(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2003/0157475A1
`(43) Pub. Date:
`Aug. 21, 2003
`Schenk
`
`US 2003O157475A1
`
`(54) CRYOPRESERVATION OF SELECTED
`SPERM CELLS
`
`(75) Inventor: John L. Schenk, Fort Collins, CO (US)
`Correspondence Address:
`Santangelo Law Offices, P.C.
`Third Floor
`125 South Howes
`Fort Collins, CO 80521 (US)
`(73) Assignee: XY, Inc., Fort Collins, CO
`(21) Appl. No.:
`10/266,562
`(22) Filed:
`Oct. 7, 2002
`Related U.S. Application Data
`(60) Continuation of application No. 09/478,299, filed on
`Jan. 5, 2000.
`Division of application No. 09/577,246, filed on May
`24, 2000.
`(60) Provisional application No. 60/167,423, filed on Nov.
`24, 1999.
`
`Publication Classification
`
`(51) Int. Cl." ....................................................... A01N 1/02
`(52) U.S. Cl. .................................................................. 435/2
`
`(57)
`
`ABSTRACT
`
`The present invention provides a method of cryopreserving
`Sperm that have been Selected for a specific characteristic. In
`a preferred embodiment, the method is employed to freeze
`Sex-Selected Sperm. Although the cryopreservation method
`of the invention can be used to freeze Sperm Selected by any
`number of Selection methods, Selection using flow cytom
`etry is preferred. The present invention also provides a
`frozen Sperm Sample that has been Selected for a particular
`characteristic, Such as Sex-type. In preferred embodiments,
`the frozen Sperm Sample includes mammalian Sperm, Such
`as, for example, human, bovine, equine, porcine, Ovine, elk,
`or bison Sperm. The frozen Selected Sperm Sample can be
`used in a variety of applications. In particular, the Sample
`can be thawed and used for fertilization. Accordingly, the
`invention also includes a method of using the frozen Selected
`Sperm Sample for artificial insemination or in Vitro fertili
`Zation.
`
`Exhibit 1035
`Select Sires, et al. v. ABS Global
`
`

`

`US 2003/O157475 A1
`
`Aug. 21, 2003
`
`CRYOPRESERVATION OF SELECTED SPERM
`CELLS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`0001) The applications claims the benefit of U.S. Provi
`sional Application No. 60/167,423, filed Nov. 24, 1999.
`
`FIELD OF THE INVENTION
`0002 The invention relates to a method for freezing
`Sperm Selected for a particular characteristic, as well as to a
`frozen Selected Sperm Sample and methods of using Such a
`Sample. The invention is particularly useful for preserving
`Sex-Selected Sperm.
`
`BACKGROUND OF THE INVENTION
`0.003 Over half a century ago, artificial insemination was
`introduced in the United States as a commercial breeding
`tool for a variety of mammalian Species. Although artificial
`insemination was initially limited to regions relatively close
`to the Site of Sperm collection, advances in the cryopreser
`Vation and Storage of Sperm have facilitated widespread
`distribution and commercialization of Sperm intended for
`artificial insemination or in vitro fertilization.
`0004 Further improvements in mammalian sperm col
`lection, Selection, cryopreservation, Storage, and handling
`techniques have enhanced the ability of breeders to produce
`animals having desired traits. For example, advances in
`Selection of mammalian Sperm based on Slight differences in
`physical characteristics has made it possible to Separate
`Sperm based on SeX-type, that is, to Select for cells contain
`ing either the X or Y chromosome. This technique allows the
`breeder to manipulate the relative percentage of X- or Y-type
`Sperm in a Sample and thereby determine offspring SeX. The
`ability to Select Sperm based on SeX-type or any other
`desirable characteristic provides an important tool for accel
`erating genetic progreSS, increasing production efficiency,
`and achieving greater flexibility in livestock management.
`Full exploitation of this tool, however, depends on the ability
`to freeze and Store Selected Sperm.
`0005) A variety of methods are available for selecting
`cells, however, the Selection and Subsequent processing of
`Sperm presents unique challenges because Sperm are inca
`pable of DNA repair and because of Sperm morphology.
