Regulation 3.2 AUSTRALIA PATENTS ACT, 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant: XY', LLC Actual Inventors: SCHENK,John L Address for service in A J PARK, Level 11, 60 Marcus Clarke Street, Canberra ACT Australia: 2601, Australia [nvention Title: Methods and apparatus for reducing protein content in sperm cell extenders The following statement is a ful] desciption of this invention, including the best method of performing it known to us. 3542441 WO 2007/016090 PCT/US20)06/028846 METHODS AND APPARATUS FOR REDUCIG PROTEIN CONTENT IN SPERM CELL EXTENDERS This is an international patent application claiming priority to and the benefit of 5 'United States provisional application number 60/704,001, filed July 29, 2005 and United States nonprovisional application number 11/219,607, filed September 2, 2005, each said application hereby incorporated herein by reference. TECHNICAL FIELD 10 Generally, the inventive technology disclosed herein relates to meth6cls and apparatus for reducing protein content in sperm cell extenders. More specifically, this inventive technology may involve eliminating the protein content of a cryoprotectant component used in a multiple step sperm cell extension process. The inventive 15 technology may be particularly suited for use in sorted sperm applications. BACKGROUND Sperm cell extenders may be commonly used in a variety of biological disciplines 20 that require working with sperm cells. For example, one discipline that may make extensive use of sperm cell extenders is the field of artificial insemination. Whereas natural insemination may involve direct male to female insemination, artificial insemination may typically involve collecting sperni cells from a male, performing a degree of human manipulation of such spenm cells removed from their natural 25 environment, and then inserting the manipulated sperm cells into a female. The precise degree of human manipulation may vary depending on the precise natum of the particular application. For example, some human manipulation may simply involve dividing a collected sperm sample into multiple doses for use in multiple insemination events, possibly with multiple female animals. However, other applications may require more 30 intensive human manipulation. For example, human manipulation in some applications may involve sorting sperm cells into populations based on characteristics exhibited by spean cells. One such application may include the use of flow cytometery to separate spenn cells into a (followed by page 2) WO 2007/016090 PCTUS2006/028846 populations of X-chromnosome-beaing and Y-chromosone bearing sperm cells. A flow cytomreter may typically accomplish such separation by flowing spein cells entrained in a (Ulid stream one at a lie through an interrogation region, where information about each sperm cell may be obtained. Inerrogation may typically be accomplished through the use 5 of optics, ikzr example perhaps by inteasecting a laser beam with a sperm cell and measuring the resulting light scatter or fluorescence. The determination of a sex characteristic perhaps may be made by staining the spenm cells with a fluorescent dye that binds to the DNA within individual sperm cells. When a laser illuminates individual sperm cells, the dye may fluoresce. Sorting of sperm cells according to a sex 10 characteristic may then be accomplished, perhaps by recognizing that sperm cells bearing an X chromosome have more DNA than sperm cells bearing a Y chromosome, thus possibly emitting more fluorescent light when excited by a laser and perhaps allowing the cell to be identified and separated. 15 Another example of human manipulation may involve perhaps freezing rspern cells for use at a later time. Freezing sperm cells may often be critical to the effective use of sperm cells, because freezing may preserve at least some degree of the viability of sperm cells for a period of time extended beyond a point at which such viability otherwise may typically become compromised. Such extension of sperm cell viability may be 20 accomplished in freezing techniques pediaps by slowing the inetabolism of sperm. cells and perhaps extending their useful life accordingly. In particular, it perhaps may be known that sperm cell metabolism may be slowed by about 50% approximately [or every 10 degrees Celsius to which a sperm cell is cooled, Moreover, frozen sperm cells may be packaged in formats convenient for particular applications, for example perhaps as frozen 25 straws, frozen pellets, or other forms of frozen artificial samples. Frozen sperm cells also may lend themselves well to transportation over large distances, for example as where a spern cell collection facility, sperm cell extension facility, and artificial insemination facility may be widely dispersed at different locations. 30 It may be appreciated that the removal of' sperm from their natural environment may remove them from natural support ncchanisms that maintain their viability. Sperm ccll cxtenders may act to restore at least a degree of such support to sperm cells. For example, ono function of sperm cell extenders may be to buffer speri cells, for example perhaps by adjusting the pH or osmoality of a medium into which spenn cells may be 2 WO 2007/016090 PCUf/US2006f028S46 placed. Another function of sperm ' lls extenders peiaps may be to provide nutrients to sperm cells or to serve as a sperm cell energy sowme. In freezing applications, a further function may be to serve as a cryoprotectant to minimize the adverse ei~eols of freezing upon sperm cells, It may be appreciated that such functions of sperm cell extenders may 5 be accomplished at least to sime degree by the constituent paris that make up any individual sperm cell extender. For example, protein content may be a frequently used constituent part of various types of sperm cells extenders. Protein content may serve one or more functions in a 10 sperm cell extender. A primary purpose of protein content may be to provide nutrients and perhaps serve as an energy source for sperm cells. However, some kinds of protein may also have a cryoprotectant :imction, for example, perhaps the use of lipoproteins to replace lipids lost from sperm cell membranes fhat may be due to a freezing process. Moreover, protein content in sperm call extenders rmay take a variety of forms. Some 15 protein content may be plant-based, for example lecithin derived from soy. Other protein content may be animal-based, for example, perhaps egg yolk derived from sources including common baen's eggs. Cryoprotectants also may be an example of a frequently used constituent part of 20 various types of sperm cell extenders. Moreover, cryoprotectants may take a variety of fonns in sperm cell extenders, One commonly used cryoprotectant may be glycerol. Glycerol may protect sperm cells during a freezing press, perhaps by binding to water contained witlinu and surrounding a sperm cell, perhaps dehydrating the sperm cell as a result, and accordingly perhaps mducing the formation of intracellular ice that may cause. 25 damage to the spenn cell. However, using glycerol to cryoprotect sperm cells also may entail certain disadvantages. For example, glycerol may poso at least a degree of toxicity to sperm cells, the effect of which may become more pronounced with larger amounts of glycerol. Further, glycerol may be hyperosmotic to sperm cells, which may result in a degree of shock to -perm cells to which glycerol has been added. In particular, such 30 byperosmotq properties of glycerol may cause a spenu cell coming into contact with glycerol to rapidly shrink or expand as a result of a difference in solute concentration across the sperm cell's membraii. Such rapid shrinking and expanding may perhaps cause damage to a sperm cell. 3 WO 2007/016090 PCT/1JS2006/02S846 Accordingly, certain procedures may have been developed for sperm cell extenders to minimize the adverse effects of glycerol on sperm cells. For example, as a practical matter it may perhaps be recognized that combining glycerol with sperm cells at reduced temperatures may reduce the toxic effects of glycerol on sperm cells. 5 Accordingly, sperm cell extenders using glycerol often may be prepared in a multiple step process involving two or more extender fractions. More particularly, certain sperm cell extenders may contain an "A" fraction without glycerol and a "B" fraction with glycerol, This may allow a sperm cell extender to be prepared in two or more steps, for example, a first step in which sperm cells may be added to the A fraction of a sperm cell extender at 10 perhaps room temperature, followed by a second step in which the sperm cells added to the A fraction are cooled to a lower temperature, and the B fraction containing glycerol added at such a lower temperature. Moreover, to mitigate the hyperosmotic effects of glycerol on sperm cells, the B fraction perhaps may be added in multiple steps, possibly so as to reduce the shock to sperm cells by subjecting sperm cells to lowered amounts of 15 glycerol at each added glycerol step. The number of steps in which glycerol may be added may vary from perhaps as few as two stops or four steps to perhaps a great number of steps, including perhaps adding glycerol drip-wise over a period of time. However, the interaction of glycerol with other sperm cell extender components in 20 such procedures may entail significant drawbacks. In particular, protein components of sperm cell extenders such as egg yolk may pose complications for the handling of such extenders when present in the B fraction. This may be due to the volumetric bulk that such protein components create in a sperm cell extender. This phenomenon perhaps may be highlighted by the use of egg yolk in the B fraction of a sperm cell extender requiring 25 centrifugation, Centrifugation may be a commonly used technique in various sperm cell applications to concentrate sperm cells. For example, in flow cytometery applications, the passage of spenn cells through a flow cytometer may tend to dilute the concentration of sperm cells to a lower concentration than that found in nature. This may be because flow cytometers typically may require entraining sperm cells in a sheath fluid, which may 30 add to the volume of material in which sperm cells are contained. Centrifugation may return sperm cells to a higher concentration perhaps by subjecting them to centrifugal forces and concentrating them accordingly. However, centrifuging the B fraction of a sperm cell extender containing egg yolk may be problematic because the volumetric bulk of lipoproteins contained in the egg yolk may tend to compact any sperm cells that may 4 WO 2007/11490 PCTt1S2006/02846 be present in the P. niaction, perhaps with the result of cushing or otherwise damaging such spenu cells. As a result, it may perhaps be necessary to clarify the B fraction of a sperm cell 5 extender containing protein content sueh as egg yolk. The goal of clarification may be to confer a lower and more uniform degree of density to such a protein-containing extender, perhaps in particular by removing clunps or other locally dense regions due perhaps to protein concentrations such as lipoprotein components of egg yolk, so that centrifugation perhaps may be accompliAed