in the present) varies greatly, naturally or due to the classification or both, and thus, the cells exhibit a hypersensitive nature with respect to that chemical composition. By selecting certain metabolic chemical compositions, most notably citrates or chemicals that are within the citric acid cycle, major advances seem possible. Thus, for the application of bovine sperm, the coating fluid (3) is selected and coordinated so as to present approximately 2.9 percent sodium citrate composition. Specifically, 2.9 percent sodium citrate solution can be created as follows: 1. Place 29.0 grams of sodium citrate dihydrate (Na_jC6Hs07 »2H20) in a 1,000 ml volumetric flask. to. Dissolve the sodium citrate in 3/4 of the water batch, then add water to the volume. 2. Add deionized water or Nanopura to make a final volume of 1,000 ml. 3. Transfer to bottles and autoclave at 6.81 kg (15 pounds) of pressure (245 ° F) for at least 30 minutes a. Treat the solution in the autoclave using conditions to minimize evaporation (loose cover) b. Take care that the water does not go beyond boiling. 4. Cool slightly at room temperature. 5. Store it sealed in a room cooled to 5 ° C. In addition, for a coating fluid, the sodium citrate solution can be filtered. 6. Filter with a .22 micron filter using aseptic techniques. Interestingly, for equine sperm cells, such a composition does not work very well. Instead it has been found that for equine sperm cells, a regulated hepes medium such as a medium of hepes bovine gametes - particularly HBGM3 as previously created by J.J. Parrish for an application in bovines-works well. This means is described in the article "Bovine Sperm Training by Heparin", ("Capacitation of Bovine Sperm by Heparin") 38 Biology of Reproduction 1171 (1988) incorporated herein by reference. Not only is this surprising because it is not the same type of substance used for bovine sperm, but the current regulator was originally developed for an application in bovines. Thus in the application of equines the coating fluid that is selected contains the hepes regulator. This solution can have a pH at room temperature of about 7.54 (pH at 39 ° C = 7.4) with the following composition: Chemistry Dry weight (g / 500ml) CaCl- 0.145 KC1, 0.115 MgCl2 »6H20 0.004 NaH2P04» H20 0.018 NaCl 2.525 Na pyruvate 0.011 Lactic acid (60%, 1.84 ml HEPES 4.765 NaHC03 0.420 BSA (fraction V) 3.0 Another aspect that may interact in the present invention is the fact that the cells involved may experience unusual sensitivities. In one aspect this may be due to the fact that the sperm cells are in a class of cells that are not repairable cells. That is, they do not have the ability to restore themselves and therefore may need to be treated much more sensibly than usual by flow cytometers or other handling equipment. Thus, it may be appropriate for the improvement to be particularly applicable when the flow cytometer or other separation device acts to establish a source of sperm cells. Another potentially related aspect that may be unique to a class of cells such as sperm cells, is the fact that their DNA is non-restorable, non-replicable and non-transcribable. Any of these factors can intervene and therefore can be relevant either individually or together. Thus, it may be that the teachings of the present invention are applied to all gamete cells or even to viruses and the like that they are non-repairable, non-translatable, non-transcribable cells. A separate aspect of flow cytometry processing that may also be important is the proper treatment of cells both chemically and physically after classification. As shown in Figure 4, since the cells within the drops (8) are deposited in the collector (14), it may be important that the container that integrates the collector is appropriately sized so that it acts as a medium to avoid the impact between the cells and the container by itself. Although it is known to place an initial collector fluid (17) in the bottom of the container to collect the cells so that they do not hit the bottom of the container, it appears that a simple widening of the container can be used to direct variations in the presentation of the current as well as the inevitable splash due to the impact of the cells in the container to improve the result. In one aspect this may act as a buffer element so that cells that may be sensitive to mechanization, that is, may be broken or damaged by an impact, may be treated appropriately. Thus, when the source of the cytometer establishes cells that are physically delicate cells such as the cells to be sorted, it may be important to provide some type of damping element such as a wide collection tube for which the amplitude of the opening (18) serves to position the walls of the container in a manner that avoids contact with the cells. Thus, the tube does not have side walls so close that there is any significant probability of making contact between the cells to be classified and the walls of the tube. In this way, in addition to the collection fluid (17), it may also be desirable to include a wide collection tube. Perhaps, providing only a wide opening to the container that serves as part of the collector (14) may be sufficient. For applications using the classification of the sperm cells at high speed, it has been found that providing a container having an opening with an internal diameter of at least 15 millimeters is considered sufficient.

Specifically when using a 14 ml Falcon test tube in such an application, minimal physical damage to the cells has been discovered as a result of the collector (14). It should be noted that even the 14 ml Falcon test tube may not be optimal. Specifically, it is considered that it may be more optimal to design a collection container that matches the geometry of the stream (i.e., a container adapted to the stream). This current-adapted vessel can have any or all of the following characteristics: a relatively wide orifice, an elliptically shaped orifice, a smaller height-to-amplitude ratio than currently involved, an angled or otherwise coordinated display such as that which they can present the side walls that are parallel to the downdrafts and the like. It may also be desirable to provide a mounting member such as a movable member or similar means to a ball bearing or the like to allow variable orientation of the tube in order to equalize the desired downdraft to be collected. In addition, the physical characteristics for the class of vessels such as the existing tube (described as a "Falcon type" test tube) can include not only the width of the tube but also the material from which it is made (such as polystyrene to which it does not cells stick) and the like. (These material options are well known for the 14 ml Falcon tube). Thus, the container and its collection fluid can also serve as a buffer to minimize physical damage to the cells. This can also serve, by its size, to facilitate the collection of the appropriate numbers of sperm without a significant dilution effect. Another aspect of the collector fluid (17) may be the fact that it may also serve to minimize chemical stresses in the cells. In one aspect, since this may be important in providing a nutrient to the cells both before and after sorting, the collecting fluid (17) may be selected in order to provide a coordinated level of nutrients so that the levels are balanced. both before and after the classification. For bovine sperm in which a yolk citrus nutrient is used at a two percent egg yolk level, it has been found that using a six percent egg yolk citrate level (ie , content of egg yolk at six percent in a solution of, citrate) provides good results. This is as a result of the volumes that exist before and after the classification event. The collecting fluid (17) can initiate (before the sorting) with approximately 2 ml of volume. The classification event can add approximately twice this volume (ending in three times the initial volume) with very little egg yolk citrate in solution (due to 'obstruction' and other considerations of the flow cytometer). Thus the final result in terms of the level of the amount of egg yolk citrate present, can be equivalent to the initial result, that is, the content of egg yolk at two percent in a citrate solution due to the volumes involved. . Thus, the collection fluid (17) may be selected in order to create a final collector fluid environment that is balanced with the initial nutrient or other fluid environment. In this way, it can serve to minimize the time and the modified level of the composition to which the cells are subjected. Naturally these fluid environments can be presented within the flow cytometer or they can exist in some other previous time, being simply the important point, to minimize the stress to which the cells are subjected at any time in their life cycle. Also, since the content of the initial chemical substance may vary (for example, the percentage of egg yolk content in the citrate can vary upwards or downwards), likewise the fluid initial collection environment or various - - volumes, can also vary so that the final result is the same. Thus, before starting the classification process, the collection fluid exists with a six percent content of egg yolk in the citrate solution and after finishing the classification event, the collection fluid - with the specific sperm from sex - can result in two percent egg yolk content in the citrate solution similar to the initial nutrient content. Note that in the previous use, these sperm cells can be treated at a 20% egg yolk content in the citrate fluid for other reasons, however these changes are not considered to provide stress to the cells since they are only a known part of the total insemination process. Although, of course the levels can vary according to those easily understood by those skilled in the art, the 20% egg yolk citrate regulator can be constituted as follows: I. FINAL COMPOSITION 80% sodium citrate solution (72mM) 20% egg yolk (vol / vol) II. PREPARATION FOR A LITER: A Sodium citrate solution 1. Place 29.0 grams of sodium citrate dihydrate (Na3C6H507 »2H20) in a 1,000 ml volumetric flask. 2. Add deionized water or Nanopura to make 1,000 ml of final volume. 3. Transfer to bottles and autoclave at 6.81 kg (15 pounds) of pressure (245 ° F) for at least 30 minutes. to. Treat the solution in an autoclave using conditions to minimize evaporation (loose cover). b. Be careful that the water does not go beyond boiling. 4. Cool slowly to room temperature. 5. Store sealed in a room cooled to 5 ° C. B. Egg preparation 1. Obtain fresh chicken egg from a good commercial source. 2. Wash the egg, leaving it free of dirt (do not use too much detergent) and rinse. 3. Immerse the egg in 70% ethanol for 2-5 minutes. 4. Remove the egg and let it dry (or dry) and store it in a clean towel.