`Each Sperm has an acroSome overlying the head and a tail,
`which are important for fertility and which are relatively
`Susceptible to physical injury. In addition, Sperm fertility
`decreases with increasing time between collection and use.
`As most of the available selection methods involve physical
`Stresses and take time, Selected Sperm are typically Some
`what compromised compared to non-Selected cells. Fertility
`may be further reduced if the Selection technique involves
`Significant dilution. It has been Suggested that this "dilution
`effect” may be due to the loss of protective components in
`Seminal plasma.
`0006 Flow cytometry is a particularly efficient selection
`method that has been employed for Sorting Sperm by Sex
`type. However, Sorted Sperm are Subject to stresses beyond
`those normally encountered in Standard artificial insemina
`tion or in vitro fertilization protocols. In particular, flow
`cytometry is time consuming, and, because of the physical
`
`constraints of flow cytometers, Sperm must be diluted for
`Sorting to levels that are not optimal for Storage (usually to
`on the order of 10-10°/ml). Furthermore, sorted sperm
`intended for artificial insemination must be concentrated So
`that conventional packaging and delivery equipment can be
`used. The need for a concentration Step thus exposes already
`Somewhat compromised Sperm to additional physical
`StreSSeS.
`0007. The freezing of sperm also invariably reduces
`fertility, motility, and/or viability, and, although techniques
`for freezing unselected Sperm are well known, no technique
`for cryopreservation of Selected Sperm has been described.
`
`SUMMARY OF THE INVENTION
`0008. The present invention provides a method of cryo
`preserving Sperm that have been Selected for a specific
`characteristic. The method is particularly useful for cryo
`preserving Sperm Selected by a method that results in
`dilution of the sperm, since the method provides for the
`isolation of Sperm from a Selected Sperm Sample, followed
`by addition of a final extender to the isolated sperm to
`produce a Suspension having a desired concentration of
`Sperm. In a preferred embodiment, the method is employed
`to freeze Sex-Selected Sperm. Although the cryopreservation
`method of the invention can be used to freeze Sperm Selected
`by any number of Selection methods, Selection using flow
`cytometry is preferred.
`0009. The present invention also provides a frozen sperm
`sample that has been selected for a particular characteristic,
`Such as Sex-type. In preferred embodiments, the frozen
`Sperm Sample includes mammalian Sperm, Such as, for
`example, human, bovine, equine, porcine, Ovine, elk, or
`bison Sperm. Also within the Scope of the invention is a
`container including a frozen Sperm Sample according to the
`invention.
`0010. The frozen selected sperm sample can be used in a
`variety of applications. In particular, the Sample can be
`thawed and used for fertilization. Accordingly, the invention
`also includes a method of using the frozen Selected Sperm
`Sample for artificial insemination or in vitro fertilization.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`0011. The present invention allows cryopreservation of
`Sperm that have been Selected for a particular characteristic,
`facilitating Storage and/or shipment of Selected Sperm
`Samples to Sites distant from the collection site. Thawing
`yields viable Sperm that can be used in procedures Such as
`artificial insemination (“AI”) and in vitro fertilization
`(“IVF). This result was surprising because of the well
`documented fragility of Sperm. Prior researchers had dem
`onstrated that the Stresses associated with various Selection
`methods or with cryopreservation resulted in Significant
`losses in fertility and/or viability. The present inventors have
`demonstrated, for the first time, that pregnancies can be
`achieved with Sperm that have been Selected and then
`frozen.
`0012. The invention represents an important advance in
`livestock management, where Selection of Sperm for use in
`Such procedures can be used to increase the production of
`offspring having desirable traits. For example, Selection to
`
`Exhibit 1035
`Select Sires, et al. v. ABS Global
`
`

`

`US 2003/O157475 A1
`
`Aug. 21, 2003
`
`obtain Sperm carrying either the X or the Y chromosome
`allows control over offspring Sex, which is advantageous for
`producers of animals. Such as dairy or beef cattle. SeX
`Selection also finds application in breeding valuable (e.g.,
`show or race horses) or endangered animals. The ability to
`freeze Selected Sperm, which the invention provides, will
`enable widespread use of Such Selection methods to, e.g.,
`increase livestock production efficiency as well as quality.