without adversely compacting sperm cells. Clarification 10 may be accomplished by any of various suitable methods, for example pedps by filtration. However, all forms of clarification may require a dedication of resources to accomplish. For example, clarification may entail material costs such as filters or other required devices, labor costs which may tie up personnel resources that otherwise could be dedicated elsewhere, time costs which bmay slow down a sperm cell extension process, 15 and financial costs related to all of the foregoing. Moreover, simply preparing a B fraction of a spern cell extender to contain protein content such as egg yolk may entail a degree of inherent drawbacks. Similarly to clarification, preparation of such a B fraction may entail material costs, labor costs. time 20 costs, and financial costs. Moreover, the tendency toward spoliation over time due to the protein content of such a B faction may further complicate its use. hi particular, because such a B fraction may not keep well, it may require preparation on an as-needed basis, perhaps disrupting schedules and reducing efficiencies that could be realized if the B fraction otherwise could be prepared in large quantities ahead of time. This drawback 25 may be particularly acute in situations where a sperm cell application may require a relatively high ratio of B faction to A fraction. The spoliation tendencies of such a B fraction may also pose a contamination risk, for example as where the B fraction may perhaps become contaminated with bacteria due to spoliation, which may adversely affect a sperm cell application in which the contaminated B faction inadvertently may be used. 30 Such spoliation tendencies also may limit the use of such a B fraction in situations where environmental conditions cannot be closely monitored, for example as where it may be desired to transport the B fraction from one location to another perhaps over a large distance. 5 WO 2007/016090 PCT/US2006/028846 The foregoing problems regarding conventional sperm cell extenders may represent a long-felt need for an effective solution to the same. While implementing elements may have been available, actual attempts to meet this need may have been lacking to some. degree. This may have been due to a failure of those having ordinary 5 skill in the art to fully appreciate or understand the nature of the problems and challenges involved. As a result of this lack of understanding, attempts to meet these long-felt needs may have failed to effectively solve one or more of the problems or challenges here identified. These attempts may even have led away from the technical directions taken by the present inventive technology and may even result in the achievements of the present 10 inventive technology being considered to some degree an unexpected result of the approach taken by some in the field. DISCLOSURE OF INVENTION 15 The inventive technology relates to methods and apparatus for reducing protein content in sperm cell extenders and may include one or more of the following features: techniques for reducing protein content in a sperm cell extender, techniques for reducing protein content in a cryoprotectant-containing B fraction of a sperm cell extender; techniques for preparing sperm cell extenders that do not require clarification; techniques 20 for preparing low density gradient sperm cell extenders suitable for centrifugation; techniques for reducing protein content between individual steps in preparing a sperm cell extender, and techniques for establishing novel values of reduced protein content in sperm cell extenders. Accordingly, the objects of the methods and apparatus for reducing protein content in sperm cell extenders described herein address each of the foregoing 25 problems in a practical manner. Naturally, further objects of the invention will become apparent from the description and drawings below, BRIEF DESCRIPTION OF DRAWINGS 30 Fig. 1 is a depiction of various nascent substances in admixture relation. Fig.2a is a depiction of a prior art sperm cell extender. Fig.2b is a depiction of an unclarified centrifugation medium. 6 WO 2007/016090 PCT/US2006/028846 Fig. 3 is a representation of an intermediate sperm cell extender. MODE(S) FOR CARRYING OUT THE INVENTION 5 The present inventive technology includes a variety of aspects, which may be combined in different ways. The following descriptions are provided to list elements and describe some of the embodiments of the present inventive technology. These elements are listed with initial embodiments, however it should be understood that they may be 10 combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present inventive technology to only the explicitly described systems, techniques, and applications. Further, this description should be understood to support and encompass descriptions and claims of all the various embodiments, systems, techniques, methods, 15 devices, and applications with any number of the disclosed elements, with each element alone, and also with any and all various permutations and combinations of all elements in this or any subsequent application. Now referring primarily to Fig. 1, some embodiments may include a method for 20 freezing sorted sperm cells compromised by a sorting event. The term sorting event may be understood to include any of a variety of events in which sperm cells are sorted based on a discrimination of characteristics retained by such sperm cells, which may include in various embodiments immunosexing techniques, buoyancy techniques, or perhaps even flow cytometery techniques. The term compromised may be understood to include any 25 effect of a sorting event that may tend to adversely affect any desired aspect for which the spenn cells may be used, including for example sperm cell viability, sperm cell fecundity, or perhaps even sperm cell longevity. The term freezing may be understood to include any technique for preserving sperm cells that includes depressing their temperature below 0 degrees Celsius at least at some point in the technique. 30 Moreover, embodiments may involve obtaining a plurality of sperm cells, subjecting such a plurality of sperm cells to sorting stresses, and selecting such a plurality of sperm cells for a desired characteristic. By the term obtaining, it may be understood that any of various known techniques for obtaining sperm cells may be used, for example 7 S W(O 2007/110090 PCT/US2006/028846 perhaps including manual techniques or techniques involving an artificial vagina. The term sorting stresses niay be understood to include stresses that sperm cells may experience as a result of a sorting event, and subjecting sperm cells to sorting stresses may include perhaps merely accomplishing a sorting event. 5 A protein-containing sperm cell extender may be added to a plurality of selected sperm cells in some embodiments to form a first extended spenn cell mixture. The term sperm cell extender may be understood to include a substance that confers at least some degree of maintenance function to sperm cells. Such kinds of maintenance function may 10 include, for example, serving to buffer sperm cells, providing nutrients to sperm cells, or perhaps even acting as a cryoprotectant for sperm cells, It may be appreciated that various kinds of sperm ce-l extenders may be known, perhaps including egg yolk based extenders, milk based extenders, cittate containing extenders, sodium-citrate containing extenders, Tris containing extenders, and TEST containing extenders, Moreover, the 15 term first extended sperm cell mixture may be understood to include the combination of such a protein-containing sperm cell extender with such a plumlity of selected sperm cells. Various embodiments may further involve cooling such a first extended spenrn 20 cell mixture. to create a first cooled extended sperm cell mixture. The tenn cooling may be understood to include reducing the temperature of a first extended spenn cell mixture to any tempenture above 0 degrees Celsius. Cooling may perhaps be accomplished by any of various well-known techniques, such as perhaps refrigeration, water baths. or ice baths. In various embodiments, a first extended sperm cell mixture may be cooled 25 perhaps to less than about 10 degrees Celsitis, less than about 9 degrees Celsius, less than about 8 degrees Celsius, less than about 7 degrees Celsius, less than about 6 degrees Celsius, less than about 5 degrees Celsius, less than about 4 degrees Celsius, less than about 3 degrees Celsius, less than about 2 degrees Celsius, or perhaps even less than about I degree Celsius, Certain embodiments may involve cooling a first extended sperm 30 cell mixture to about 5 degrees Celsius. Moreover, a protein-free cryoprotectant containing spenmi cell extender may be added to such a first cooled extended spern cell mixture in some embodiments to form a second cooled extended sperm cell mixture. The term second cooled extended sperm cell mixtumi may be understood to include the combination of such a first cooled extended sperm. cell mixture with such a protein-free 8 WO 2007/0160% PCT/US2006/02g846 cryoprotectant-containing sperm cell extender. Various embodiments may also include freezing such a second cooled extended sperm cell mixture. A first extended sperm cell mixture in some embodiments may contain a 5 percentage.of egg yolk. This may be a function, for example, of the amount of protein contained within a protein-containing sperm cell extender added to a plurality of selected spenn cells, wherein such a protein may be egg yolk. In various embodiments, the percentage of egg yolk contained within a first extended sperm cell mixture may include iore than about .8 percent egg yolk, more than about 1.6 percent egg yolk, more than 10 about 3.2 percent egg yolk, or perhaps even more than about 6.4 percent egg yolk. Some embodinicats may include a puvecixtage of egg yolk contained within a first extended sperm cell mixture of about 3.2 percent. Moreover, a second cooled extended sperm cell mixture in some embodiments 15 also may contain a percentage of egg yolk. This may be a fiction, for example, of perhaps a dilution effect of adding a protein-free cryoprotoctant-containing sperm cell extender to a. first cooled extended sperm cell mixture. In various embodiments, the percentage of egg yolk contained within a second cooled extended spenn cell inixhre may include more thau about .4 percent egg yolk, more than. about .8 percent egg yolk, 20 more- than about 1.6 percent egg yolk, or perhaps even more than about 3.2 percent egg yolk. Some embodiments may include a percentage of egg yolk contained vith a second cooled extended spern cell mixture of about 1.6 pewent. In some emboditnents, a second cooled extended sperm cell mixture may be 25 maintained in un unclarified state. .In maintaining a second cooled extended sperm cell mixture in an unclatified state, it may be understood that such a second cooled extended spenn cell mixture may not be subject to clarification prior to any step of centrifuging. Moreover, certain embodiments may involve subjecting such a second cooled extended sperm cell mixture to centrifugation. This centrifugation. may perhaps serve to 30 concentrate sperm cells contained within such a second cooled extended sperm cell mixture, perhaps by separating such sperm cells from other components of the second cooled extended sperm cell mixture on a density basis due to the application of centrifugal force to the spern clls. Various embodiments may further involve decanting a portion of such a centrifuged second cooled extended sperm cell mixture. For example, WO 2007/016091) PCT/US2006/028846 sperm cells concentrated by centrifugation may be concentrated largely within one area, and removing a volumetric section perhaps may include removing the volumetric section containing such concentrated spem cells or' perhaps even removing all volumetric sections not including such concentrated sperm cells. 