C. Preparation of the diluent 1. Use clean, sterile glassware. 2. Fraction-A (fraction without glycerol) a. Place 800 ml of 2.9% sodium citrate solution 5 in a 1,000 ml graduated cylinder. b. The antibiotic levels for the fraction that does not contain glycerol (Fraction-A) of the diluent can be as follows: i. Tylosin = 100 μg / ml 10 ii. Gentamicin = 500 μg / ml iii. Lineo-spectin = 300/600 μg / ml c. Add 200 ml of fresh egg yolk as summarized below (Section D) i. Mix thoroughly. 15 d. This provides the diluent of Fraction A based on 2.9% sodium citrate, with 20% egg yolk and antibiotics at known concentrations to be non-toxic for bull sperm. 20 e. The diluent can be stored overnight at 5 ° C. F. Decant the supernatant (greater than 800 ml) the next day. g. Heat at 37 ° C before using the next day. D. To add the egg yolk to the buffer solution, the following procedure works well. 1. Wash the egg and clean the eggs (see B above) 2. Open the egg and separate the yolk from the albumin using a yolk separator. Alternately empty the yolk 2-3 times between the two shell halves from one side to the other. Without breaking the membrane surrounding the yolk. 3. Place the yolk on a piece of 15 cm sterile filter paper. 4. Hold the filter paper on the graduated cylinder containing the regulator and squeeze the yolk (breaking the membrane) and allowing the yolk to leave the golden filter paper towards the cylinder. Typically about 12-15 ml of yolk can be obtained from an egg. Another aspect that can interact in the various factors of the present invention is that of using low dose quantities of sperm for artificial insemination or the like. Additional background in the aspect of artificial insemination, sexed, can be found in "Prospects for the Classification of Mammalian Sperm" by Rupert P. Amman and George E. Seidel, Jr., Associated Press of the University of Colorado (1982) incorporated herein by reference. As mentioned, natural insemination involves amounts of sperm in the order of billions of sperm. The typical artificial insemination is currently conducted with millions of sperm for the bovine species and hundreds of millions of sperm for the equine species. By the term "low dose" is meant that the dose of sperm used in the event of insemination is less than half or preferably even less than about 10% of the typical number of sperm provided in a typical artificial insemination event. Thus, the term "low dose" is to be contemplated in the context of typical artificial insemination doses or also as an absolute number. For bovine sperm, which currently provides one to ten million sperm, a low-dose process can be considered an absolute number of approximately 500,000 sperm or perhaps as low as 300,000 sperm or lower. In fact through the use of the techniques of the present invention, artificial insemination has been shown with good success rates with sperm insemination levels at 100,000 and 250,000 sperm (41% and 50% respectively of pregnancies rates). ), as shown in the article "Insemination of Uterine Tubes of Calves with Very Low Numbers of Sexed and Non-Frozen Sperm" (Uterine Horn Insemination of Heifers With Very Low Numbers of Non-frozen and Sexed Spermatozoa) as published in the 48 of Teriogenology 1255 (1997) incorporated herein by reference Since the sperm cells appear to display a sensitivity to dilution, these results may exhibit particular interdependence in the use of low dose sperm sample with respect to the various techniques of the present invention The absolute numbers may be dependent on the species. e less than about twenty-five, ten, five or even a million sperm can be considered a low-dose process. Another aspect that may be important is the fact that sperm sexed through the techniques of the present invention or otherwise, are used in an artificial insemination system. Thus, when, for a flow cytometer technique, the harvester (14) is used to provide sperm for artificial insemination, the techniques of the present invention may be particularly relevant. In addition, it is possible that the combination of both the use of artificial insemination and the use in a low dose environment can create together synergies that make the various techniques of the present invention particularly suitable. Naturally, sexed sperm can be used not only in an artificial insemination mode, but in other techniques such as in vitro fertilization and the like. The process of collecting, sorting and eventually inseminating an animal through the use of flow cytometry or another separation technique involves a variety of stages. In the context of bovine insemination, semen is first collected from the bull through the use of an artificial vagina. This occurs at rates of approximately 1.5 billion sperm per ml. This pure semen can be verified through the use of a spectrometer to assess the concentration and can be evaluated microscopically to ensure that it meets the appropriate motility and viability standards. Then antibiotics can be added. As a result the initial sample may have approximately 60 to 70 percent of the progressively mobile sperm per ejaculate. For processing, a dilution through some type of TALP (thyroid albumin pyruvate lactate) can be used to achieve the number of sperm at a manageable level (for flow analysis) of approximately 100 million per ml. TALP not only nourishes sperm cells, but can make them hyper-activated for the dyeing stage. Before dyeing in some species such as equine species, centrifugation can be carried out. The dyeing can be carried out according to a multiple dyeing or single dyeing protocol, the latter being the subject of the Johnson Patent and related technology. The dyeing can be carried out while also adjusting the diluent to create the proper nutrient environment. In bovine applications this may involve adding approximately 20% egg yolk content in a citrate solution immediately after dyeing. Furthermore, in the dyeing of the sperm cells, it has been found that by using larger amounts of dye, better results than might be expected to some extent can be achieved. This high concentration of dyeing may involve using amounts of dye in the dozens of micro-molar content as discussed in the subsequent examples where the micro-molar 38 content of Hoechst 33342 dye is used. After adding the dye, use an incubation period such as one hour incubation at 34 ° C until ink absorption is precipitated with concentrations of approximately 100 million sperm cells per ml. Filtration can then be carried out to remove groups of sperm cells and then dilution can be carried out up to the desired classification concentration of approximately 100 million sperm cells per ml. Classification according to the various techniques discussed above can then be carried out from which the sperm cells can be recovered in the collection phase. As mentioned above, the collection can result in samples with approximately 2% of the content of egg yolk citrate concentrate (for bovine species). This sample can also be concentrated to approximately 3-5 million sperm cells per ml through the use of centrifugation after which the coating fluid and the preservation fluid can be removed. A final dilution can then be carried out either with 20% egg yolk or a Cornell Universal Diluent or the like. The Cornell Universal Diluent can have the following composition for 1,000 ml: 14.5 g of sodium citrate dihydrate 2.1 g of NaHCO3 0.4 g of KCl 3.0 g of glucose 9.37 g of glycine 0.87 g of citric acid For the composition of egg yolk 20%, 800 ml of the above preparation and about 200 ml of egg yolk can be used. After this last dilution, 3 to 5 million sperm may result from me (for bovine species). This sample can then be cooled to decrease sperm metabolism and to allow use over longer periods of time. In equine species the sample can then be used in oviductal insemination processes or other processes such as those that those skilled in the art will well understand. In bovine sperm, the sample can still be diluted one more time up to the desired dose level. It has been found that dilution can create an effect on the viability of sperm cells and therefore it may be appropriate to avoid too large dilution levels by providing a smaller sample. Regardless of the separation technique used, at present, low doses of approximately 300,000 sperm per 0.184 ml can be achieved. In addition it may be desirable to maintain a plasma-seminal level of approximately a level of 5%, although the results of this requirement are currently mixed. The eSperm cell specimen can then be placed in a straw for use in artificial insemination and can be transported to the cows or calves to be inseminated. In order to achieve artificial insemination conveniently synchronized, the estrus of the calves or cows can be synchronized using known techniques such as the use of prostaglandin F2a according to the techniques well known in the art. This last substance may be particularly valuable in that it has been reported to potentially achieve improved fertility in calves as discussed in the article "Is Prostaglandin F2a - A Defecitic Drug in Dairy Cattle?", ("Prostoglandin F2a - A Fertility Drug in Dairy Cattle? ") In 18 Theriogenology 245 (1982) incorporated herein by reference. Although recent results have not maintained this premise, it may be that the present invention demonstrates its particular viability in low dose, sexed insemination situations. For bovine species, artificial insemination can then be carried out through the use of embryo transfer equipment with placement of the sperm cells deep within the uterine tubes. This can be carried out not at peak time as is typically used in artificial insemination, but rather at any later time such as 12 hours after that time since there is some possibility that fertility for sexually inseminated insemination may occur slightly later. Embryo transfer equipment can be used because it can be highly sensitive to the uterine wall for such low-dose sex inseminations. In addition, techniques can also be combined to achieve high efficiency production. Particularly the now invented processes that allow high-speed classification and low-dose insemination of sexed embryos are also possible in a superovulated animal. Superovulation can be achieved through the use of a superovulatory pharmaceutical technique or through any other. Pharmaceutical superovulatory can act directly or indirectly, such as through a sequence of reactions to achieve a higher than normal production of ovules. The combination with superovulation is surprising because it was previously considered that superovulation obstructed such a combination. Sperm transport is included in superovulated cattle, so that animals are artificially inseminated frequently on multiple occasions and / or with multiple doses of semen. Also the previous procedures for sexed semen were relatively slow, therefore it was of interest to determine the rates of fertilization after a single insemination of the pharmaceutical superovulatory, such as cattle treated with FSH (follicle stimulating hormone) with only 600,000 total non-frozen sperms sexed using these combinations of newer techniques. For example, twelve Angus hybrid calves were superovulated using standard procedures: 6,6,4,4,2,2,2 and 2 mg of FSH were injected intramuscularly at half-day intervals beginning between days 9 and 12 of the cycle of estrus; and 25 and 12.5 mg of prostaglandin F-2 alpha were injected intramuscularly with the sixth and seventh injections of FSH. The sperm of bulls of unknown fertility were stained with Hoechst 33342 and then classified using a flow cytometer MoFlo® / 700-800 live sperm cells producing the cell sorter of each sex / second. The average purity of the classification was 89% of the desired sex. The classified spermatozoa were concentrated at 3.36 x 106 sperm / ml by centrifugation at 650 g for 10 minutes, cooled to 5 ° C and stored for 4 hours. Then 184 ul were loaded into 0.25 ml plastic straws; half of the dose was inseminated in each uterine tube 20 to 24 hours post-onset of estrus using automatic lateral opening embryo transfer liners. The embryos were collected by standard non-surgical procedures at 7 or 16 days post-estrus. The results were similar between the collections of day 7 and 16 and among the spermatozoa classified X and Y. The embryos were recovered from 9 calves. There were 52 embryos (ie, 4.3 ± 5.3 / donor) in normal stages of development, 13 delayed embryos and 31 unfertilized ovules. Forty-six embryos were sexed by PCR using primers for a specific Y chromosome DNA sequence; 43 (93%) were of the proposed sex. Although this study involved few animals, surprisingly, insemination of superovulated calves with only 600,000 nc frozen (total) sexed spermatozoa gave similar results to conventional procedures. Variations in the above can also be achieved, including, but not limited to, classification through different flow cytometric media, achieving superovulation in other ways that increase fertility in other ways and the like. In addition to the congruence of the methods of sexing sperm on the basis of DNA content, the high-speed flow cytometer / cell sorting and the procedures of insemination of calves with less than 500,000 total sperm without compromising fertility, has resulted the possibility of a viable sexed semen industry in cattle within a few years. There will be countless applications of sexed semen with accuracy > 85% Perhaps the most obvious is the insemination of a subset of cattle (both dairy and meat) for replacements of herds of females, and having the reverse subset (both dairy and meat) spawned for completely different types of bulls to produce males for meat . A very important subset of the above is the insemination of calves with sperm that carries the X chromosome to produce female calves that have a lower incidence of dystocia than that of male calves, mainly due to the smaller size. In addition it would be much more efficient to provide young dairy parents with a preponderance of calf calves. Having more than 85% of calves calves also makes it possible to manage dairy cows in order to average less than two surviving calves per life time, which is attractive because it reduces the problems associated with pregnancy and childbirth. Single-sex systems of meat production would also be feasible in that each female replaces itself and is slaughtered between 2 and 3 years of age, thus utilizing a much higher percentage of nutrients in the system for the development and a lower percentage for maintenance. Sexed semen would be useful for in vitro fertilization and for inseminating superovulated cows for embryo transfer. Frequently one sex of calves is considerably more valuable than the other and although exact methods of sexing embryos are available, they are delayed and half of the embryos produced are of the less valuable sex. It is assumed that exactly sexed semen would be widely adopted for artificial insemination of cattle., if the overload of sex was low and fertility was only minimally compromised. The percentage of beef cattle artificially inseminated would probably increase substantially with sexed semen. Interestingly, better results may be achieved than in place of insemination within the uterine body where such insemination is usually placed, by insemination deep into the uterine tube. Perhaps - it is also surprising that the samples studied so far have shown no difference between ipsi- and against lateral inseminations when carried out deep inside the uterine tube. It must be understood deeply that the insertion is placed deep inside the uterine tube using the embryo transfer kit. The fact that the results do not appear to be significantly different using ipsi and contra lateral inseminations has led the present inventors to propose the use of insemination in both, so that the process of identifying the proper uterine tube may no longer be necessary . As a result of the insemination, it is of course desired that an animal of the desired sex be produced. This animal can be produced according to the systems previously treated through the use of the sexed sperm specimen. It should also be understood that the techniques of the present invention can find application in other techniques, such as laparoscopic insemination, oviductal insemination, or the like. As examples, the following experiments have been conducted. Although not everyone uses every aspect of the inventions described herein, they show the possible performance improvements through the different aspects of the invention.