`
`Definitions
`0013 The term “acrosome” or “acrosomal cap” refers to
`the cap that covers the anterior half of the head of Sperm and
`that contains enzymes necessary for ovum penetration.
`0.014. The term “sex-type” refers to the type of sex
`chromosome present in the Sperm (i.e., the X or Y chromo
`Some).
`0.015 The term “capacitation” refers to the specific
`changes a Sperm undergoes to develop the capacity to
`fertilize ova, Such as enzyrnic changes on the Surface of the
`acroSome that lead to release of acroSomal enzymes that
`facilitate penetration of the Sperm into the OVum.
`0016. As used with reference to sperm, the term “cryo
`protectant” refers to a molecule that protects Sperm during a
`freeze-thaw cycle, promoting Survival and retention of fer
`tilizing capacity.
`0017. The term “dilution effect” refers to the rapid
`decline in motility and/or viability of sperm when highly
`diluted.
`0.018. As used herein, the term “selection” refers to a
`method whereby a Sample is Subdivided based on presence
`or absence of a specific characteristic (unless context dic
`tates otherwise). Thus, a “selected Sperm Sample” is a
`Sample obtained by Subjecting a Source Sample to Selection
`for the Specific characteristic. A Selected Sperm Sample is
`therefore enriched, relative to the Source Sample, in Sperm
`having the Specific characteristic.
`0019. The term "sorting” is used herein to describe a
`Selection method carried out using a fluorescence-activated
`cell sorter (FACS).
`0020. The term “extender” refers to any medium that
`tends to preserve sperm viability. The term “extension”
`refers to the dilution of sperm with extender.
`0021. The term “initial extender” refers to a medium used
`to extend Sperm prior to the isolation Step of the method of
`this invention.
`0022. The term “final extender” refers to a medium used
`to extend Sperm prior to the freezing Step of the method of
`this invention.
`0023. An “organic Substance” in an extender described
`herein is any organic Substance that tends to reduce cold
`Shock and preserve fertility of Sperm.
`0024. An “energy source” in an extender described herein
`is any Substance or Substrate that Sperm can utilize for cell
`maintenance and/or motility.
`0.025 The term “osmolality,” as used herein, is a measure
`of the OSmotic pressure of dissolved Solute particles in a an
`aqueous Solution (e.g., an extender). The Solute particles
`include both ions and non-ionized molecules. OSmolality is
`
`expressed as the concentration of OSmotically active par
`ticles (i.e., osmoles) dissolved in 1 kg of water.
`Cryopreservation Method
`0026. The invention provides a method of cryopreserving
`Selected Sperm includes the following Steps:
`0027 (1) obtaining a selected sperm sample;
`0028 (2) cooling the selected sperm sample;
`0029 (3) isolating sperm from the selected sperm
`Sample,
`0030 (4) adding final extender to the isolated sperm
`to produce a Suspension of Sperm; and
`0031 (5) freezing the suspension of sperm.
`0032) Obtaining a Selected Sperm Sample
`0033. The first step in the cryopreservation method of the
`invention encompasses obtaining a previously Selected
`Sperm Sample, as well as Subjecting a Source Sample to any
`Suitable Selection method. Sperm from any species can be
`Selected and frozen according to the method of the inven
`tion. The method can be carried out with sperm from
`domesticated animals, especially livestock, as well as with
`Sperm from wild animals (e.g., endangered species). Pref
`erably, the Selected Sperm Sample contains mammalian
`Sperm. Human Sperm, bovine, equine, porcine, Ovine, elk,
`and bison Sperm are particularly preferred.
`0034 Generally, the selected sperm sample contains nor
`mal, viable Sperm. To this end, the ejaculate from which the
`Sperm are obtained typically has at least about 50%, and
`preferably at least about 75% morphologically normal
`Sperm. In these embodiments, generally at least about 40%,
`and preferably at least about 60% of the sperm in the
`ejaculate exhibit progressive motility.
`0035) A wide variety of methods for selecting cells from
`a mixed populations are available, including, for example,
`Selection based on binding of cells or cell components with
`antibodies, antibody fragments, or other binding partners
`and differential Staining.