5 Now referring primarily to Fig. 2, some embodiments may include a method for. processing sorted sperm cells compromised by a sorting event. The term processing may be understood to include any event in which sperm cells are treated to change at least one characteristic of such sperm cells by at least some degree. In various embodiments, 10 processing may include for example freezing, thawing, or perhaps oven centrifuging such sperm cells. Moreover, embodiments may involve obtaining a plurality of sperm cells, subjecting such a plurality of sperm cells to sorting stresses, and selecting such a plurality 15 of sperm cells for a desired characteristic. Some embodiments further may include providing a protein-free sperm cell extender, providing a protein-free cryoprotectant containing sperm cell extender, and combining such a protein-free sperm'cell 'xtender with such a protein-free cryoprotectant-containing sperm cell extender to form a cryoprotectant-containing centrifugation medium. The term centrifugation medium may 20 be understood to include any medium conducive to sperm cells that at some point is subjected to centrifugation. In some embodiments, combining such a protein-free sperm cell extender with such a protein-free cryoprotectant-containing sperm cell extender to form a 25 cryoprotectant-containing centrifugation medium may be accomplished at a cool temperature. Such a cool temperature may include less than about 10 degrees Celsius, less than about 9 degrees Celsius, less than about 8 degrees Celsius, less than about 7 degrees Celsius, less than about 6 degrees Celsius, less than about 5 degrees Celsius, less than about 4 degrees Celsius, less than about 3 degrees Celsius, less than about 2 degrees 30 Celsius, or perhaps even less than about I degree Celsius. Certain embodiments may involve combining such a protein-free sperm cell extender with such a protein-free cryoprotectant-containing sperm cell extender to form a cryoprotectant-containing centrifugation medium at about 5 degrees Celsius. 10 WO 200o70 16i09 PCT/IS206:02I846 A cryoprotectant-containing centrifugation medium in some embodiments may be maintained in an unclurified state. An unclarified state may be understood to include a state in which a substance may exist wherein such a substance has not been clarified. Clarification may be understood to include conferring a lower and more uniform degree 5 of density to a substance, and perhaps may involve techniques such as filtering or straining. In maintaining a cryoprotectant-containing centrifugation medium in an unclarified state, it may be understood that such a cryoprotectant-containing centrifugation medium may not be subject to clarification prior to any tep of centrifuging. 10 Various embodiments may futher involve adding a plurality of aperm cells subjected to sorting stresses and selected for a desired characteristic to such an unclarified cryoprotectant-containing centriftigation iiedium, perhaps to form an unclarified ci:yoprotec.tait-containing sperm cell centrifugation inedimn. The rmin unclarified 15 cryoprotcctant-containing sperm cell centrifugation niediurn may be understood to include perhaps simply an unclarified cryoprotectant-containing centrifugation mediuni to vrhich such a plurality of sperm cells has been added. Moreover, certain cmnbodiments may involve subjecting such an unclarified cryoprotectant-containing spend cell centrifugation medium -to centrifugation. The term centrifugation may be understood to 20 include applying a centrifugal. force to a substance in order to separate at least two constituent components of that substance based on density. This centrifugation may perhaps serve to concentrate spenn cells contained within such an unclarified cryoprotectant-containing spern cell centrifugation nedimn, perhaps by separating such spenr cells from other components of the unclarified cryopotectant-containing sperm 25 cell centrifugation medium on a density basis due to the application of centrifugal force to the sperm cells. Various embodiments may further involve decanting a portion of such a centrifuged unclarified cryoprotectant-containing sponrm cell centrifugation medium. 30 Decanting may be understood to include removing at least some volumetric section of a substance, perhaps such a centrifuged unclarified cryoprotectant-containing sperm cell centrifugation medium, which perhaps may be achieved 'by any of various well-inown techniques, including for example pouring off such a volumetric section. Significantly, it may be possible to select such a voltuetric section to be removed based on the amount of WO 2007/016090 PCTIUS20061028946 a centrifuged component present in such a volumetric section. For example, sperm cells concentrated by centrifugation may be concentrated largely within one area, and removing a volumetric section perhaps may include removing the volumetric section containing such concentrated sperm cells or perhaps even removing all volumetric 5 sections not including such concentrated spenn cells. Certain embodiments may further involve providing a protein-containing sperm cell extender and combining such a protein-containing sperm cell extender with a cryoprotectant-containing centrifugation medium to create .a protein-containing 10 cryoprotectant-containing centrifugation medium. Moreover, various embodiments may further include adding a plurality of sperm cells subjected to sorting stresses and selected for a desired characteristic to such a protein-containing cryoprotectant-containing centrifugation medium to create a protein-containing cryoprotectant-containing sperm cell centrifugation medium. Some embodiments may further involve maintaining such a 15 protein-containing cryoprotectant-containing sperm cell centrifugation medium in an unclarified state. Moreover, such a protein-containing cyroprotectant-containing sperm cell centrifugation medium in certain embodiments may contain a percentage of egg yolk. 20 This may be a function, for example, of the amount of protein contained with a protein containing sperm cell extender combined with a cryoprotectant-containing centrifugation medium, wherein such a protein may be egg yolk. In various embodiments, the percentage of egg yolk contained within a protein-containing cyroprotectant-containing sperm cell centrifugation medium may include more than about .4 percent egg yolk, more 25 than about .8 percent egg yolk, more than about 1.6 percent egg yolk, or perhaps even more than about 3.2 percent egg yolk. Some embodiments may include a percentage of egg yolk contained within a protein-containing cyroprotectant-containing sperm cell centrifugation medium of about 1.6 percent. 30 In some embodiments, subjecting a protein-containing cyroprotectant-containing sperm cell centrifugation medium to centrifugation perhaps may include centrifuging such a protein-containing cyroprotectant-containing sperm cell centrifugation medium having a fraction of the percentage of egg yolk as compared to a centrifugation medium utilized in a typical method for centrifuging sorted sperm cells. Typical methods for 12 WO 20071016090 PCT/US2006/028846 centrifuging sorted sperm cells may be understood to include perhaps all methods for centrifuging sorted sperm cells not utilizing the novel techniques disclosed herein, and particularly may include perhaps those methods for centrifuging sorted spenn cells that may be well known in the art. Moreover, the term fraction may be understood to include 5 an amount of egg yolk less than that contained in a centrifugation medium utilized in such a typical method. In some embodiments, such a fraction may include perhaps less than about 50 percent as compared to a centrifugation medium utilized in a typical method for centrifuging sorted sperm cells, less than about 25 percent as compared to a centrifugation medium utilized in a typical method for centrifuging sorted sperm cells, less than about 10 10 percent as compared to a centrifugation medium utilized in a typical method for centrifuging sorted sperm cells, less than about 5 percent as compared to a centrifugation medium utilized in a typical method for centrifuging sorted sperm cells, less than about 4 percent as compared to a centrifugation medium utilized in a typical method for centrifuging sorted sperm cells, less than about 3 percent as compared to a centrifugation 15 medium utilized in a typical method for centrifuging sorted sperm cells, less than about 2 percent as compared to a centrifugation medium utilized in a typical method for contrifuging sorted sperm cells, or perhaps even less than about 1 percent as compared to a centrifugation medium utilized in a typical method for centrifuging sorted sperm cells. In some embodiments, such a fraction of the percentage of egg yolk as compared to a 20 centrifugation medium utilized in a typical method for centrifuging sorted sperm cells may be a protein-containing cryoprotectant-containing sperm cell centrifugation medium having less than about 3 percent egg yolk. Some embodiments perhaps may even include centrifuging a protein-containing cryoprotectant-containing sperm cell centrifugation medium containing glycerol and having less than about 11 percent egg yolk 25 Now referring primarily to Fig. 3, some embodiments may involve a method for extending sorted sperm cells compromised by a sorting event. The term extending may be understood to include conferring to sperm cells at least one or more of the functionalities of a sperm cell extender. 30 Moreover, various embodiments may include obtaining a plurality of sperm cells, subjecting such a plurality of sperm cells to sorting stresses, and selecting such a plurality of sperm cells for a desired characteristic, Embodiments may further include establishing a protein-containing sperm cell extender having a first protein content value. Such a first 13 S2007/0609US206246 protein--conte t value may be understood to include the protein content olf such an established protein-containing sperm cell extender prior to any subsequent events that may alter such a protein content, 5 Various embodiments may also include adding a plurality of spern cells subjected to sorting stresses and selected for a desired characteristic to a protein-containing sperm cell extender having a first protein content value and also adding a protein-free sperm cell extender, perhaps including a protein-free cryoprotectant-containing spenn cell extender, to said protein-containing sperm cell extender having a first protein contenL value, In 10 various embodiments, adding a protein-free cyroprotectant-containing sperm cell extender to a protein-con)taiintg sperma cell e~xtender having a first protein content value may be accomplished at a cool temperature. Such a cool temperature may include less tharn about 10 degrees Celsius, less than about 9 degrees Celsius, less than about 8 degrees Celsius, less than about 7 degrees Celsius, less than about 6 degrees Celsius, less 15 than about 5 degrees Celsius, less than about 4 degrees Celsius, less than about 3 degrees Celsius, less than about 2 degrees Celsius, or perhaps even less than about I degree Celsius. Certain embodiments may involve adding a protein-free sperm cell extender to a proteini-containing sperm cell extender having a 5irst protein content value at about 5 degrees Celsius. 