In addition, a summary of some experiments is contained in the article "Insemination of Uterine Tubes of Terns with Very Low Number of Sexed and Non-Frozen Sperm" ("Uterine Horrn Insemination of Heifers UIT Very Low Numbers of Non-frozen and Sexed Spermatozoa" ) as referred to above. This article summarizes some of the data that show the efficacy of the present invention. As for the experiments, one was conducted with unfrozen sperm cells, sexed with high success as follows: EXAMPLE 1 Angus calves, 13-14 months old and under moderate body conditions, were synchronized with 25 mg of prostaglandin F- 2 alpha at 12-day intervals and were inseminated 6-26 hours after the establishment of estrus was observed. Freshly collected semen from three bulls, 14-26 months old, was incubated in 38 μM of Hoechst 33342 at 75 x 106 sperm / ml in TALP medium for 1 hour at 34 ° C. Spermatozoa were classified by sex chromosomes on the basis of epifluorescence from laser excitation at 351 and 364 nm at 150 mW using a MoFlo® flow cytometer / cell sorter operating at 50 psi and using 2.9 percent citrate of Na as coating fluid. The spermatozoa carrying the X-chromosome (-90% purity as verified by the reclassification of aliquots of sonicated spermatozoa) were collected ~ 500 live sperm / second in 2 ml Eppendorf tubes containing 100 μl of Cornell Universal Diluent (CTJE) ) with 20% egg yolk. The collected sperm were centrifuged at 600 x g for 10 minutes and resuspended at 1.63 x 106 live sperm / ml in CUE. For a non-sexed liquid semen control; spermatozoa stained with Hoechst 33342 were diluted with the coating fluid to 9 x 105 sperm / ml and centrifuged and resuspended to 1.63 x 106 of progressively mobile sperm / ml in CUE. The sexed semen and the liquid control semen were cooled at 5 ° C for 75 minutes and loaded into 0.25 ml straws (184 ul / straw). The straws were transported from 3 to 5 ° C in a temperature controlled beverage cooler at 240 km for insemination 5 to 9 hours after sorting.

• Sexed semen and liquid control semen were inseminated using blue lateral opening (IMV) coatings, half of each straw in each uterine tube (3 x 105 live sperm / calf). As a standard control, the semen from the same bulls that had been frozen in 0.5 ce straws by standard procedures (means 15.6 x 106 mobile sperm / post-thaw dose), was thawed at 35 ° C for 30 seconds, and It inseminated in the uterine body. The treatments were balanced on the 3 bulls and 2 inseminators in a ratio of 3: 2: 2 inseminations per sexed semen and two controls. Pregnancy was determined ultrasonically 31-34 days after insemination and confirmed 64-67 days later when the fetuses were also sexed (blind). The data is presented in the table. a-13 The proportions of sex values with different indices differ (P <0.02).

Although the pregnancy rate with sexed semen was only 80% of the controls, this difference was not statistically significant (> 0.1). One pregnancy was lost at 64-67 days in each of the sexed and frozen control groups; 18 of 19 fetuses (95%) were females in the sexed group and 20 of 30 (67%) were females in the control groups. The control of liquid semen produced a pregnancy rate virtually identical to the control of semen - frozen containing 50 times more mobile sperm (over 120 times more than total sperm), demonstrating the effectiveness of low dose insemination in the uterine tubes. We have significantly modified the proportion of sex in cattle, using flow cytometer technology and artificial insemination. In an analogous way, an experiment was conducted with non-sexed sperm cells, without freezing and were reported as follows: EXAMPLE 2 The objective was to determine pregnancy rates when calves were inseminated with extremely low numbers of frozen sperm, under ideal field conditions. The semen of three Holstein bulls of above-average fertility was diluted in homogenized milk, 7% glycerol diluent (CSS) plus 5% seminal homologous plasma at 2 x 105, 5 x 105 or 10 x 106 (control) of total sperm per French straw of 0.25 ml and frozen in moving liquid nitrogen vapor. The semen was thawed in water at 37 ° C for 20 seconds. Holstein calves 13-15 months old weighing 350-450 kg were injected with 25 mg of prostanglandin F-2-alpha (Lutalyse®) twice at 12-day intervals and were inseminated with a straw gun. Embryo transfer and lateral opening lining, half of the semen deep into each uterine tube 12 or 24 hours after estrus detection. The experiment was carried out in five replicates during five months and was balanced by two insemination technicians. The ambient temperature in the gestation was frequently -10 to -20 ° C, thus care was taken to keep the insemination equipment warm. Pregnancy was determined by detecting a le fetus using ultrasound at 41-44 days post-estrus and confirmed at 55-62 days post-estrus.; 4 to 202 embryos were lost between these times. Pregnancy rates on days 55-62 were 55/103 (53%), 71/101, (70%) and 72/102 (71%) for 2 x 105, 5 x 105 and 10 x 106 of total sperm / insemination (P < .1). Pregnancy rates were different (P <.05) between bulls (59, 62 and 74%), but not between technicians (64 and 65%) or post-estrus insemination times (65% for 12 hours and 64% for 24 hours, N = 153 at each time). With the methods described, pregnancies in calves were similar with 5 x 105 and 10 x 106 of total sperm 'by insemination. An experiment was also conducted on sexed, non-frozen sperm cells and can be reported as follows EXAMPLE 3 Semen was collected from bulls at the Atlantic Breeders Cooperative, diluted 1: 4 with a regulated HEPES diluent + 0.1% BSA, and transported at 160 km (~ 2 hours) to Beltsville, Maryland where it was classified at room temperature by flow cytometry in a TEST diluent producing (20%) using the methods described above (Biol Reprod. 41: 199). Classification rates of up to 2 x 106 of sperm from each sex were achieved for 5-6 hours at ~ 90% purity. The spermatozoa were concentrated by centrifugation (300 g for 4 min.) To 2 x 106 sperm / ml. Some spermatozoa were classified in the diluent containing homologous seminal plasma (final concentration, 5%). The classified sperm were shipped by air to Colorado (~ 2,600 km) and were classified at any ambient temperature or 5 ° C (cooled during boarding for 6 hours in an Equitainer, an isolated device with a compartment containing ice). Calves or dry cows detected in estrus from 11 to 36 hours previously were inseminated within 9 to 29 hours of the end of the sperm classification session. The spermatozoa (1 to 2 x 105 in 0.1 ml) were deposited deep in the uterine tube ipsilateral to the ovary in - the largest follicle as detected by ultrasound at the time of insemination. None of the 10 females became pregnant when inseminated with the sperm on board and stored at room temperature. Of 29 females inseminated with sperm cooled to 5 ° C during shipment, 14 were pregnant at 4 weeks of gestation and 12 (41%) at 8 weeks. Eleven of the 22 inseminated within 10 hours of the end of the classification were pregnant at 8 weeks, but only 1 of 7 inseminated from 17-24 hours after the classification were pregnant. There were no significant effects of the addition of seminal plasma. One of the 12 fetuses was not of the predetermined sex, 1 was not clear and 10 were of predetermined sex as determined by ultrasonography at 60-70 days of gestation. Subsequently, 33 additional calves were inseminated with 0.05 ml (semen diluted as described above) in each of the uterine tubes without using ultrasonography; only 3 were pregnant at 4 weeks after insemination and only 1 remained pregnant at 8 weeks. However, different bulls from the previous group were used and all inseminations were made 18-29 hours post-classification. An additional 38 calves were similarly inseminated (-22 hours post-classification) 200 km from our laboratory with classified sperm coming from another bull; none of these were pregnant 8 weeks after insemination. To summarize, it is possible to achieve pregnancies in cattle through the artificial insemination of sperm classified by sex chromosomes through flow cytometry and the sex rates of the fetuses are close to those predicted by the re-analysis of sperm classified by the DNA content (90%). However, the pregnancy rates varied greatly in those preliminary experiments that required the shipment of sperm at great distances. Fertility decreased dramatically at 17 hours post-classification but were somewhat confusing because different bulls were used at different times. In addition, studies are needed to determine if the observed variation in pregnancy rates was due to differences in bulls, to insemination techniques, to the interval between classification and insemination or other factors. Finally, an experiment was also conducted with non-sexed sperm cells, without freezing and can be reported as follows: > - EXAMPLE 4 The objective was to determine pregnancy rates when calves were inseminated with very low numbers of sperm, under ideal experimental conditions. The semen from 3 Holstein bulls was diluted in Cornell Universal Diluent plus 5% seminal plasma homologous to 1 x 105 or 2.5 x 105 sperm per 0.1 ml; it was used as a control 2. 5 x 106 total sperm per .25 ml Fully diluted sperm were packed in French plastic 0.25 ml straws modified to deliver insemination doses of 0.1 or 0.25 ml. Semen was cooled to 5 ° C and used 26-57 hours after collection. Holstein calves 13-15 months old weighing 350-400 kg were injected with 25 mg of prostaglandin F-2-alpha (Lutalyce®) at 12-day intervals and inseminated with an embryo transfer straw gun. and lateral opening lining in a uterine tube 24 hours after estrus detection. The insemination was ipsilateral on the side with the major follicle determined by ultrasound 12 hours after estrus; the side of ovulation was verified by the detection of a corpus luteum by ultrasound of 7-9 days post-estrus. Pregnancy was determined by detecting an ultrasound fetus of 42-45 days post-estrus. The experiment was done in 4 replicates and balanced by 3 insemination technicians. The side of ovulation was correctly determined in 205 out of 225 calves (91%); Surprisingly, pregnancy rates were almost identical to ipsilateral and inseminated collaterally. Pregnancy rates were 38/93 (41%), 45/87 (52%) and 25/45 (56%) for 1 x 105, 2.5 x 105 and 2.5 x 106 sperm / insemination (P> .1 ). There was a significant difference in the pregnancy rate (P <.05) among the technicians, but not among the bulls. With the described methods it is possible to reduce the sperm numbers by insemination, enough that the sperm classified by sex with a flow cytometer have commercial application. As mentioned and as can be seen from the various experiments, the field is statistically based and thus a variety of additional experiments can be conducted to show the appropriate combination and limiting strategies. In this way the synergies between the different effects will be further identified, such as the cases in which the effects of the ink and the effects of the ink combined with laser excitation can be studied.