`0036) The invention is exemplified herein with selection
`based on SeX-type, and Sex-Selected Sperm for use in the
`invention can be obtained using any Selection Strategy that
`takes advantage of slight differences in characteristics
`between X- and Y-type Sperm. Exemplary Sex-Selection
`methods include magnetic techniques (see, e.g., U.S. Pat.
`No. 4,276,139), columnar techniques (see, e.g., U.S. Pat.
`No. 5,514,537) gravimetric techniques (see, e.g., U.S. Pat.
`No. 3,894,529, reissue Pat. No. 32350, U.S. Pat. Nos.
`4,092.229, 4,067,965, and 4,155.831). Sex-selection based
`on differences in electrical properties is disclosed in U.S.
`Pat. No. 4,083,957, and techniques that select based on
`differences in electrical and gravimetric properties are dis
`cussed in U.S. Pat. Nos. 4,225,405, 4,698,142, and 4,749,
`458. U.S. Pat. Nos. 4,009,260 and 4,339.434 describe selec
`tion based on differences in motility. Biochemical
`techniques relying on antibodies are disclosed in U.S. Pat.
`Nos. 4,511,661, 4,999,283, 3,678,806, 4,191,749, 4,448,
`767, whereas U.S. Pat. Nos. 5,021,244, 5,346,990, 5,439,
`362, and 5,660,997 describe selection based on differences
`in membrane proteins.
`
`Exhibit 1035
`Select Sires, et al. v. ABS Global
`
`

`

`US 2003/O157475 A1
`
`Aug. 21, 2003
`
`0037 Flow cytometry is a preferred method for separat
`ing cells from mixed populations based on differential
`Staining with fluorescent dyes or binding to fluorescently
`labeled molecules, Such as antibodies or nucleic acids. In
`fluorescence activated cell sorting (“FACS”), cells are
`“sorted” into different populations based on the fluorescence
`intensity upon irradiation. FACS can be used for sex
`Selection of Sperm because the X chromosome contains
`slightly more DNA than the Y chromosome. When sperm are
`stained with a fluorescent DNA-binding dye, X-chromo
`Some bearing Sperm absorb more dye than Y-chromosome
`bearing Sperm and the two populations can therefore can be
`separated by FACS. This strategy was discussed in U.S. Pat.
`No. 4,362,246 and significantly expanded upon in U.S. Pat.
`No. 5,135,759 (issued to Johnson). Separation has been
`enhanced through the use of high-speed flow cytometers,
`such as the MoFlo(R) flow cytometer produced by Cytoma
`tion, Inc. (Ft. Collins, Colo.) and described in U.S. Pat. Nos.
`5,150,313, 5,602,039, 5,602,349, and 5,643,796, as well as
`in PCT Publication No. WO 96/12171
`0.038. The selection method used to obtain the selected
`Sperm Sample is preferably one that preserves Sperm viabil
`ity. Because of the relative fragility of Sperm, normal flow
`cytometry techniques should generally be modified for Sort
`ing Sperm. More specifically, the flow cytometry entails
`Staining, dilution, and interrogation of cells with light. All of
`these StepS represent Stresses that can reduce Sperm viability.
`The Sensitivity of Sperm to these Stresses can vary between
`Species and even between individuals within Species. Such
`Sensitivities have either been documented or can readily be
`determined by empirical Studies, Such as those described in
`Examples 1-5.
`0.039
`Modifications that enhance viability are described
`the patent publications discussed above. For instance, pro
`cedures that provide improved sheath and collector Systems
`for sorting sperm are disclosed in PCT Publication No. WO
`99/33956 (Application No. PCT/US98/27909). Further,
`Examples 1-7 below describe exemplary procedures for
`Staining and Sorting Sperm. Example 3 describes a study of
`the effects of laser intensity and dye concentration of post
`thaw motility of sorted frozen sperm. This study indicates
`that the use of lower laser intensities during Sorting can
`increase post-thaw motility.
`0040. The selected sperm sample can contain a variety of
`components besides Sperm and will often contain compo
`nents added to protect the Sperm during the Selection pro
`ceSS. In the case of FACS, the Selected Sperm Sample can
`contain component(s) of the Solutions used for Staining and
`Sorting (e.g., the sheath fluid and the catch buffer).