20 Moreover, sorae embodiments may further include reducing a total protein content of such a protein-containing sperm cell extender having a first protein content value to a second protein content value below such a Oirst protein content value. A total protein content perhaps simply may be the total amount of protein in a protein-containing sperm 25 cell extender at any given lime, and the term reducing a total protein content may be understood to inchido any of various suitable methods for accomplishing such a reduction, including perhaps directly removing, protein content or perhaps simply increasing the amount of non-protein components in such a protein-containing sperm cell extender. 30 A first protein content value in some embodiments may include a percentage of egg yolk, for example, wherein such a protein may be egg yolk. in various enbodiments, such a percentage of egg yolk may include more than about .8 peentii egg yolk, more than about 1.6 percent egg yolk, more than about 3.2 percent egg yolk, or perhaps even 14 WO 20)7/ 016090 PCT/US2itJ6I/028846 more than about 6.4 percent egg yolk. Some embodiments may include a percentage of ogg yolk of about 3.2 percent Moreover, reducing a total protein content to a second protein content value below 5 such a first protein content value in some embodiments may include reducing a percentage of egg yolk of such a protein-containing sperm cell extender. This may be a function, for example, of perhaps a dilution effect of adding a plurality of spenm cells and adding a protein-free sperm cell extender to such a protein-contttining sperm cell extender. In various embodiments, such a pomentage of egg yolk of a second protein. 10 content value may include more than about .4 percent egg yolk, more than about ,8 percent egg yolk, more than about 1.6 percent egg yolk, or perhaps even more than about 3.2 percent egg yolk, In some embodiments such a percentage of egg yolk of a second protein content value may be about 1.6 percent. 15 -1 some embodiments, such a protein--containing spean cell extender having a second protein content value may be maintained in an utciarified state. In maintaining a protein-containing sperm cell extender having a second protein content value in an unclarified state, it may be understood that such a protein-containing sperrn cell extender having a second protein content value may not be subject to clarification prior to any step 20 of centrifuging, Moreover, certain embodiments may involve subjecting such a protein containing sperm cell extender having a second protein content value to cenitrifugation. This centrifligation may perhaps serve to concentrate sperm cells contained within such a protein-containing sperm cell extender having a second protein content value, perhaps by separating such sperm cells from oiher components of a protein-containing sperm cell 25 extender having a sccond protein content value on a density basis due to the application of centrifugal force ti the sperm cells. Various embodiments may further involve decanting a portion of such a centrifuged protein-containing spern cell extender having a second protein content value. For example, sperm cells concentrated by centrifugation may be concentrated largely within one area, and removing a volumetric section perhaps 30 may include removing the volunettic section. containing such concentrated spenn cells or perhaps even removing all volumetric sections not including such concentrated spend cells. 15 WO 2007/016090 PCTIS2EJU288 4 6 Various embodiments may also include adding a supplemental protein-containing sperm cell extender to a protein-containing sperm cell extender having a second protein content value and increasing a total protein content of such a protein-containing sperm cell extender to a third protein content value higher than a first protein content value. The 5 term supplemental protein-containing sperm cell extender may be understood to include any additional protein-containing sperm cell extender that supplements a protein containing sperm cell extender having a second protein content value. The term supplement may be understood to include adding additional spern cell extender components, for example perhaps adding an additional amount of protein. 10 Further, increasing a total protein content to a third protein content value in some embodiments may include increasing a percentage of egg yolk of such a protein containing sperm cell extender. This may be a function, for example, of perhaps adding a supplemental protein-containing sperm cell extender to a protein-containing sperm cell 15 extender having a second protein content value. In various embodiments, such a percentage of egg yolk of a third protein content value may include more than about I percent egg yolk, more than about 5 percent egg yolk, more than about 10 percent egg yolk, more than about 15 percent egg yolk, more than about 20 percent egg yolk, more than about 25 percent egg yolk, or perhaps even more than about 50 percent egg yolk. In 20 some embodiments such a percentage of egg yolk of a third protein content value may be about 16.5 percent. Moreover, various embodiments may include freezing a protein containing sperm cell extender having a third protein content value higher than a first protein content value. 25 Now'referring to Figs. 1-3, in various embodiments a plurality of sperm cells may include perhaps a plurality of mammalian sperm cells, including fbr example perhaps a plurality of bovine spern cells, a plurality of equine sperm cells, a plurality of porcine sperm cells, a plurality of ovine sperm cells, a plurality of cervid sperm cells, a plurality of canine sperm cells, or perhaps even a plurality of delphinidae sperm cells. Moreover, 30 some embodiments may involve selecting such a plurality of sperm cells for a desired characteristic. The term selecting may be understood to include identifying individual sperm cells based on a determination as to whether or not they possess a desired characteristic being sought. In some embodiments, selecting sperm cells for a desired characteristic may include sorting sperm cells. The term sorting may be understood to 16 WO 2007/416090 PCT/US2OO6/028846 nicimO actmg 11 separate selected sperm cells having a desired characteristic from those sperni cells not having such a desired. characteristic, perhaps including into populations of sperm cells with such a desired characteristic and populations of spern. cells without such a desired characteristic, Sorting sperm cells may be accomplished by any of a variety of 5 suitable techniques, including perhaps immunosexing techniques, buoyancy techniques, or perhaps even flow cytometery techniques, Additionally, the term desired characteristic may be understood to include any identifiable characteristic of a spenn cell desired for a given spenn cell application. For example, in some embodiments a desired characteristic may include a sex characteristic of a sperm cell., perhaps even an X-chromosome-bearing 10 characteristic of a spern cell or a Y-chromosome-bearing chamctistic of a sperm cell. The term protein-containing sperm cell extender in various embodiments may be understood to include any spenn cell extender containing at least some degree of protein content. Moreover, in various embodiments a protein-containing sperm cell extender 15 may include perhaps plant-based protein content or perhaps animal-based protein content, which may be understood to include proteins derived from plant sources and animal sources respectively. In certain embodiments, an animal-based protein-containing spern cell extender perhaps may include a lipoprotein-containing sperm cell extender. It may be appreciated that lipoproteins may be perhaps a subclass of proteins in which at least 20 one component of such a protein is a lipid. Such a lipoproiein content may perhaps be derived from various animal sources, including perhaps egg yolk collected from various kinds of animal eggs, perhaps including hen's eggs. Accordingly, in certain enibodiments a protein-containing spenn cell extender 25 perhaps may include an egg-yolk-containing sperm cell extender. It may be appreciated that the precise egg yolk content of such an egg-yolk-containing sperm cell extender may be varied depending on the needs of a particular application for which sperm cells may be used. However, in some embodiments an egg yolk content of an egg-yolk-containing spern cell extender may include less than about 50 percent egg yolk, less than about 45 30 percent egg yolk, loss than about 40 percent egg yolk. less than about 35 percent egg yolk, less than about 30 percent egg yolk, less than about 25 percent egg yolk, less than about 20 percent egg yolk, less than about 15 percent egg yolk; less than about 10 percent egg yolk, or perlaps even less than about 5 percent egg yolk. In certain embodiments, an 17 WO 2007/016090 PCT/IlS2006/028846 egg yolk content of an egg-yolk-containing sperm cell extender may be about 20 percent egg yolk. A cryoprotectant may be included as a constituent part of a sperm cell extender in 5 various embodiments. Accordingly, a sperm cell extender in various embodiments may include perhaps a protein-free cryoprotectant-containing sperm cell extender or perhaps even a protein-containing cyroprotectant-containing sperm cell extender. It may be appreciated that various well-known cryoprotectants perhaps may be appropriate for such addition to a sperm cell extender. In various embodiments, such a cryoprotectant perhaps 10 may include glycerol. It may be appreciated that the precise glycerol content of such a glycerol-containing sperm cell extender may be varied depending on the needs of a particular application for which sperm cells may be used. However, in some embodiments a glycerol-containing sperm cell extender may have more than about 3 percent glycerol, more than about 6 percent glycerol, more than about 12 percent 15 glycerol, or perhaps even more than about 24 percent glycerol. In certain embodiments, a glycerol content of a glycerol-containing spern cell extender may be about 6 percent. Moreover, in certain embodiments a protein-free cryoprotectant-containing sperm cell extender may be added to a substance in an equal volume to that substance, perhaps 20 including accomplishing such an addition in multiple steps. For example, adding a protein-free cryoprotectant-containing sperm cell extender to a first cooled extended