The exhibit included in this application is intended to serve as a basic description. The reader should be aware that the specific exposition may not explicitly describe all possible modalities; Many alternatives are implicit. This may also not fully explain the generic nature of the invention and may not explicitly show how each feature or element may currently be representative of a broad function or of a wide variety of alternatives or equivalent elements. Again, these are included implicitly in this exhibition. Where the invention was described in the device-oriented terminology, each device element implicitly performs a function. The claims of the apparatus may not only be included for the device described, but also the claims of the method or process may be included to direct the functions of the invention and each performance of the element. Neither the description nor the terminology tries to limit the scope of the claims that may arise. It will be understood that a variety of changes can be made without departing from the essence of the invention. Such changes are also included implicitly in the description. They also fall within the scope of the invention. A broad exposition that includes both the explicit modality (s) shown, the great variety of implicit alternative modalities and the amplitude of the methods or processes and the like, is included in this exposition. In addition, each of the various elements of the invention and claims can also be achieved in a variety of ways. This disclosure will be understood to comprise each of such variations, whether it is a variation of one embodiment or any modality of the apparatus, a method or method of process or even only a variation of any element thereof. Particularly, it should be understood that as the disclosure relates to the elements of the invention, the words for each element can be expressed by terms of equivalent terms or methods of methods - even if only the function or the result is the same. Such equivalent terms, broader or even more generic, should be considered to be included in the description of each element or action. Such terms may be substituted when it is desired to make explicit the implicitly broad coverage for which this invention is entitled. As an example, it must be understood that all actions can be expressed as a means to take such action or as an element that causes that action. Similarly, each physical element described should be understood as comprising an exposure of the action that that physical element facilitates. As an example of this aspect, the description of a "collector" should be understood as comprising the description of the act of "collecting" - whether treated explicitly or not - and, conversely, when there is only the description of the act of "collecting", such a description should be understood as comprising the description of a "collector". Such alternative changes and terms will be understood to be explicitly included in the description. Furthermore, it should be understood that in addition to the initially filed claims, the claims may vary at least to more expansive directions: i) to the devices as set forth and described herein, ii) the related methods discussed and described, iii) the similar, equivalent and even implicit variations of each of these devices and methods, iv) those alternative designs that fulfill each of the functions shown as they were described and described, v) those alternative designs and methods that meet each of the functions shown as implicit to fulfill what is exposed and described, vi) each characteristic, component and stage shown as separate and independent inventions and vii) the various combinations and permutations of each of the above. To assist in the understanding of the invention a variety of published references may be aided. These are listed below and are incorporated herein for reference; however, to the extent that the exposures may be considered inconsistent with the patentability of this invention (s) such exposures are not considered as expressly made by the applicant (s). References potentially useful include: Patents of the United States of America Nos .: 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 references may also include the following publications: "Insemination of Holstein Calves with Very Low Numbers of Thawed Sperm" 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 Bovinc Sperm that Carries the X and Y", G.E. Seidel, Jr., L.A. Johnson, C.A. Alien, G.R. Welch, M.D. Holland, Z. Brink and M.B. Cattell, Laboratory of Biotechnology and Animal Reproduction, Colorado State University, Fort Collins, CO; Physiological Laboratory of - - Germiplasma and Gametos, ARS, USDA, Beltsville, MD; Coop de Criadores Atlantic, Lancaster, PA; DÚO Dairy, Loveland, CO, USA January 1996; "Insemination of Calves with Very Low Frozen Sperm Numbers" 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; "Lamb Production by Low Dose Intrauterine Insemination with Cytometrically Classified Flow and Unclassified 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 College of Agriculture, Craibstone, Bucksburn, Aberdeen. AB21 9YA, UK, Teriogenology, Page 267; "Uterine Tubal Insemination of Calves with Very Low Numbers of Sexed and Non-Frozen Sperm" 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 Biotechnology and Reproduction Laboratory, Colorado State University, Atlantic Breeders Cooperative, Lancaster, PA 17601, Germplasma Physiology Laboratory and ARS Gametes, USDA, Beltsville, MD 20705, DUO Diary, Loveland, CO 80538, Teriogenology 48: 1255-1264, 1997; "Training of Bovine Sperm Using Heparin", J.'J. Parrish, J. Susko-Parrish, M.A. Winer and N.L. First, Department of Meat and Animal Science, University of Wisconsin, Madison, Wl 53706, Biology of Reproduction 38, 1171-1180 (1988); "Is Prostaglandin F2a- a Fertility Drug in Dairy Cattle?", K.L. Macmillan and A.M. Day, Ruakura Animal Research Station, Prívate Bag, Hamilton, New Zealand, Teriogenology, September 1982, VOL. 18 No. 3, pages 245-253; "Prospects for Sexing Mammal Sperm," Associated Press of the University of Colorado, 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 Diluents and Milk on the Chromatin Structure and Viability of Cryopreserved Bull Sperm", J. Dairy Sci 74: 3836, D.S. Karabinus and D.P. Evenson and M.T. Kaproth; "Valuation of Ram Sperm and Verraco during Cell Sorting by Flow Cytometry", Reprod. Anim. Dom. 32: 251; "Goat superovulation with Purified pFSH supplemented with defined amounts of pLH", Therio 43: 797, M.A. Nowshari, J.F. Beckers and W. Holtz; "Preselection of Sex in Mammals: Summary", Dtsch. Tierarztl Wschr. 103: 285, L.A. Johnson. Through this specification, unless otherwise required by the context, the word "understand" or variations such as "comprises" or "comprising" shall be understood to imply the inclusion of an established element or whole or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Claims

CLAIMS 1. A method for producing a mammal having a predetermined sex comprising the steps of: a. collect sperm cells from a male of a mammalian species; b. determining the sex characteristic of a plurality of sperm cells; c. classify the sperm cells according to the determination of their sex characteristic; d. establish a sample of classified artificial insemination having from about 10% to about 50% of the number of sperm cells relative to a typical unclassified artificial insemination sample. and. insert the artificial insemination sample classified in 'a female of the mammalian species. F. fertilize at least one egg within the female of the mammalian species at statistically comparable successful levels (p> 0.1) at the typical unclassified artificial insemination dose; and g. produce a mammalian progeny of the desired sex. -
2. A method for 'producing a mammal having a predetermined sex as described in claim 1, wherein the step of collecting sperm cells from a male of a mammalian species, comprises the step of collecting male sperm cells of the mammalian species selected from the group consisting of bovines and equines.
A method for producing a mammal having a predetermined sex as described in claim 1 or 2, wherein the step of inserting the artificial insemination sample classified in a female of the mammalian species and fertilizing at least one ovule within from the female of the mammalian species to statistically comparable successful levels (p> 0.1) at a typical unclassified artificial insemination dose are each carried out in a field environment.
A method for producing a mammal having a predetermined sex as described in claim 3, wherein the step of inserting the artificial insemination sample classified in a female of the mammalian species and fertilizing at least one ovule within the female of the mammalian species at statistically comparable successful levels (p> 0.1) at the typical artificial insemination dose not classified in a field environment, comprises the steps of repeatedly inserting a significant number of artificial insemination samples classified in a significant number of female of the mammal species in rapid succession and in farm or ranch conditions.
A method for producing a mammal having a predetermined sex as described in claim 1, wherein the female of the mammalian species has uterine tubes and wherein the step of inserting the artificial insemination sample classified in the female of the mammalian species, comprises the step of inserting the artificial insemination sample classified both ipsi and contra lateral within the uterine tubes of the female of the species of the mammal.
6. A method for producing a mammal having a predetermined sex as described in the claim 1 or 2, wherein the female of the species of the mammal has at least one uterine tube and wherein the step of inserting the artificial insemination sample classified in the female of the mammalian species comprises the step of inserting the artificial insemination sample classified deep inside the uterine tube.
A method for producing a mammal having a predetermined sex as described in claim 5, wherein the step of inserting the classified artificial insemination sample into a female of the species of the mammal further comprises the step of inserting the sample of artificial insemination classified deep within the uterine tubes.
A method for producing a mammal having a predetermined sex as described in claim 6, wherein the step of inserting the classified artificial insemination sample into a female of the mammalian species further comprises the step of inserting the sample from classified artificial insemination within the uterine tube through the use of embryo transfer equipment.
9. A method for producing a mammal having a predetermined sex as described in claim 7, wherein the step of inserting the artificial insemination sample classified into a female of the mammalian species further comprises further comprising the step of inserting the classified artificial insemination sample into the uterine tubes through the use of embryo transfer equipment. .
A method for producing a mammal having a predetermined sex as described in claim 5, wherein the step of inserting the classified artificial insemination sample into a female of the mammalian species comprises the step of inserting the insemination sample artificially classified twelve hours after the time generally considered optimal for a single artificial insemination.
A method for producing a mammal having a predetermined sex as described in claim 9, wherein the step of establishing a sample of classified insemination comprises the step of establishing a sample of non-frozen classified artificial insemination, wherein the stage of classifying the sperm cells according to the determination of their sex characteristic occurs in a sorting time, and wherein the step of inserting the artificial insemination sample classified in a female of the mammalian species occurs not after approximately Seventeen hours of qualifying time.
12. A method for producing a mammal having a predetermined sex as described in the claim 9, wherein the stage of establishing a sample of classified insemination comprises the stage of establishing a sample of artificial insemination classified not frozen, where the stage of classifying the sperm cells according to the determination of their sex characteristic occurs in a classification time, and wherein the step of inserting the artificial insemination sample classified in the female of the mammalian species occurs not after approximately ten hours of the classification time.
A method for producing a mammal having a predetermined sex as described in claim 1, wherein the step of determining the sex characteristic of a plurality of sperm cells comprises the step of dyeing sperm cells with at least approximately 38 micro-molar content of dye.
14. A method for 'producing a mammal having a predetermined sex as described in the claim 1 or 3, wherein the step of determining the sex characteristic of a plurality of sperm cells and classifying the sperm cells according to the determination of their sex characteristic comprises the steps of: a. establish a source of cells that supplies the sperm cells to be classified; b. chemically coordinating a coating fluid to create a fluid coating environment for the sperm cells which are coordinated with a fluid environment of both pre-sorting and post-sorting sperm cells; c. detect a property of the sperm cells; d. discriminate between sperm cells that have a desired sex characteristic; and e. Collect the sperm cells that have the desired sex characteristic.
15. A method for producing a mammal having a predetermined sex as described in the claim 1, wherein the step of determining the sex characteristic of a plurality of sperm cells and classifying the sperm cells according to the determination of their sex characteristic comprises the steps of: a. establish a source of cells that supply the bovine sperm cells to be classified; b. establish the coating fluid for bovine sperm cells containing approximately 2.9% sodium citrate; c. detect a property of bovine semen cells; d. discriminate between bovine sperm cells that have a desired sex characteristic; and e. Collect the bovine sperm cells that have the desired sex characteristic.
16. A method for producing a mammal having a predetermined sex as described in the claim. 1, wherein the steps of determining the sex characteristic of a plurality of sperm cells and classifying the sperm cells according to the determination of their sex characteristic comprises the steps of: a. establish a source of cells that supply equine sperm cells to be classified; b. establish the coating fluid for equine sperm cells containing a regulated hepes medium; c. detect a property of equine sperm cells; d. discriminate between equine sperm cells that have a desired sex characteristic; and e. Collect the equine sperm cells that have the desired sex characteristic.