`0041. In addition, the selected sperm sample typically
`contains an extender or extender fraction. For example,
`“two-step’ extenders including an "A fraction' lacking
`glycerol and a "B fraction' containing glycerol are well
`known. The A fraction is added to sperm first, followed by
`addition of an equal volume of the B fraction. For this step,
`the B fraction is often divided into at least two aliquots and
`added Sequentially; e.g., the Second B fraction aliquot is
`added 15 minutes after the first.
`0042. If no extender components are present, an extender
`or extender fraction is typically added to the Selected Sperm
`Sample before the Sperm are isolated from the Sample. If
`only Some extender components are present, additional
`
`components can optionally be added So that Selected Sperm
`Sample includes a complete extender or an extender fraction
`before the isolation Step. In exemplary embodiments, bovine
`Sperm are flow-Sorted So as to produce a Selected Sperm
`Sample including the A fraction of an extender (see
`Examples 2, 3, and 4). If desired, the B fraction can then be
`added to the Selected Sperm Sample before the isolation Step
`(see Example 5). The pre-isolation step extender (or
`extender fraction) is termed “the initial extender” to distin
`guish it from the “final extender' employed for the extension
`of isolated Sperm before freezing. If the Selected Sperm
`Sample was Selected using FACS, the initial extender can be
`matched to the Sheath fluid employed for Sorting. Exemplary
`matched sheath fluids and extenders are described in detail
`in Example 4.
`0043. An extender suitable for use in the selected sperm
`Sample includes a physiologically acceptable carrier. The
`physiologically acceptable carrier is typically aqueous, and,
`in preferred embodiments, includes deionized water. Suit
`able extenders commonly comprise one or more of the
`following additional components: a cryoprotectant, a com
`ponent that maintains oSmolality and buffers pH, an organic
`Substance that prevents cold shock and preserves fertility of
`Sperm, a detergent that acts Synergistically with certain
`organic Substances to enhance preservation of Sperm, an
`energy Source that can be readily utilized by Sperm, an
`antioxidant, which protects Sperm from cold shock. a Sub
`stance that facilitates Sperm capacitation, and one or more
`antibiotics.
`0044 Although cryoprotectants useful in the invention
`are not limited to those acting by a particular mechanism,
`most conventional cryoprotectants act, at least in part, by
`reducing intracellular dehydration. Specifically, freezing is
`accompanied by an increase in Solute concentration in the
`medium Surrounding Sperm. This increase in Solute concen
`tration draws water out of the cells, which increases intra
`cellular electrolyte concentration. Exemplary cryopro
`tectants include glycerol, dimethyl Sulfoxide, ethylene
`glycol, propylene glycol, and the like. The cryoprotectant
`Suitable for use in a given extender can vary, depending on
`the Species from which Sperm are derived. For example,
`glycerol is Suitable for use in cryopreservation of human and
`bovine Sperm, but is generally not used for cryopreservation
`of porcine or rabbit Sperm. Such preferences are well known
`for many commercially valuable Sperm and can readily be
`determined empirically for other types of Sperm.
`004.5 The extender useful in the invention optionally
`includes one or more components that help maintain OSmo
`lality and provide buffering capacity. In preferred embodi
`ments of the invention, the osmolality of the extender
`approximates that of physiological fluids. More preferably,
`the osmolality of the extender is in the range of about 280
`mOsm to about 320 mOsm. The pH is also preferably within
`a physiologically acceptable range, more preferably in the
`range of about 6.5 to about 7.5.
`0046) Substances helpful in maintaining omolality and
`pH within these ranges are well known in the art and can be
`added to the extender as a Solid or already in Solution. A
`buffer containing a Salt, a carbohydrate, or a combination
`thereof can be employed for this purpose. Specific examples
`include Sodium citrate, Trishydroxymethylaminomethane,
`and TES (N-Tris Hydroxymethylmethyl-2-aminoethane
`
`Exhibit 1035
`Select Sires, et al. v. ABS Global
`
`

`

`US 2003/O157475 A1
`
`Aug. 21, 2003
`
`Sulfonic acid), and monosodium glutamate buffers; milk,
`HEPES-buffered medium; and any combination thereof. The
`component employed to help maintain OSmolality and pro
`vide buffering capacity in a particular application can vary
`depending on the other components of the extender and, in
`Some cases, on the Species from which the Sperm are
`derived. The Selection of Such a component for use in the
`present invention is, however, within the level of skill in the
`art.