sperm cell mixture may involve adding such a protein-free cryoprotectant-containing sperm cell extender having a volume equal to a volume of such a first cooled extended sperm cell mixture, perhaps in two or more steps. Similarly, combining a protein-free 25 cryoprotectant-containing sperm cell extender and a protein-free sperm cell extender may involve adding such a protein-free cryoprotectant-containing sperm cell extender in a volume equal to a volume of such a protein-free sperm cell extender, perhaps in two or more steps. Further, adding a protein-free cryoprotectant-containing sperm cell extender to a protein-containing sperm cell extender may involve adding such a protein--free 30 cryoprotectant-containing sperm cell extender having a volume equal to a volume of such a protein-containing sperm cell extender, perhaps in two or more steps. A protein-free cryoprolectant-containing sperm cell extender in certain embodiments may include a low density gradient cryoprotectant-containing sperm cell 18 va) 2~O7~tI16ll9UPCTHIl US20)'I/'i8Mi 6 U10 2)07/116090 extender. Accordingly, in sone embodiments adding a protein-free cryoprotectant containing spenu cell extender to a first cooled extended sperm cell mixture may involve adding a low density gradient cryoprotectant-containing spon cell extender to such a first cooled extended spern cell mixture. Further., in some embodiments providing a 5 protein-,free cryoprotectat--containing sperm cell extender may involve providing a low density gradient cryoprotectant-containing sperm cell extender. Also, in some embodiments adding a protein-free ciyoprotectant-containing spemi cell extender to a protein-containing sperm cell extender may involve adding a low density gradient cryoprotectant-containing sperm cell extender to such a protein-containing spern cell 10 extender. The temi. low density gradient may be understood to include a spern cell extender Itaving mi(oimaml density variations throughout its volume. In sone embodiments, a low density gmdient cryoprotectant-containing sperm cell extender may perhaps include a 15 substantially liquid cryoprotcltant-cmntaining sperm cell extender. The tern substantially liquid may be tndestood to include a ciyoprotectant-containing sperm call extender wherein all constituent parts of such a cryoprotectant-containing sperm cell extender arC in a substantially liquid state. In various embodiments, a low density gradient cryoprotectant-containing sperm cell extender may perhaps include a centrifugation 20 efficient cryoprotectant-containiig sperm cell extender. The term centrifugation-efficient may be understood to include a cryoproteciant-containing sperm cell extender having properties conducive to centrifugation, for example perhaps including clearly deim-arcated density diferences in its constituet parts or perhaps even a lack of localized higher density regions that may pose a compaction risk to certain of its constituent parts. in 25 various embodiments, a low density gradient cryoprotectant-containing spern cell extender hay perhaps include a substantially uniform density cryoprotectant-containing sperm cell extender. which may be understood to inchide a minimal number of localized areas of higher density, perhaps even approaching no areas of localized higher density. In various embodiments, a low density 'gradient cryoptectant-containing sperm cell 30 extender may perhaps include. a cryoprotectant-containing sperm cell extender with substantially no sperm cell compaction particles. The term sperm cell compaction, particle may be understood to include any particle or group of particles joined together that may tend to compact sperm cells to a damaging degree when subjected to various types of forces., perhaps including centrifugal forces, In various embodiments, a low 19 WO 2007/416090 PC1YUS20M%028846 density gradient cryoprotecmt-containing sperxt cell extender may perhaps inchide a low viscosity cryoprotectant-containing epern cell extender. The term low viscosity may be understood to include a viscosity of a cryoprotectAnt-containing Speri cell extender sufficient to pennit its constituent paits to slip past each other without tending toward the 5 compaction of any one constituent pait by any other constituent part. Various embodiments may include adjusting a sperm cell concentration of a substance to a pre-freeze sperm cell concentration. The term pre-feeze sperm cell concentration may be understood to include a concentration of sperm cells at which such 10 sperm cells may subsequently be fxozeri. For example, some embodinents may involve adjusting a spern cell concentration of a second cooled extended sperm cell mixture to a pre-freeze sperm cell concentmtion, while other embodiments may involve adjusting a sperm cell concentration of a protein-containing sperm cell extender having a second protein content value to a pre-freeze spern cell concentration. In viious embodiments, 15 adjusting to such a pre-freeze spenn cell concentration may include adding a protein containing sperm cell extender, including for example perhaps adding a protein containing sperm cell extender to a second extended sperm cell mixture or perhaps adding a supplemental protein-containing spem1 cell extender to a protein-containing spenn coll extender having a second protein content 'value. 20 In various embodiments, adjusting a sperm cell concentration to a pie-freezo spenn cell concentration may involve adjusting a sperm cell concentration to a species appropriate pre-freeze concentration. The term species-appropriate pre-freeze sperm cell concentration my be understood to include a concentration of sperm cells at which such 25 t2perm cells may subsequently be frzen that is appropriate for the species of animal from which such spenn cells were obtained. It may be appreciated that a species-appropriate pre-feeze sperm cell concentration may be known for a variety of animal species, or perhaps even may detennined through routine empirical observation over a number of freezing events. Tn some enibodiments, a species-appmpriate pre-&eeze sperm cell 30 concentration may include a bovine pre-freeze sperm cell concentration, an equine pre freeze sperm cell concentration, a porcine pre-&eeze sperm coll concentration, an ovine pre-freeze sperm cell concentration, a cervid pre-freeze sperm cell concentration, a canine pre-freeze sperm cell concentration, or perhaps even a delphinidae sperm cell concentration. Moreover, in various embodiments a species-appropriate pre-freeze sperm 20 wo 2007/I16090 pcTh82u06I42L846 cell concentration may include less than about 100 million sperm cells per milliliter, less than about 50 million sperm cells por milliliter, less than about 40 million sperm cells por milliliter less than about 30 million sperm cells per milliliter, loss than about 20 million sperm cells per milliliter, less than about 15 million sperm cells per milliliter, less than 5 about 10 million sperm cells per milliliter, less than about 5 million sperm cells per milliliter, or perhaps even less than about 2 million sperm cells per milliliter. In certain embodiments, perhaps including those involving a bovine predAeeze sperm cell concentration, a specios-appropriale pre-freeze concentration may be about 10 million sperm cells per milliliter. 10 Adjusting a sperm cell concentration to a pre-freeze spenn cell concentmtion in some embodiments may include establishing a pre-freeze egg yolk content. For example, adjusting a sperm cell. concentration of a second cooled extended sperm cell mixture to a pre-freeze sperm cell concentration may include establishing a pre-freeze egg yolk 15 contact of such second extended sperm cell mixture. Similarly, adjusting a sperm cell concentration of a protein-containing sperm cell extender having a second protein content value to a pre-freeze sperm cell concentration may include establishing a pre-freeze egg yolk content of such a protein-containing sperm cell extender. A prx-freeze egg yolk content may be understood to include perhaps simply an egg yolk content of a substance 20 at a pre-feeze concentration. In various embodiments, a pre-freeze egg yolk content may include an egg yolk content within a percentage of the egg yolk content of a typical method for freezing sorted sperm cells. Such a typical method for freezing soiled sperm cells may be understood to include perhaps all methods for freezing sorted sperm cells not utilizing the novel techmiques disclosed herein, and particularly may include perhaps 25 those methods for freezing sorted spem cells that may be well known in the art. In various embodiments, a pre-freeze egg yolk content may include may include perhaps within about 50 percent of the pre-freeze egg yolk content of a typical method for freezing sorted spenn cells, within about 25 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells, within about 20 percent of the pre-freeze 30 egg yolk content of a typical method for freezing sorted sperm cells, within about 15 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells, within about 10 percent of the pre-freeze egg yolk content of a typical method for freezing sorted sperm cells, within about 5 percent of the pre-freze egg yolk content of a typical method for freezing sorted sperm. cells, within about 2 percent of the pre-freeze 21 WO 20O7016090 PCT/1JS7tMf6N28846 egg yolk content of a typical method for forezing sorted spenin cells, and perhaps even within about I percent of the pre-feeze egg yolk content of a typical method for freezing sorted spomi cells. 5 Moreover., in certain embodiments a pre-freeze egg yolk content perhaps may be tablished at an absointe value, of le.a than about 50 percent egg yolk, less than about 45.. percent egg yolk, less than about 40 percent egg yolk, less than about 35 percent egg yolk, less than about 30 percent egg yolk, less than about 25 percent egg yolk, less than about 20 percent egg yolk, less than about 15 percent egg yolk, or perhaps even less than 10 about 10 percent. egg yolk. In some embodimenis, a pre-freeze egg yolk content may be established at about 16.5 percent The use of a sterile sperm cell extender may be involved in certain embodiments. For example, in various embodinents a protein-free sponn poll extender may include C 15 sterile protein-free sperm cell extender, and a protein-free cryoprotectant-containirng sperm cell extender may include a sterile protein-free cryoprotectant-containing sperm cell extender. Now with further reference pdmarily to Fig. 1, various embodiments may include 20 an incipient admixtrre (1), which in various embodiments perhaps may include an incipient compromised sorted spenn cell admixture. An admixture may be understood to include two or more substances in a state of being mixed, and an incipient admixturc (T) may be understood to include an admixture that is less than completely saturated with respect to any two constituent components capable of being mixed. In various 25 embodiments, an incipient admixture (1) may include perhaps an admixture that has achieved less than 50 percent saturation, less than 25 percent saturation, less than 10 percent saturation, less than 5 percent saturation, less than 2 percent saturation, or perhaps even less than 1 percent saturation. 