17. A method for producing a mammal having a predetermined sex as described in claim 1, wherein the steps of determining the sex characteristic of a plurality of sperm cells and classifying the sperm cells according to the determination of its sex characteristic comprises the stage of: a. establish a source of cells that supplies the sperm cells to be classified; b. establish the coating fluid for the sperm cells; c. detect a property of sperm cells; d. discriminate between sperm cells that have a desired sex characteristic; and e. collecting the sperm cells having the desired sex characteristic while buffering the impact sperm cells with a collector and wherein the buffer element comprises an initial collection fluid at the bottom of the collector and where the collector has a configuration Large enough to avoid the impact of the sperm cells with the collector.
18. A method for producing a mammal having a predetermined sex as described in the claim 1, wherein the steps of determining the sex characteristic of a plurality of sperm cells and classifying the sperm cells according to the determination of their sex characteristic comprises the steps of: a. establish a source of cells that supplies the sperm cells to be classified; b. establish the coating fluid for the sperm cells; c. detect a property of the sperm cells; d. discriminate between sperm cells that have a desired sex characteristic; and e. Collect the sperm cells that have the desired sex characteristic in a citrate collection fluid that contains approximately six percent of egg yolk before starting the collection stage.
19. A method for producing a mammal of a desired sex as described in claim 15, 16, 17 or 18, and further comprising the step of classifying the sperm cells at a rate of at least 500 classifications per second.
20. A method for producing a mammal having a predetermined sex as described in claim 1 and further comprising the step of using a pharmaceutical ovulatory to cause multiple ovules to be produced, and wherein the step of fertilizing at least one ovule within the female of the mammalian species at statistically comparable successful levels (p> 0.1) at the typical unclassified artificial insemination dose, comprises the step of fertilizing a plurality of ovules to produce multiple embryos having a predetermined sex, in where the pharmaceutical ovulatory is injected increasing every half day between any of the days 2 and 18 of the estrus cycle.
21. A method for producing a mammal having a predetermined sex as described in the claim 20, wherein the step of using a pharmaceutical ovulatory to cause multiple ovules to occur, comprises the step of injecting a dose of follicle-stimulating hormone.
22. A method for producing a mammal having a predetermined sex as described in the claim 21, wherein the step of injecting a dose of follicle-stimulating hormone in about half of the day's increments comprises a dose level of 6,6,4,4,2,2,2, and 2 mg between days 9 and 12 inclusive of the estrus cycle and further comprises the step of injecting 25 and 12.5 mg of prostaglandin F-2-alpha in the sixth and seventh doses, respectively, of the follicle-stimulating hormone.
23. A method for producing a mammal having a predetermined sex as described in claim 19, and wherein the step of determining the sex characteristic of the plurality of sperm cells and the step of classifying the sperm cells according to to the determination of its sex characteristic also includes the stages of: a. dye the sperm cells of the male mammal with at least about 38 micro-molar dye content; b. classify the sperm cells of the male mammal at a rate of at least 500 classifications per second, - and c. concentrate the classified sperm cells of the male mammal.
24. A method for producing a mammal having a predetermined sex as described in claim 22, wherein the step of determining the sex characteristic of a plurality of sperm cells comprises the step of dyeing the cells with a microcontainer. -molar dye.
25. A method for producing a mammal having a predetermined sex as described in claim 1, and further comprising the step of chemically coordinating a fluid coating environment for sperm cells that coordinates with fluid environments both pre-sorting and post-sorting. -classification.
26. A method for producing a mammal having a predetermined sex as described in claim 25, wherein the step of chemically coordinating a coating fluid to create a fluid coating environment for the cells that coordinates with both pre-fluid environments. -classification as post-classification comprises the stage of establishing a source of cells which supplies bovine sperm cells, and the step of establishing a coating fluid containing approximately 2.9% sodium citrate.
27. A method for producing a mammal having a predetermined sex as described in claim 25, wherein the step of chemically coordinating a coating fluid to create a fluid coating environment for the cells that coordinates with both pre-fluid environments. -classification as post-classification comprises the step of establishing a source of cells which supplies equine sperm cells, and the step of establishing a coating fluid containing a regulated hepes medium.
28. A method for producing a mammal having a predetermined sex as described in the claim 27, wherein the step of classifying the sperm cells according to the determination of their sex characteristic further comprises the step of buffering the cells of the impact with a harvester in which buffering the sperm cells comprises collecting the sperm cells in a initial collection fluid in the lower part of the collector and where the collector has a configuration large enough to avoid the impact of the sperm cells with the collector.
29. A method for producing a mammal having a predetermined sex as described in the claim 28, wherein the step of damping the impact cells with the collector comprises the step of providing a collection vessel having a diameter of at least fifteen millimeters.
30. An improved flow cytometer system for isolating sperm cells to produce a mammal according to the method of claim 1, comprising: a. a source of cells that supplies sperm cells to be analyzed by the flow cytometer; b. a source of coating fluid that creates an environment of coating fluid for the sperm cells; c. a nozzle through which the sperm cells pass while undergoing the fluid coating environment; d. an oscillator acting on the coating fluid as it passes through the nozzle; and. a sperm cell detector system that is sensitive to sperm cells; F. a classifying discrimination system that acts to classify sperm cells according to the determination of their sex characteristic at a speed of at least 500 classifications per second; and g. a collector in which the classified sperm cells that have a desired sex characteristic are placed.
31. An improved flow cytometer system for isolating the sperm cells to produce a mammal as described in claim 30, wherein the source of coating fluid that creates an environment of coating fluid for the sperm cells contains a citrate regulator.
32. An improved flow cytometer system for isolating the sperm cells to produce a mammal as described in claim 30, wherein the source of coating fluid that creates a coating fluid environment for the sperm cells contains a hepes regulated.
33. An improved flow cytometer system for isolating sperm cells to produce a mammal as described in claim 32, wherein the cell source comprises equine sperm cells.
34. An improved flow cytometer system for isolating sperm cells to produce a mammal as described in claim 31, wherein the cell source comprises bovine cells.
35. An improved flow cytometer system for isolating sperm cells to produce a mammal as described in claim 34, wherein the source of coating fluid contains 2.9% sodium citrate buffer.
36. An improved flow cytometer system for isolating sperm cells to produce a mammal as described in claims 30, 31, 32, 33, 34 or 35 wherein the collector is used to provide a sample of artificial insemination classified having from about 10% to about 50% of the number of sperm cells relative to the typical unclassified artificial insemination sample.
37. An improved flow cytometer system for isolating the sperm cells to produce a mammal as described in claim 36, wherein the collector further comprises a damping element wherein the damping element comprises an initial collection fluid in the lower part of the collector and where the collector has a configuration large enough to avoid the impact of the sperm cells with the collector.
38. An improved flow c-trometer system for isolating sperm cells to produce a mammal as described in claim 37, wherein the configuration large enough to avoid the impact of the sperm cells with the collector comprises a collection vessel having a diameter of at least fifteen millimeters.
39. A method for producing a bovine mammal having a predetermined sex comprising the steps of: a. collect bovine sperm cells from a male of a bovine mammal species; b. determining the sex characteristic of a plurality of bovine sperm cells; c. classify bovine sperm cells according to the determination of their sex characteristic, wherein an environment of coating fluid for bovine sperm cells contains approximately 2.9% sodium citrate; d. establish a sample of artificial insemination classified as bovine having between no more than approximately one hundred thousand and no more than three hundred thousand bovine sperm cells classified; and. insert the artificial insemination sample in a female of the bovine mammal species; F. fertilize at least one egg within the female species of the bovine mammal at successful levels of at least 50%; and g. produce a progeny of bovine mammal of the desired sex.
40. A method for producing a mammal having a predetermined sex as described in claim 39, wherein the step of fertilizing at least one egg within the female of the bovine mammal species comprises the step of fertilizing at least one ovule within the bovine mammal female species at successful levels of at least 90%.
41. A method for producing a mammal having a predetermined sex as described in claim 39 or 40 wherein the steps of inserting the artificial inseminated bovine sample into a female of the bovine mammal species and fertilizing at least one Ovum within the female of the bovine mammal species at successful levels of at least 50% is carried out in a field environment.
42. A method for producing a mammal having a predetermined sex as described in claim 39, wherein the steps of inserting the artificial insemination sample of bovine into a female of the bovine mammal species and fertilizing at least one The ovule within the female of the bovine mammal species at successful levels of at least 50% in a field environment comprises the steps of repeatedly inserting a significant number of artificial insemination samples classified as bovine into a significant number of females of the species of the bovine mammal in rapid succession and in farm or ranch conditions.
43. A method for producing a mammal having a predetermined sex as described in the claim - 39, 40 or 41, wherein the bovine mammal has uterine tubes and wherein the step of inserting the artificial insemination sample of bovine into the female of the species of the bovine mammal comprises the step of inserting the artificial insemination sample classified from bovine in both ipsi and contra lateral within the uterine tubes of the bovine mammal.
44. A method for producing a mammal having a predetermined sex as described in claim 39, 40 or 41, wherein the bovine mammal has at least one uterine tube and wherein the step of inserting the artificial insemination sample as described in The bovine in the female of the bovine mammal species comprises the step of inserting the artificial inseminated bovine insemination sample deep into the uterine tube.
45. A method for producing a mammal having a predetermined sex as described in claim 42, wherein the step of inserting the artificial insemination sample of bovine into the female of the species of the bovine mammal further comprises the step of inserting the sample of inseminated bovine insemination deep inside the uterine tubes.
46. A method for producing a mammal having a predetermined sex as described in claim 43, wherein the step of inserting the artificial insemination sample of bovine into the female of the species of the bovine mammal further comprises the step of inserting the artificial insemination sample of bovine inside the uterine tube through the use of embryo transfer equipment.
47. A method for producing a mammal having a predetermined sex as described in the claim 44, wherein the step of inserting the artificial insemination sample of bovine into the female of the species of the bovine mammal further comprises the step of inserting the artificial insemination sample of bovine into the uterine tube through the use of equipment of embryo transfer.
48. A method for producing a mammal having a predetermined sex as described in claim 42, wherein the step of inserting the artificial insemination sample of bovine into the female of the species of the bovine mammal comprises the step of inserting the sample of artificial insemination of cattle bovine twelve hours after the time in which it is generally considered as optimal for a simple artificial insemination of bovine.
49. A method for producing a mammal having a predetermined sex as described in claim 46, wherein the step of establishing a sample of artificial insemination of bovine animals comprises the step of establishing a sample of artificial insemination of non-frozen bovine, wherein the stage of classifying the bovine sperm cells according to the determination of their sex characteristic occurs at a time of classification and wherein the stage of inserting the artificial insemination sample of bovine in the female of the species of the Bovine mammal occurs no later than approximately seventeen hours of the time of classification.
50. A method for producing a mammal having a predetermined sex as described in claim 46, wherein the step of establishing a classified insemination sample comprises the step of establishing a non-frozen classified insemination sample, wherein the step of classifying the sperm cells according to the determination of their sex characteristic occurs at a time of classification and wherein the stage of inserting the sample of insemination into a female of the mammalian species occurs no later than approximately ten hours of the memento. of classification.