30 Some embodiments may include two or more nascent substances in incipient admixture relation. An incipient admixture relation may be. understood to include two or more substances related by existing together in an incipient admixture (1). A nascent substance may be understood to include a substance that exists in a less than saturated combination with another substance in incipient admixture relation. Moreover, an 22 WO 207/01090 .206IO2846 admixture in some enibodirments may include two or more nascent components, wherein suclh a component may be understood to be a component of an incipient adinixtuma (1). For example, various embodiments may include a nascent plurality of sperm cells selected for a desirod chbameteristic (2) in intcipient admixturo rotation, a nascent protein~ 5 free sperm cell extender component in incipient admix.ture relation (3), a nascent protein free cryoprotectant-containing sperm cell extender component in incipient admixture relation (4), or perhaps even a nascent protein-cantaining sperm cell extender component in incipient admixture relation (5). 10 In some embodiments, a nascent substance may include a substance proximately located in a substantially uncombined state to at least one component of an incipient adnixtNre (1). The temi proximately located may be understood to include a location of such a nasceut substance near enough to such a component of an incipient admixture (1) so as to permit a combination of the two. The term substantially uncombined state may 15 C understood to include the existence of such a nascent substance in a state of mostly unsaturated combination with such a component of an incipient admixture (1), which may include perhaps existing as more than 50 percent uncombined, existing as more than 75 percent uncombined, existing as more than 90 percent uncombined, existing ar more than 95 percent uncombined, or perhaps even existing as more than 99 percent uncombined. 20 For example, various embodiments may include a plurality of sperm cells selected for a desired characteristic prmximately located in a substantially uncombined state to at least one component of an incipient admixture (1), a protein-free sperm cell extender component proximately located in a substantially uncombined state to at least one component of an incipient admixture (1), a protein-free cryoprotectant-containing sperm 25 celli extender component proximatcly located in a substantially uncombined state to at least one component of an incipient admixtum (1), or perhaps even a protein-containing spern cell extender component proximately located in a substantially uncombined state to at least 0o component of an incipient admixture (1). 30 Moreover, certain embodiments may include a barrier-free .zone between a nascent substance and a component of an incipient admixture (1). Such a barier free zone may be understood to include a zone containing no elements ihat may tend to prevent the combination of such a nascent substance and such a component of an incipient admixture (1). Some embodimtents may even involve an induced combination 23 WO 20071010090 PCIUS2006/028846 force to which a nascent substance and a component of an incipient admixture (1) may be responsive. Such an induced combination force may be understood to include any force tending to induce a combination of two or more substances in incipient admixture relation. Examples of an induced combination force may include perhaps a density 5 related force, a concentration-related force, or perhaps even simple hydrodynamic forces generated by placing various liquids in a container. The term responsive may be understood to include any effect on such a nascent substance or component of an incipient admixture (1) caused by such an induced combination force. 10 Moreover, an incipient admixture (1) in some embodiments may contain an egg yolk content This may be a function, for example, of the amount of protein in a nascent protein-containing sperm cell extender component, perhaps wherein such protein may be egg yolk. In various embodiments, the percentage of egg yolk contained within an incipient admixture may include more than about .4 percent egg yolk, more than about .8 15 percent egg yolk, more than about 1.6 percent egg yolk, or perhaps even more than about 3.2 percent egg yolk. Some embodiments may include a percentage of egg yolk contained With an incipient admixture (1) of about 1.6 percent. In various embodiments, an incipient admixture (1) may include an unclarified 20 incipient admixture (1). -Various embodiments also may include a cool incipient admixture. In some embodiments, a cool incipient admixture may be an incipient admixture (1) at a temperature of less than about 10 degrees Celsius, less than about 9 degrees Celsius, less than about 8 degrees Celsius, less than about-7 degrees Cclsius Cless. than about 6 degrees Celsius, less than about 5 degrees Celsius, less than about 4 degrees 25 Celsius, less than about 3 degrees Celsius, less than about 2 degrees Celsius, or perhaps even less than about 1 degrcc Celsius. Moreover, in somc embodiments a cool incipient admixture may be an admixture at about 5 degrees Celsius. Now with further reference primarily to Fig. 2, some embodiments may include a 30 compromised sorted sperm cell processing medium. The term processing medium may be understood to include any medium conducive to sperm cells in which spern cells may - - be placed to undergo processing. Moreover, embodiments also may include a plurality of sperm cells selected for a desired characteristic, an unclarified protein-free sperm cell extender component, an unclarified protein-free cryoprotectant-containing sperm cell 24 WO 2007/016090 PCT/S2061028846 extender component, and an unclarified centifugation medium (7) in which said plurality of sperm cells selected for a desired characteristic, said unclarified protein-free sperm cell extender component, and said unclartiied protein-free cryoprotectant-containing spermu cell. extender component am suspended. The term unclarified may be understood to 5 include a substaucc maintained in an unclarified state, and the teni unclarified state may be understood to include a state in which a substance may exist wherein such a substance has not been clarified. The tem ceitrifugation medium may be understood to include any medium conducive to sperm cells that at some point is subjected to centrifugation. 10 Certain embodiments may further include an. unclarified protein-containing spenn cell extender component suspended in an unclarified centrifugation medium (7). Morover, such an unclarifiod cecutrifugation medium (7) in some embodiments may contain an egg yolk content. This may be a function, for example, of perhaps the amount of protein in such an unclarified protein-containing sperm cell extender component, 15 perhaps wherein such protein may be egg yolk, In various embodiments, the percentage of egg yolk contained within an unclarified ceintrifugation medium (7) may include more than about .4 percent egg yolk, more than about ,8 percent egg yolk, mom than about 1.6 percent egg yolk, or perhaps even more than about 3.2 percent egg yolk. Some mboim may include it percenktgo of cgg yolk cunmained with an unclarifled 20 centrifugation medium (7) of about 1.6 percenL Moreover, in certain. embodiments an unclarified conttifugation medium (7) may contain at least some glycerol and have less than about 11 percent egg yolk. Moreover, in certain embodiments an unclarified centrifugation medium (7) may 25 perhaps include a cool unclarified centrifugation medium (7). Such a cool unclarified centiifaigation median (7) perhaps may include an unclarified centrifugation medium (7) at a temperatum of less than about 10 degees Celsius, less than about 9 degrees Celsius, less than about 8 degros Celsius, less than about 7 degrees Celsius, less than about 6 degrees Celsius, less than about 5 degrees Celsius, less than about 4 degrees Celsius, less 30 (han about 3 degrees Celsius, less than about 2 degrees Celsius, or perhaps even less than about I degree Celsius. In some embodiments, a cool unclarified centrifugation medium (7) may include an unclarified centrifugation medium (7) at a temperature of about 5 degrees Celsius. 25 W(O 200710.16090 PCT/US2006!028846 In some embodiments, an unclarified centrifugation medium (7) may perhaps have a minimized number of localized high density regions (6), including perhaps even no localized high density regions (6). The term localized may be understood to include a region of an unclarified centrifugation medium (7) that may be concentrated within a 5 small volume of such an unclaritied centriflugation medium (7). including perhaps a. volume of less than 3 percent, less that 2 percent, less 1 percent, less than .05 percent, or perhaps even less than .01 percent of the total volume of an unclarified centrifugation medium (7). The term localized high density region (6) may be understood to include localized regions of an unclarified centrifugatioT medium (7) having a substantially 10 higher density than surrounding legions, including perhaps more than 10 % of a surrounding density, mow than 20 % of a surrounding density, more than 30 % of a surrounding density, more than 40 % of a surrounding density, more than 50 % of a surrounding density, more than 100 % of a surrounding density, more than 200 % of a surrounding density, more than 300 % of a surrounding density, more than 400 % of a 15 sunoimding density, or perhaps even more than 500 % of a aurounding- density - Now with further reference primarily to Fig. 3, certain emboxiincats may include an intermediate compromised sorted sperm cell extension medium. A sperm coll extension medium may be understood to include any medium conducive to sperm cells in 20 which sperm cells may be placed for extension. The term intermediate may be understood to include a. sperm cell extension medium representing an intermediate step in a process of treatin sperm cells. For example, in various embodiments such an inteinediate step perhaps may include adding a cryoprotectant to a previously prepared sperm cell medium, adding protein content to a previously prepared sperm cell medium, 25 or perhaps centrifuging a previously prepared sperm cell medium. Moreover, further embodiments may inchide a plurality of sperm cells selected for a desired characteristic (8), a protein-free spenn cell extender component (9), a protein free cryoprotectant-containing sperm cell extender component (10), and a protein 30 containing sperm cell extender component (1). Certain ombodiments alsomnay include a - - total protein content not exceeding about 1.6 percent (12). This may be a function, for example, of perhaps the amount of protein in a protein-containing spend cell extender coiponent of such an intermediate sperm cell extension medium. In some embodiments, the protein in a protein-containing sperm cell extender perhaps may be egg yolk. 26 WO 2007/016090 Accordingly, a total protein content not exceeding about 1.6 percent perhaps may include an egg yolk content not exceeding about 1.6 percent. Certain embodiments may also include a cooled intermediate extension medium in 5 which a plurality of sperm cells selected for a desired characteristic (8), proteia-fire sperm cell exiter component (9), a protein-free cryoprotectant-containing sperm cell extender component (10), and a protein-containing sperm cell extender component (11) may be suspended. In some embodiments, such a cooled intermediate extension medium may include an intermediate extension medium at a temperature of perhaps less than 10 about 10 degrees Celsius, less than about 9 degrees Celsius, less than about 8. degrees Celsius, less than about 7 degrees Celsius, less than about 6 degrees Celsius, less than about 5 degrees Celsius, less than about 4 degrees Celsius, less than about 3 degrees Celsius, less than about 2 degrees Celsius, or perhaps even less than about I degree C(elsius. In cerLtain embodiments such a cooled intenediate extension medium may have 15 a temperature of about 5 degrees Ce)sius. Sevenl advantages may attend the inventive technology. In particular, the use of a potein-free cryoprotectant-contaiing sperm cell extender in various enibodimentis may represent a significant improvement over previous spermn cell extenders. For example, the 20 addition of such a protein-free cryoprotectant-containing sperm cell extender to other spem1 cell extenders may reduce clumps or other locally dense regions due perhaps to lower concentrations of such proteins, perhaps including egg yolk. This may reduce or perhaps even eliminate the compaction of spend cells in certain applications, for example centrifuging, that may cause damage to sperm cells. Additionally, the reduction of such 25 clumps in a sperm cell extender may eliminate the need to clarify such an extender, resulting in related materials savings, labor savings, time savings, and financial savings. Further, a protein-free cryoprotectant-containing spenn cell extender may be less susceptible to the effects of spoliation. This may allow such a sperm cell extender to be prepared in large quantities ahead of time, rather than on an as-needed basis. 30 Additionally, mitigating the effects of spoliation may reduce the risk of contamination by bacteria. Further, because such a sperm cell extender perhaps may be less sensitive to environnmential conditions, it may be more able to be transported over large distances where environmental conditions may vary. 27 WO 2007/016090 PC/US2006/028846 Accordingly, such a protein-free coprttant-contining sperm cell. extender may be an effective B fraction of a sperm cell extender in various applications. Moreover, it may be that use of such a protein-fmte. cryoprotectnt-containing spern cell extender may not significantly adversely impact the effectiveness of a spern cell extender in which 5 it used. For example, in embodiments relating to artificial insemination techniques, the use of such a spenn cell extender perhaps may yield results that are not significantly different than those achieved with the use of typical spenn cell extenders. In particular, pregnancy rates achieved with such a sperm cell extender in various embodiments... ....... perhaps may be comparable to those achieved with typical sperm cell extenders, 10 including perhaps even being statistically comparable (P > 0.05) in various embodiments. Several examples may be reported using the inventive technology as herein described. Importantly, these examples should be understood to represent only some embodiments of the inventive technology. Accordingly, it may be appreciated that these 15 examples should not be construed as limiting the scope of the inventive technology herein described. Example I 20 One possible procedure for collecting and processing sorted sperm involving a B fraction of an extender containing egg yolk may be as follows, Ths-A catch medium (2 ml) may be deposited in a 50-int Falcon tubu. Sorted sperm may be collected into the 50 ml Falcon tube over the course of approximately 1 hour for a total sorted volume of 12.5 mL This volume xnay be non-glycerol containing and may be referred to as the A-fmetion..... ...... 25 The percent of egg yolk in the A-fraction for this example is 3.2% [(2-ml "Catch") / (12.5-1il total volume) x (20% egg yolk "CatW) =3.2%)]. The 3.2% egg yolk admixture may be cooled to 5 *C over perhaps 90-min. Following the cooling period, an equal volume of glycerol-containing 20% egg yolk extender (B-fraction; 12% glycerol) may be added stepwise as 2 equal fractions at perhaps 15-in intervals, Cooled sorted sperm, 30 now contained in this example in an 13 .6% egg yolk ABextender [((12.5-ml A-fraction) x (32% ogg yolk) - (12.5-41d B4-iauivtion) 1 (20% egg yolk)) / 25-ril total volume = 11.6% egg yolk] may be centrifuged for concentration. This method of adding the glycerol containing extender to cooled sperm may avoid over extension of sperm pellets that may occur when non-spenn containing droplets are perhaps collected in the sorting process, if 28 WO 2007/016090 PCTIUS2006/028846 the sperm pellet is left in too much volume, and may assure that the final glycerol content is always 6%. A 200-pl sperm pellet may remain after removal of the supernatant. Sperm pellets from the same male may be pooled and total volume may be determined by weight. The number of total sorted sperm may be determined, perhaps with multiple 5 liemacytometer counts, and the sperm concentration of the pellet may be adjusted to a desired freezing concentration, perhaps with 20% egg yolk AB extender. In this example, if the sperm concentration in the 12.5-ml sorted volume is I x 106 sperm/ml, representing 12.5 x 106 total sperm, and the post-centrifuge recovery rate is 10 85%, then the sperm concentration in the 200-, sperm pellet is - .53 X 106 spernil (10.6 x 106 total sperm). If a freezing concentration of 10 x 106 sperm/ml is desired for this methodology, then 860-pu1 of 20% egg yolk AB extender may be added to the 200-ILl sperm pellet. Based on this model, the final egg yolk percent for i-eezing sorted sperm is 18.4% [((0.200-ml sperm pellet) x (11.6% egg yolk) + (0.860-ml AB extender) x (20% 15 egg yolk)) / 1.06-ml total volume = 18.4% final egg yolk]. Using the above example, but substituting 0% egg yolk B-extender in place of 20% egg yolk B-extender, the percent of egg yolk contained in the 25-ml volume to be centrifuged is 1.6% [((12.5-mI A-fraction) x (3.2% egg yolk) + (12.5-ml B-fraction) x 20 (0% egg yolk)) / 25-ml total volume = 1.6% egg yolk]. If the sperm pellet in this example is adjusted to a final freezing concentration of 10 x 106 sperm/ml with 20% egg yolk A1B extender, the final egg yolk percent is 16.5% [((0.200-mi sperm pellet) x (1.6% egg yolk) + (0.860-mi AB-extender) x (20% egg yolk)) + 1.06-ml total volume = 16.5% egg yolk]. 25 Accordingly, it may be seen that the final egg yolk percentage may perhaps vary only slightly between the two different B-fraction extenders, When using the 0% egg yolk B-extender, the final egg yolk percentage may be less when higher sperm concentrations (> 10 x 106 sperm/ml) are desired. 30 Example 2 A further example may be reported as follows. Sperm were studied from first -ejaculates obtained from 6 bulls and the study was replicated three times. Sperm for this 29 WO 2007/016090 PCT/U S2006/02R846 study were not sorted but were subjected to Hoechst 33342 staining and extreme dilution as occurs during sorting. The objective was to compare post-thaw motility of speim that received glycerol-containing extender (B-fraction) with or without egg yolk. An additional objective was to identify the optimal glycerol content needed for sorted sperm. 5 Therefore, 3, 4, 5 and 6% final glycerol content was studied in both 0 and 20% egg yolk containing B-fraction extender. Sperm frozen in 0.25-ml straws were thawed in a 37 0 C water bath for 30 sec and were incubated at 37*C. Visual estimates of total motility were determined by 2 observers, blind to treatment, at 30 and 120 min of incubation. 10 The exclusion of egg yolk in the B-fraction extender did not adversely affect post thaw sperm motility. In fact, motility was statistically higher for sperm processed in B fraction extender without egg yolk as compared to that with 20% egg yolk (P<0.05) at both incubation times. See Chart 1. Chart 1. Effect of Egg Yolk on Total Motility Means within treatments differ P<0.05 40 35 30 25 - 0 2 20 15 # 10 - 5- - 0.5-h 2-h Incubation Time (h) at 37 0 C [l 0% EY-B E 20% EY-B 15 A final glycerol concentration of 3 % resulted in statistically lower motilities at 30-min and 120-min after thawing, while motilities were higher and did not differ as a 30 W(O 20070I6090 PCTUS2006/022846 fuIinction of 4-6% glycerol. See Chart 2. From this example, it perhaps may be concluded that 3% glycerol did not provide adequate cryoprotection for sorted spern. Since motilities did not differ between 4-6% glycerol, and the provision of adequate cryoprotection may be desirable (which may differ between bulls), a final concentration 5 of 6% glycerol for sex sorted sperm cryopresetvation may be appropriate, Chart 2. Effect of Glycerol on Total Motility Means with different superscripts within incubation times differ P<0.05 35 31 30 26 250 254 25" 25 - --- 23" 20 - - S15.----1- - - I.-I 10 - -'- V 1 5 - -... . -------------- - -- - 5. 10 3 4 5 6 % Glycerol 0 0.6-h incubation (37"C) M 2-h incubation (37 0 C) 10 Example 3 Another example may be reported as follows. An objective was to compare 30 day pregnancy rate in Ilolstcin heifors insiinated with X-chromoome bearing sperm that were processed with 0% egg yolk glyomol (12%) containing extender to that 15 containing 20% egg yolk. X-chromosome bearing sperm from each of 2 bulls ware isolated on the basis of 31 P(cT/US20061028846 wO 20107/U 160190 CfJ2t6O54 DNA content using a flow (ytomneter, Sorted sperm were collected in 50-ml plastic tubes containing 2-mi of 20% egg yolk-TRIS extender without glycerol until each tube contained 12.5-nil and approximately 12 million sperm. Sorted spenm were cooled (5*C) over 90 minutes. After cooling, spen-containing sort tubes (50-ml Falcon) were evenly 5 separated and glycerol-containing extcader (B-fraction) added. -Coolcd..spe.n received either B-ftaction extender containing 0% egg yolk or 20% egg yolk. Tubes containing sorted sperm (25-mi total volume) were then centri-fuged at 850 x g for 20-minutes at 50C. Supernatant was removed, leaving sorted spenn in approximately 200-pl pellets. Like sperm pellets were pooled and adjusted to 10 x 106 spermln with 20% egg yolk!.AB 10 medium (6% final glycerol content). Final egg yolk percentage in the product varied by sorting day (range: 16.5 - 18.2%). Sperm (2 x. 10') were packaged. into 0.25-mi coded straws to ensuro treatments were blind to A[ technicians, placed on freezing racks and cryopreserved in LN 2 vapor. An equal number of straws from each bull and treatment were placed into goblets and attached to aluminum canes. 15 Sperm post-thaw motility was determined using "Track" motility after 30-min of incubation at 37*C. The mean percentage of progressively motile sperm for the fiveze codes processed with 20% egg yolk. B-ftaction extender was 44% and that for the 0% egg yolk 1--fraction extender was 13%. zo 119 non-syncbronized Holstein hoiters were balanced across the different egg yolk-media and 2 Holstein bulls, Insemination occurred 12 or 24 hours after observed standing estrus. Three inseminators were used in this example. Approximately I month alter insemination, pregnancy was detennined using ultrasound. Data were subjected to 25 ANOVA. Pregnancy rate did not differ (P>0.05) between sorted spern processed with 0% egg yoilk-"B" fraction extender to that for 20% egg yolk-"B" fraction extender. See Table 1. 