51. A method for producing a mammal having a predetermined sex as described in claim 1 or 50, wherein the step of determining the sex characteristic of a plurality of bovine sperm cells comprises the step of dyeing the cells of bovine sperm with at least about 38 micro-molar content of colorant.
52. A method for producing a mammal having a predetermined sex as described in claim 39, wherein the step of determining the sex characteristic of a plurality of bovine sperm cells and classifying the bovine sperm cells according to the determination of their sex characteristic, wherein an environment of coating fluid for the cells of bovine semen contains approximately 2.9% sodium citrate comprises the stages of: a. establish a source of cells that supply bovine sperm cells to be classified; b. chemically coordinate the environment of coating fluid for bovine sperm cells that coordinate with both a fluid environment of pre-sorting and post-classification cells; c. detect a property of bovine sperm cells; d. discriminate between bovine sperm cells that have a desired sex characteristic; and e. Collect the bovine sperm cells that have the desired sex characteristic.
53. A method for producing a mammal having a predetermined sex as described in claim 40, wherein the step of determining the sex characteristic of a plurality of bovine sperm cells and classifying bovine sperm cells according to to the determination of its sex characteristic, wherein a coating fluid environment for bovine sperm cells contains approximately 2.9% sodium citrate comprising the steps of: a. establish a source of cells that supply bovine sperm cells to be classified; b. chemically coordinate the environment of coating fluid for bovine sperm cells that coordinate with both a fluid environment of pre-sorting and post-classification cells; c. detect a property of bovine sperm cells; - d. discriminate between bovine sperm cells that have a desired sex characteristic; and e. Collect the bovine sperm cells that have the desired sex characteristic.
54. A method for producing a mammal having a predetermined sex as described in claim 39, wherein the step of determining the sex characteristic of a plurality of bovine sperm cells and classifying bovine sperm cells according to to the determination of its sex characteristic, wherein a coating fluid environment for bovine sperm cells contains approximately 2.9% sodium citrate comprising the steps of: a. establish a source of cells that supply bovine sperm cells to be classified, - b. establish the environment of coating fluid for bovine sperm cells, - c. detect a property of bovine sperm cells; d. discriminate between bovine sperm cells that have a desired sex characteristic; and e. collecting the bovine cells having the desired sex characteristic while buffering the bovine sperm cells from the impact with a harvester wherein a damping element comprises initial harvesting fluid at the bottom of the harvester and where the harvester has a configuration large enough to avoid the impact of the sperm cells with the collector.
55. A method for producing a mammal having a predetermined sex as described in the claim 39, wherein the step of determining the sex characteristic of a plurality of bovine sperm cells and classifying the bovine sperm cells according to the determination of their sex characteristic, wherein an environment of coating fluid for the Bovine sperm cells contain approximately 2.9% sodium citrate comprising the stages of: a. establish a source of cells that supply bovine sperm cells to be classified; b. establish the environment of coating fluid for bovine sperm cells, - c. detect a property of bovine sperm cells; d. discriminate between bovine sperm cells that have a desired sex characteristic; and e. Collect bovine sperm cells that have the desired sex characteristic in a citrate collection fluid that contains approximately six percent egg yolk before starting the collection stage.
56. A method for producing a mammal having a predetermined sex as described in the claim 39 and further comprising the step of classifying bovine sperm cells at a rate of at least 500 classifications per second.
57. A method for producing a mammal having a predetermined sex as described in the claim 39 and further comprising the step of using a pharmaceutical ovulatory to cause multiple ovules to be produced by the female of the bovine mammal species and wherein the step of fertilizing at least one ovule within the female of the bovine mammal species to Successful levels of at least 50% comprise the step of fertilizing a plurality of ovules produced by the female of the bovine mammal species to produce multiple sexed embryos having a predetermined sex.
58. A method for producing a mammal having a predetermined sex as described in the claim 57, wherein the step of using a pharmaceutical ovulatory to cause multiple ovules to be produced by the female of the species of the bovine mammal, comprises the step of injecting a dose of follicle stimulating hormone a plurality of times a day where the ovulatory Pharmaceutical is injected increasing every half day between any of days 2 and 18 of the estrus cycle.
59. A method for producing a mammal having a predetermined sex as described in the claim 58, wherein the step of injecting the dose of follicle stimulating hormone a plurality of times a day comprises the step of injecting the follicle stimulating hormone at approximately increasing every half day a dose level of 6,6,4,4, 2,2,2, and 2 mg between days 9 and 12 inclusive of the estrus cycle and also includes the step of injecting 25 and 12.5 mg of prostaglandin F-2-alpha in the sixth and seventh doses, respectively, of the stimulating hormone of the follicle.
60. A method for producing a mammal having a predetermined sex as described in claim 56, wherein the step of sorting the cells at a rate of at least 1200 classifications per second comprises operating a flow cytometer in the range of about 5 kilohertz to approximately 50 kilohertz.
61. A method for producing a mammal having a predetermined sex as described in claim 54, wherein the step of buffering bovine sperm cells from impact with the collection vessel comprises a collection vessel having equal physical characteristics. to the current.
62. A method for producing a mammal having a predetermined sex as described in the claim 54, wherein the step of buffering bovine sperm cells from impact with the collection vessel comprises the step of providing a collection vessel having a diameter of at least 15 millimeters.
63. An improved flow cytometer system for isolating bovine sperm cells to produce a mammal according to the method of claim 39, comprising: a. a source of cells that supplies bovine sperm cells for analysis by the flow cytometer; b. a coating fluid source that creates a coating fluid environment for bovine sperm cells containing approximately 2.9% sodium citrate; c. a nozzle through which the bovine sperm cells pass while they are subjected to the fluid coating environment, - d. an oscillator acting on the coating fluid as it passes through the nozzle; and. a sperm cell detector system that is sensitive to bovine sperm cells; F. a classifying discrimination system that acts to classify bovine sperm cells that have a desired sex characteristic; and g. a collector in which the bovine sperm cells that have the desired sex characteristic are placed.
64. An improved flow cytometer system for isolating bovine sperm cells as described in claim 63 wherein the collector is used to provide a sample of artificial bovine inseminated insemination.
65. An improved flow cytometer system for isolating bovine sperm cells as described in claim 63 or 64 wherein the nozzle, the oscillator, the cell detector system and the classifier discriminator system are part of the flow cytometer system and wherein the flow cytometer system comprises a cell sorter that classifies bovine sperm cells at a rate of at least 500 classifications per second.
66 An improved flow cytometer system for isolating bovine sperm cells as described in claim 64 wherein the classified bovine sperm cells are used to provide bovine sperm cells for artificial insemination.
67. An improved flow cytometer system for isolating bovine sperm cells as described in claim 65 wherein the cell sorter sorts the bovine sperm cells for analysis at a rate of about 1200 classifications per second.
68. An improved flow cytometer system for isolating bovine sperm cells as described in claim 67 wherein the cell sorter operates in a range of about 5 kilohertz to about 50 kilohertz.
69. An improved flow cytometer system for isolating bovine sperm cells as described in claim 63 wherein the collector comprises an initial collection fluid at the bottom of the collector and wherein the collector has a sufficiently large configuration to avoid the impact of the sperm cells with the collector.
70. An improved flow cytometer system for isolating bovine sperm cells as described in claim 69 wherein the collector comprises a collection vessel having a diameter of at least fifteen millimeters.
71. An improved flow cytometer system for isolating bovine sperm cells as described in claim 63 wherein the collector comprises a collection vessel having physical characteristics matched to the current.
72. An improved flow cytometer system for isolating bovine sperm cells as described in claim 63 wherein the bovine sperm cells are stained with at least one micro-molar content of dye.
73. An improved flow cytometer system for isolating bovine sperm cells as described in claim 63 wherein the source of coating fluid that creates a coating fluid environment for bovine sperm cells containing approximately 2.9% sodium citrate also comprises a fluid coating environment that coordinates with the fluid environments of pre-sorting and post-classification bovine sperm cells.
74. A method for producing a mammal having a predetermined sex comprising the steps of: a. collect equine sperm cells from a male of an equine mammal species; b. determining the sex characteristic of a plurality of equine sperm cells; c. classify equine sperm cells according to the determination of their sex characteristic, wherein an environment of coating fluid for equine sperm cells contains half regulated hepes; d. establish a sample of artificial insemination classified as equine ranging from no more than about one million to no more than twenty-five million equine sperm cells classified; and. insert the equine insemination specimen into a female of the equine mammal species; F. fertilizing at least one egg within the female of the equine mammal species at successful levels of at least 35% of at least 41%, of at least 50% and of at least 90%; and g. produce a progeny of equine mammal of the desired sex.
75. A method for producing a mammal having a predetermined sex as described in the claim 74 wherein the steps of inserting the equine artificial insemination sample into a female of the equine mammal species and fertilizing at least one ovule within the female of the equine mammal species at successful levels of at least 35% is carried out in a field environment.
76. A method for producing a mammal having a predetermined sex as described in claim 74, wherein the steps of inserting the equine artificial insemination sample into a female of the equine mammal species and fertilizing at least one ovule within the female of equine mammal species at successful levels of at least 35% in a field environment comprising the steps of repeatedly inserting a significant number of equine inseminated samples into a significant number of females of the mammalian species equine in rapid succession and ranch conditions.
77. A method for producing a mammal having a predetermined sex as described in claim 74, wherein the equine mammal has uterine tubes and wherein the step of inserting the artificial insemination sample of equine into a female of the species of the equine mammal comprises the step of inserting the artificial inseminated animal sample classified both ipsi and contra lateral within the uterine horns of the equine mammal.
78. A method for producing a mammal having a predetermined sex as described in claim 74, wherein the equine mammal has at least one uterine tube and wherein the step of inserting the equine artificial insemination sample into a female of the equine mammal species comprises the step of inserting the equine insemination sample deep into the uterine tube.
79. A method for producing a mammal having a predetermined sex as described in claim 77, wherein the step of inserting the equine artificial insemination sample into a female of the equine mammal species further comprises the step of inserting the artificial insemination sample classified as equine deep within the uterine tubes.
80. A method for producing a mammal having a predetermined sex as described in claim 78, wherein the step of inserting at least a portion of the equine artificial insemination sample into a female of the equine mammal species comprises In addition, the stage of inserting the equine artificial insemination sample into the uterine tube through the use of embryo transfer equipment.
81. A method for producing a mammal having a predetermined sex as described in claim 79, wherein the step of inserting the equine artificial insemination sample into the female of the equine mammal species further comprises the step of insert the equine artificial insemination sample into the uterine tube through the use of embryo transfer equipment.
82. A method for producing a mammal having a predetermined sex as described in claim 77, wherein the step of inserting the classified artificial insemination sample into a female of the mammalian species comprises the step of inserting the insemination sample. artificially classified twelve hours after the time in which it is generally considered as optimal for a simple artificial insemination.