30 Actual pregnancy rates were similar for bulls and Al technicians (P>0.05), and there were no statistical interactions. Numerically, the pregnancy rate for bull 52110039 was higher than for bull 52H0038. See Table 2. A largersampling of the population may have resulted in a significant difference in pregnancy rate between the two bulls. It may 32 WO 2007/016090T he important to note for this example that the 95% confident intervals (C) are large. Table 1 5 0% Ep Yolk v. 20% gg Yolk "R" Extender Field Trial by Trentrent Treatment (n) Prognant (%) i S.E.M. 95% C1 0% Egg Yolk-"W' 59 56 i 0.065 43 - 68 10 20% Egg Yolk-"'Y 60 55 i 0.065 42- 67 Actual means arc presented. Bulls (M2), Al technicians (n3) and Treatments (n=2) were similar (V>0,05), 15 Table 2 0% Er Yolk v-. 20% Eg Yolk "B" Extender Field Trial by Bull 20 Bull# (n) Pregnant (%)+ S.E.M. 95% Cl 52HOO39 60 62 i 0.063 49-73 '52HOO38 59 49 i 0.065 38--62 25 Actual means are presented. Bulls (n-2) were similar (P>0.05). 30 As may be casily understood from the foregoing, the basic concepts of the present invention may be embodied in a variety of ways. It involves both sperm cell extending techniques as well as devices to accomplish the appropriate sperm cell extension.In this application, the sperm cell extending techniques are disclosed as part of the results shown to be achieved by the various devices described and as steps that are inherent to 35 utilization. ihey are simply the natural result of utilizing the devices as intended and desc-ribed. In addition, while some devices are disclosed, it should be understood that these not only accomplish certain methods but also can be varied in a number of ways. Importantly, as to all of the foregoia, all of these facets should be understood to be encompassed by this disclosure.
WO 2007/01609 PCI/US2006/028846 The discussion included herein Is intended to serve as a basic description. he reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternatives are implicit. It also may not fully explain the. 5 generic nature of the invention and may not explicitly show how each feature or element can actually be representative of a broader function or of a great variety of alternative or equivalent elements. Again, these are implicitly included in this disclosure. Where the invention is described in device-oriented terminology. each element of the device implicitly performs a function. Apparatus claims may not only be included for the device 10 described, but also method or process claims may be included to address the functions the invention and each element performs. Neither the description nor the terminology is intended to limit the scope of the claims that will be included in any subsequent patent application. 15 It should also be understood that a variety of changes may be made without departing from the essence of the invention. Such changes are also implicitly included in the description. They still fill within the scope of this invention. A broad disclosure encompassing both the explicit embodiment(s) show, the groat variety of implicit alternative embodiments, and the broad methods or processes and the like arc 20 encompassed by this disclosure and may be relied upon when drafting the claims for any subsequent patent application. It should be understood that such language changes and broader or more detailed claiming may be accomplished at a later date (such as by any required deadline) or in the. event the applicant subsequently seeks a patent filing based on this filing. With this understanding, the reader should be aware that this disclosure is 25 to be understood to support any subsequently filed patent application that may seek examination of as broad a. base of claims as deemed within the applicant's right and may be designed to yield. a patent covering numerous aspects of the invention both independently and as an overall system. 30 Further, each of the various elements of the invention and claims may also be achieved in'a variety of manners. Additionally, when used or implied, an element is to be understood as encompassing individual as well as plural structures that may or may not be physically connected. This disolosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or 34 wo 20ti101016090 PCT/0.S2006AO2m46 process enbodrient, or even merely a variation of any element of these. Particularly, it should be understood that as the disclosure relates to elements of the invention, the words for each elenent may be expressed by equivalent apparatus terms or method terms -- even if only the function or result is the same. Such equivalent, broader, or even inure ge . .. ne 5 tens should be considered to be encompassed in the description of each element or action. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this invention is entitled. As but one example, it should be understood that all actions may be expressed as a means for taking that action or as an element that causes that action. Similarly, eaub physical element disclosed should be understood to 10 encompass a disclosure of the action that that physical element facilitates. Regarding this last aspect, as but one example, the disclosure of an "extender" should be understood to encompass disclosure of the act of "extending" - whithor explicitly discussed or not and, conversely, were there effectively disclosure of the act of "extending", such a disclosure should be understood to encompass disclosure of an "extender" and even a 15 "means for extending" Such changes and alternative terms are to be understood to be explicitly included in the description. Any patents, publications, or other references mentioned in this application for patent are hereby incorporated by rfcrence. In addition, as to each term used it should be 20 understood that unless its utilization in this application is inconsistent with a broadly supporting interpretation, common dictionary definitions should be understood as incorporated for each term and all definitions, alternative terms, and synonyms such as contained in the Random House Webster's Unabridged Dictioruaiy, second edition are hereby incorporated by reference. Finally, all references listed in the Information 25 Disolosure Statement or other information statement filed with the applioation are hereby appended and hereby incorporated by reference, however, as to each of the above, to the extent that such infornation or statements incorporated by reference might be considered inconsistent with the patenting of this/these invention(s) such statements are expressly not to be considered as made by the applicantss. 30 Thus, the applicant(s) should be understood to have support to claim and make a statement of invention to at least: i) each of the sperm cell extender devices as herein disclosed and descnbed, ii) the related ruethods disclosed and described, iii) similar, equivalent, and even implicit variations of each of these devices and methods, iv) those 35 WO 2007/ 09) PCT/US2006/0328246 alternative designs which accomplish each of the ftnetions shown as are. disclosed and described, v) those alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish that which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the 5 applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, ix) each system, method, and element shown or described as now applied to any specific Ecid or devices mentioned, x) methods and apparatuses substantially as described hereinbofore and with reference to any of the accompanying examples, xi) the various combinations and permutations of each of the 10 elements disclosed, and xii) each potentially dependent claim or concept as a dependency on each and every one of the independent claims or concepts presented. With regard to claims whether now or later presented for examination, it should be understood that for practical reasons and so as to avoid great expansion of the 15 examination burden, the applicant may at any time present only initial claims or perliaps only initial claims with only initial dependencies. Support should be umdCrstood to exist to the degree required under new matter laws -- including but not limited to European Patent Convention Article 123(2) and United States Patent Law 35 USC 132 or other such laws-- to pennit the addition of any of the various dependencies or other elements 20 presented under one independent claim or concept as dependencies or elements under any other independent claim or. concept. In drafting any claims Lt any time whether in this application or in any subsequent application, it should also be understood that the applicant has intended to capture as full and broad a scope of coverage as legally available. To the exi-ent that insubstantial substitutes are made, to the extent that the 25 applicant did not in fact draft any claim so as to literally encompass any particular embodiment, and to the extent othenvise applicable, the applicant should not be understood to have in any way intended to or actually relinquished such coverage as the applicant simply may not have been able to anticipate aU eventualities; one skilled in the art, should not be reasonably expected to have drafted a claim that would have literally 30 encompassed such alternative embodiments. Further, if or when used, the use of the transitional phrase "comprising" is used to maintain the "open-end" claims herein, according to traditional claim interpretation. Thus, unless the context requires otherwise, it should be understood that the tenn 36 WO 2002/016090 PCT/US2006/028846 "comprise" or variations such as "comprises" or "comprising", are intended to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. Such tenns should be interpreted in their most expansive form so as to afford the applicant the broadest coverage legally 5 permissible. Finally, any claims set forth at any time are hereby incorporated by reference as part of this description of the invention, and the applicant expressly reserves the right to use all of or a portion of such incorporated content of such claims as additional 10 description to support any of or all of the claims or any element or component thereof, and the applicant further expressly reserves the right to move any portion- of or all of the incorporated content of such claims or any element or component thereof from the description into the claims or vice-versa as necessary to define the matter for which protection is sought by this application or by any subsequent continuation, division, or 15 continuation-in-part application thereof, or to obtain any benefit of, reduction in fees pursuant to, or to comply with the patent laws, rules, or regulations of any country or treaty, and such content incorporated by reference shall survive during the entire pendenoy of this application including any subsequent continuation, division, or continuation-in-part application thereof or any reissue or extension thereon. 37