83. A method for producing a mammal having a predetermined sex as described in claim 81, wherein the step of establishing a sample of artificial equine insemination comprises the step of establishing a sample of artificial insemination classified as non-equine. frozen, where the stage of classifying equine sperm cells according to the determination of their sex characteristic occurs at a time of classification and where the stage of inserting the sample of artificial insemination classified as equine in the female of the The equine mammal species occurs no later than approximately seventeen hours from the time of sorting.
84. A method for producing a mammal having a predetermined sex as described in claim 81, where the stage of establishing a sample of artificial equine insemination includes the stage of establishing a sample of artificial insemination classified of non-frozen equine, where the stage of classifying equine sperm cells according to the determination of their Sex characteristic occurs at a time of classification and where the stage of inserting the artificial equine insemination sample into the female of the equine mammal species occurs no later than approximately ten hours from the time of sorting.
85. A method for producing a mammal having a predetermined sex as described in claim 75, wherein the step of determining the sex characteristic of a plurality of equine sperm cells comprises the step of dyeing the sperm cells of equine with at least about 38 micro-molar dye content.
86. A method for producing a mammal having a predetermined sex as described in claim 74, wherein the steps of determining the sex characteristic of a plurality of equine sperm cells and classifying equine sperm cells according to to the determination of its sex characteristic, wherein an environment of coating fluid for ecjuino sperm cells containing regulated hepes medium comprises the steps of: a. establish a source of cells that supply equine sperm cells to be classified; b. chemically coordinating the coating fluid environment for equine sperm cells that coordinate with both a pre-sorting and a post-sorting fluid cell environment; c. detect a property of equine sperm cells, - d. discriminate between equine sperm cells that have a desired sex characteristic; and e. Collect equine sperm cells that have the desired sex characteristic.
87. A method for producing a mammal having a predetermined sex as described in claim 74, wherein the steps of determining the sex characteristic of a plurality of ecjuino sperm cells and classifying the sperm cells of ecjuino according to to the determination of its sex characteristic, wherein an environment of coating fluid for equine sperm cells contains a regulated hepes medium comprises the steps of: a. establish a source of cells that supply equine sperm cells to be classified; b. chemically coordinating the coating fluid environment for equine sperm cells that coordinate with both a pre-sorting and a post-sorting fluid cell environment; c. detect a property of equine sperm cells; d. discriminate between equine sperm cells that have a desired sex characteristic; and e. Collect equine sperm cells that have the desired sex characteristic.
88. A method for producing a mammal having a predetermined sex as described in claim 74, wherein the steps of determining the sex characteristic of a plurality of equine sperm cells and classifying the equine sperm cells according to to the determination of its sex characteristic, wherein an environment of coating fluid for equine sperm cells contains a regulated hepes medium comprises the steps of: a. establish a source of cells that supply equine sperm cells to be classified; b. establish a coating fluid for equine sperm cells; c. detect a property of equine sperm cells; d. discriminate between equine sperm cells that have a desired sex characteristic; and e. collecting the equine sperm cells having the desired sex characteristic while cushioning the impact equine sperm cells with a collector where a buffer element comprises initial collection fluid at the bottom of the. collector and where the collector has a sufficiently large configuration to avoid the impact of the sperm cells with the collector.
89. A method for producing a mammal having a predetermined sex as described in claim 74, wherein the steps of determining the sex characteristic of a plurality of equine sperm cells and classifying equine sperm cells according to to the determination of its sex characteristic, it comprises the stages of: a. establish a source of cells that supply equine sperm cells to be classified; b. establish a coating fluid for equine sperm cells; c. detect a property of equine sperm cells; d. discriminate between equine sperm cells that have a desired sex characteristic; and e. Collect equine sperm cells that have the desired sex characteristic in a citrate collection fluid that contains approximately six percent of egg yolk before beginning the collection stage.
90. A method for producing a mammal of a desired sex as described in claim 74 and further comprising the step of classifying the cells of -1 equine sperm at a rate of at least 500 classifications per second.
91. A method for producing a mammal having a predetermined sex as described in claim 74 and further comprising the step of using a pharmaceutical ovulatory to cause multiple ovules to be produced by the female of the equine mammal species comprises the steps of injecting a dose of follicle stimulating hormone a plurality of times a day where the pharmaceutical ovulatory is injected increasing every half day between any of days 2 and 18 of the estrus cycle.
92. A method for producing a mammal having a predetermined sex as described in claim 91, wherein injecting the dose of follicle stimulating hormone a plurality of times a day comprises injecting the follicle stimulating hormone in approximately increasing every half day a dose level of 6,6,4,4,2,2,2, and 2 mg between days 9 and 12 inclusive of the estrus cycle and where by supplementing the dose of follicle-stimulating hormone with prostaglandin F-2 - Alpha comprises the step of injecting 25 and 12.5 mg of prostaglandin F-2-alpha in the sixth and seventh doses, respectively, of the follicle-stimulating hormone.
93. A method for producing a mammal having - - a predetermined sex as described in the claim 92 wherein the step of classifying equine sperm cells at a rate greater than or equal to 500 classifications per second comprises operating a flow cytometer in the range of about 5 kilohertz to about 50 kilohertz.
94. A method for producing a mammal having a predetermined sex as described in the claim 93 wherein the step of collecting equine sperm cells having the desired characteristic comprises the step of buffering equine sperm cells from impact with a collection vessel wherein the buffering step comprises an initial collection fluid in the bottom of a collection container and wherein the collection container has a configuration large enough to avoid impact with equine sperm cells.
95. A method for producing a mammal having a predetermined sex as described in claim 94 wherein the step of cushioning equine sperm cells from impact with a collecting vessel having a sufficiently large configuration to avoid impact with Equine sperm cells comprises the step of providing a collecting vessel having a diameter of at least fifteen millimeters.
96. An improved flow cytometer system for isolating equine sperm cells to produce a mammal according to claim 74, comprising: a. a source of cells that supplies equine sperm cells for analysis by the flow cytometer; b. a coating fluid source that creates a coating fluid environment for equine sperm cells containing a regulated hepes medium; c. a nozzle through which the equine sperm cells pass while they are subjected to the environment of coating fluid; d. an oscillator that acts on the coating fluid as it passes through the nozzle; and. an equine sperm cell detector system that responds to equine sperm cells; F. a classifying discrimination system that acts to classify equine sperm cells that have a desired sex characteristic; and g. a collector in which classified equine sperm cells that have a desired sex characteristic are placed.
97. An improved flow cytometer system for isolating equine sperm cells to produce a mammal as described in claim 96, wherein the collector is used to provide a sample of classified artificial insemination having no more than 25 million of classified sperm cells.
98. An improved flow cytometer system for isolating equine sperm cells to produce a mammal as described in claim 97 wherein the nozzle, the oscillator, the cell detector system and the classifier discriminator system are part of the flow cytometer system and wherein the flow cytometer system comprises a classifier of cells that classify equine sperm cells at a rate of at least 500 classifications per second.
99. An improved flow cytometer system for isolating equine sperm cells to produce a mammal as described in claim 98 wherein the nozzle, the oscillator, the cell detector system and the discriminator classifier system are part of the system of flow cytometer and wherein the flow cytometer system comprises a classifier of cells that classify equine sperm cells at a rate of at least 1200 classifications per second.
100. An improved flow cytometer system for isolating equine sperm cells to produce a mammal as described in claim 99 wherein the collector is used to provide equine sperm classified for artificial insemination.
101. An improved flow cytometer system for isolating equine sperm cells to produce a mammal as described in claim 92 wherein the cell sorter classifies equine sperm cells to be analyzed at a rate of at least ( five hundred classifications per second
102. An improved flow cytometer system for isolating equine sperm cells to produce a mammal as described in claim 101 wherein the cell sorter operates in a range of about 5 kilohertz to about 50 kilohertz
103. An improved flow cytometer system for isolating equine sperm cells to produce a mammal as described in claim 99 wherein the collector comprises an initial collection fluid at the bottom of the collector and wherein the collector has a configuration large enough to avoid the impact of sperm cells with the collector
104. An improved flow cytometer system for isolating equine sperm cells to produce a mammal as described in claim 103 wherein the collector comprises a collection vessel having a diameter of at least fifteen millimeters .
105. An improved flow cytometer system for isolating sperm cells according to their sex comprising: a. a source of cells that supplies sperm cells to be analyzed by the flow cytometer; b. a chemically coordinated coating fluid source that creates a coating fluid environment for the sperm cells that is selected to coordinate with both the pre-sorted and post-classified sperm cell fluid environment; c. a nozzle through which the sperm cells pass while undergoing the coating fluid environment; d. an oscillator acting on the coating fluid as it passes through the nozzle; and. a system for detecting sperm cells that is sensitive. to the cells. F. a classifying discriminator system that acts to classify the sperm cells according to their sex; and g. a collector in which the sperm cells are placed according to their sex.
106. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 105 wherein the collector has a collection fluid, wherein the collection fluid comprises a nutrient that is coordinated to Balance the amount of the nutrient after finishing the classification of the sperm cells.
107. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 105 further comprising a collector on. which are placed sperm cells that have a desired characteristic and which comprises a citrate collection fluid containing approximately six percent egg yolk. -
108. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 105 wherein the nozzle, the oscillator, the cell detection system and the classifying discrimination system are part of the cytometer system of flow and wherein the flow cytometer system comprises a cell sorter that classifies sperm cells at a rate of at least 500 classifications per second.
109. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 105 wherein the source of chemically coordinated coating fluid that creates an environment of coating fluid for sperm cells that they are selected to coordinate with both the fluid environment of pre-sorting sperm cells and the post-classification comprising a citrate.
110. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 105 or 109 wherein the cell source comprises bovine sperm cells.
111. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 105 wherein the cell source comprises equine sperm cells.
112. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 105 wherein the collector is used to provide a sample of classified artificial insemination.
113. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 112 wherein the classified insemination sample comprises a sample of classified artificial insemination having no more than about ten percent of the typical number of sperm cells provided in a typical unclassified artificial insemination dose.
114. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 112 wherein the classified artificial insemination sample comprises a sample of inseminated bovine insemination and wherein the artificial insemination sample classified as bovine comprises a lower dose of approximately five hundred thousand bovine spermatozoa.
115. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 112 wherein the classified artificial insemination sample comprises a sample of inseminated bovine insemination and wherein the insemination sample Artificial bovine animal comprises a lower dose of approximately three hundred thousand bovine spermatozoa.
116. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 112 wherein the classified artificial insemination sample comprises an equine inseminated animal sample and wherein the artificial insemination sample Equine classified comprises a lower dose of approximately ten million equine sperm.
117. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 112 wherein the collector is used to provide a sample of artificial insemination classified for artificial insemination.
118. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 105 wherein the source of chemically coordinated coating fluid comprises a solution containing a regulated hepes medium.
119. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 118 wherein the cell source comprises equine sperm cells.
120. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 105, wherein the source of chemically coordinated coating fluid comprises a solution containing approximately 2.9% sodium citrate.
121. An improved flow cytometer system for isolating sperm cells. according to its sex as described in claim 105, wherein the collector has a damping element, wherein the damping element comprises an initial collection fluid in the lower part of the collector and in which the collector has a sufficient configuration big to avoid the impact of the sperm cells with the collector.
122. An improved flow cytometer system for isolating sperm cells according to their sex as described in claim 121, wherein the collector comprises a container comprising the buffer element.
123. An improved flow cytometer system for isolating the sperm cells as described in claim 122, wherein the container comprises a collection tube.
124. An improved flow cytometer system for isolating sperm cells as described in claim 123, wherein the collection tube is at least about fifteen millimeters wide.
125. A method for classifying sperm cells using a flow cytometer according to claim 108, comprising the steps of: a. establish a source of cells that supply the sperm cells to be classified; b. chemically coordinating a coating fluid to create a fluid coating environment for sperm cells that coordinates with a fluid environment of both pre- and post-classification sperm cells; c. detect a property of the sperm cells; d. discriminate between sperm cells according to their sex; and e. Collect the sperm cells that have the desired sex characteristic.
126. A method for classifying sperm cells as described in claim 125 wherein the step of chemically coordinating the collection fluid creates an environment of terminal collection fluid for the sperm cells which is coordinated with a fluid pre-environment. -selection.
127. A method for classifying sperm cells as described in claim 125 wherein the step of establishing a cell source comprises the step of establishing a source of bovine sperm cells.
128. A method for classifying sperm cells as described in claim 125 wherein the step of establishing a cell source comprises the step of establishing a source of ecjuino sperm cells.
129. A method for classifying sperm cells as described in claim 127 wherein the step of chemically coordinating a coating fluid to create an environment of coating fluid for sperm cells that coordinates with both pre-classification fluid environments as post-classification comprises the step of establishing a cell source which supplies bovine sperm cells to be classified and establishing a coating fluid containing approximately 2.9% sodium citrate.
130. A method for classifying sperm cells as described in claim 128 wherein the step of chemically coordinating a coating fluid to create an environment of coating fluid for sperm cells that coordinates with fluid environments both pre-sorting and post-processing. - Classification comprises the step of establishing a source of cells which supplies equine sperm cells to be classified and establishing a coating fluid containing a regulated hepes medium.
131. A method for classifying sperm cells as described in claim 125 wherein the step of establishing a cell source comprises the step of providing an initial nutrient for the cells and further comprising the step of providing a fluid nutrient for collection. for the cells and wherein the step of collecting the cells having the desired characteristic in a collection fluid comprises the step of balancing the initial nutrient and the nutrient of the collection fluid after the end of the cell collection step.
132. A method for classifying sperm cells as described in claim 125 wherein the step of collecting the cells having the desired characteristic in a collecting fluid comprises the step of establishing a citrate collection fluid containing approximately six percent of egg yolk before starting the collection stage,
133. A method for classifying sperm cells as described in claim 132 wherein the step of establishing a cell source comprises the step of establishing a source of sperm cell bovine.
134. A method for classifying sperm cells as described in claim 125 and further comprising the step of inseminating a mammal using a classified insemination sample.
135. A method for classifying sperm cells as described in claim 125 further comprising the step of collecting sperm cells having the desired sex characteristic comprising buffering the sperm cells from impact with a harvester, wherein they buffer the sperm cells it comprises collecting the sperm cells in a collection fluid at the bottom of a collector and wherein the collector has a configuration large enough to avoid the impact of the sperm cells with the collector.
136. A method for classifying sperm cells as described in claim 135 wherein the step of collecting the sperm cells in an initial collection fluid at the bottom of a collector and wherein the collector has a configuration large enough to avoid the impact of sperm cells with the collector, comprises the step of providing a collection container.
137. A method for classifying sperm cells as described in claim 136 wherein the step of providing a collection container comprises providing a collection container having a diameter of at least 15 millimeters.
138. A method for classifying sperm cells as described in claim 137 and further comprising the step of sorting the cells at a rate of at least 500 classifications per second.
139. A method for classifying sperm cells as described in claim 138 wherein the step of sorting the cells at a rate of at least 500 classifications per second comprises the step of operating a flow cytometer in a range of approximately 5 kilohertz at approximately 50 kilohertz.
140. A method for producing a sexed sperm specimen as described in claim 125 and further comprising the step of sorting the cells at a rate of at least 500 classifications per second. -
141. An improved flow cytometer system for isolating sperm cells comprising: a. a source of cells that supplies sperm cells to be analyzed by the flow cytometer; a source of coating fluid that creates an environment of coating fluid for the sperm cells; c. a nozzle through which the sperm cells pass while they are subjected to the environment of the coating fluid; d. an oscillator acting on the coating fluid as it passes through the nozzle; and. a cell detection system that is sensitive to sperm cells; F. a classifying discriminator system that acts to classify the sperm cells according to their 'sex; and g. a collector having a damping element wherein the damping element comprises initial collection fluid in the lower part of the collector and wherein the collector has a sufficiently large configuration to avoid impact of the sperm cells with the collector.
142. An improved flow cytometer system for isolating sperm cells as described in claim 141 wherein the collector comprises a container.
143. An improved flow cytometer system for isolating sperm cells as described in claim 141 wherein the collector comprises a collection tube.
144. An improved flow cytometer system for isolating sperm cells as described in claim 141 wherein the collection tube is at least about fifteen millimeters wide.
145. An improved flow cytometer system for isolating sperm cells as described in claim 142 wherein the container comprises a test tube having the physical characteristics of a vessel that equals the current.
146. A method for classifying sperm cells according to claim 141, comprising the steps of: a. establish a source of cells that supply the sperm cells to be classified; b. establish a coating fluid to create a fluid coating environment for the sperm cells; c. detect a property of the sperm cells; d. discriminate between sperm cells according to their sex; and e. collecting the sperm cells having the desired sex characteristic comprising the step of buffering the sperm cells from impact with a collector wherein a buffer element comprises initial collection fluid at the bottom of the collector and where the collector has a configuration large enough to avoid the impact of the sperm cells with the collector.
147. A method for classifying sperm cells as described in claim 146 wherein the step of buffering sperm cells from impact with a harvester comprises the step of providing a collection vessel.
148. A method for classifying sperm cells as described in claim 147 wherein the step of providing a collection container comprises providing a collection container having a diameter of at least 15 millimeters.
149. A method for producing a sexed sperm specimen as described in claim 148 and further comprising the step of providing a specimen of classified artificial insemination.
150. An improved flow cytometer system for isolating sperm cells comprising: a. a source of cells that supplies sperm cells to be analyzed by the flow cytometer; b. a source of coating fluid that creates an environment of coating fluid for the sperm cells; c. a nozzle through which the cells pass while they are subjected to the coating fluid environment, - d. an oscillator that acts on the coating fluid as it passes through the nozzle; and. a system for detecting sperm cells that is sensitive to cells; F. a classifying discriminator system that acts to classify sperm cells that have a desired characteristic; and g. a collector configured to avoid the impact between the sperm cells and the collector.
151. An improved flow cytometer system for isolating sperm cells as described in claim 150, wherein the collector configured to prevent impact between the sperm cells and the collector comprises a collector that is equal to the current.
152. An improved flow cytometer system for isolating sperm cells as described in claim 151, wherein the collector configured to prevent impact between the sperm cells and the collector comprises a collection tube having a sufficiently large diameter to avoid the impact between the sperm cells and the inner walls of the collection tube.
153. An improved flow cytometer system for isolating sperm cells as described in claim 152, wherein the collection tube has a diameter of at least 15 millimeters.
154. An improved flow cytometer system for isolating sperm cells as described in claim 150, 154, 155 or 156 wherein the nozzle, the oscillator, the cell detection system and the classifying discrimination system are part of the flow cytometer system and wherein the flow cytometer system comprises a cell sorter that classifies the sperm cells at a rate of at least 500 classifications per second.
155. A method of producing multiple embryos having predetermined sex from a female mammal comprising: a. create the superovulation in the mammal to create at least two ovules comprising the step of using a pharmaceutical ovulatory to make multiple ovules to be produced, wherein the pharmaceutical ovulatory is injected in the middle of the increments of the day between any of the days 2 and 18 of the estrus cycle; b. determine the sex of a sperm cell of a female mammal; c. classify according to sex the sperm cells; d. insert the classified sperm cells in the uterus of the female mammal after the onset of estrus; and e. fertilize a 'plurality of ovules in the uterus to produce multiple embryos that have predetermined sex.
156. A method of producing multiple embryos having predetermined sex as described in claim 155 wherein the step of injecting the pharmaceutical ovulatory by increasing each half-day comprises injecting at least seven injections and further comprising the step of incorporating a manipulation system of estrus at least in approximately the sixth and seventh injections.
157. A method of producing multiple embryos having predetermined sex as described in claim 156 wherein inserting the sperm cells classified in the uterus comprises inserting the sperm cells into both uterine tubes of the uterus.
158. A method of producing multiple embryos having predetermined sex as described in claim 157 wherein inserting into both uterine tubes comprises inserting the sperm cells - approximately between 20 to 24 hours inclusive after the onset of estrus.
159. A method of producing multiple embryos having predetermined sex as described in claim 155 wherein the step of using a pharmaceutical ovulatory to cause multiple ovules to be produced comprises the step of injecting a dose of follicle-stimulating hormone a plurality of times a day.
160. A method of producing multiple embryos having predetermined sex as described in claim 159 wherein the step of creating the superovulation in the mammal to create at least two ovules comprises also the step of incorporating a estro manipulation system comprising the step of supplementing follicle-stimulating hormone dose with prostaglandin F-2-alpha.
161. A method of producing multiple embryos having predetermined sex as described in claim 160 wherein injecting the dose of follicle stimulating hormone a plurality of times a day comprises injecting the follicle stimulating hormone at approximately increasing every half day a level of 6 doses, 6,4,4,2,2,2, and 2 mg between days 9 and 12 inclusive of the estrus cycle and where the supplement - dose of follicle stimulating hormone with prostaglandin F-2-alpha comprises the stage of injecting 25 and 12.5 mg of prostaglandin F-2-alpha in the sixth and seventh doses, respectively, of the follicle-stimulating hormone.
162. A method of producing multiple embryos having predetermined sex as described in claim 155 and further comprising the steps of: a. dye the sperm cells of a male mammal using micro-molar 38 dye; b. classify according to the sex of the sperm cells at a rate of at least 500 classifications per second; and c. concentrate the classified sperm cells.
163. A method of producing multiple embryos having predetermined sex as described in claim 155 wherein inserting the classified sperm cells comprises using a sample of classified insemination.
164. A method of producing multiple embryos having predetermined sex as described in claim 162 wherein inserting the classified sperm cells comprises using a sample of classified insemination.