`0047 One or more organic Substances that protect sperm
`against cold shock and help preserve fertilizing capacity can
`also be included in the extender. Such Substances are well
`known and are Sometimes described as "nonpenetrating
`cryoprotectants.” One skilled in the art can readily determine
`an organic Substance Suitable for a particular application of
`the cryopreservation method described herein. For example,
`organic Substances containing protective constituents (e.g.,
`lipoproteins, phospholipids, lecithin) that are believed to
`reduce the impact of cold Shock and the dilution effect can
`be included in the extender. Suitable organic Substances
`include disaccharides, trisaccharides, and any combination
`thereof. Exemplary organic Substances include egg yolk, an
`egg yolk extract, milk, a milk extract, casein, albumin,
`lecithin, cholesterol, and any combination thereof.
`0.048. The extender can also include a detergent. Alkyl
`ionic detergents, Such as Sodium dodecyl Sulfate (SDS),
`have been reported to act Synergistically with egg yolk to
`enhance protection against cold shock. Other detergents
`useful in the cryopreservation of cells can also be employed
`in the extender, and the Selection of a particular detergent for
`a specific application is within the level of skill in the art in
`light of the guidance provided herein. See, e.g., Example 5.
`0049 Preferably, the extender includes an energy source
`that is readily utilized by Sperm. In the absence of an energy
`Source, Sperm may oxidize intracellular phospholipids and
`other cellular components. Thus, the inclusion of an energy
`Source in the extender protects intracellular reserves and
`cellular components. AS is well known in the art, Sugars,
`particularly monosaccharides, provide a convenient energy
`Source, although any conventional energy Source can be
`employed in the extender. Exemplary monosaccharides use
`ful in the extender include glucose, fructose, and/or man
`OSC.
`0050. One or more antioxidants can optionally be
`included in the extender to provide additional protection
`against cold shock. Exemplary antioxidants include buty
`lated hydroxytoluene (BHT), its derivatives, and the like.
`However, other antioxidants useful in the cryopreservation
`of cells can also be employed in the extender, and the
`Selection of a particular antioxidant for a specific application
`is within the level of skill in the art in light of the guidance
`provided herein.
`0051. The extender can also contain a substance that
`facilitates Sperm capacitation. A variety of capacitation
`facilitators are known in the art and any can be employed in
`the extender. Examples include enzymes Such as alpha
`amylase, beta amylase, beta glucuronidase, which can be
`used in combination, if desired.
`0.052
`Finally, the extender preferably includes an antibi
`otic, Since Substantial bacterial growth can threaten Sperm
`viability and increase the risk of infection of the host in
`
`artificial insemination or in vitro fertilization procedures.
`Any of a variety of antibiotics useful in the cryopreservation
`of cells can also be employed in the extender. The Selection
`of a Suitable antibiotic depends on the Species from which
`the Sperm was obtained, the procedures involved in obtain
`ing and handling the Sperm Sample, and the Specific micro
`organism(s) to be targeted. Exemplary antibiotics include
`tylosin, gentamicin, lincomycin, Spectinomycin, linco-Spec
`tin (a combination of lincomycin and spectinomycin), peni
`cillin, Streptomycin, and ticarcillin, which can be used alone
`or in combination. However, one skilled in the art can
`readily determine other antibiotics suitable for use in the
`extender.
`0053 Exemplary extenders are discussed in greater detail
`below and in the examples.
`0054 The sperm concentration is typically lower in the
`Selected Sperm Sample than in the Source Sample, and, as
`indicated above, when FACS is employed, the dilution is
`Significant. A typical Sort based on SeX-type can produce a
`sample containing sperm at 6x10 cells/ml catch buffer. AS
`Such a low concentration is not optimal for Storage (at least
`for most species tested), the cryopreservation method of the
`invention generally concentrates the Selected Sperm Sample.
`0055 Cooling the Selected Sperm Sample
`0056. The second step in the cryopreservation method
`entails cooling the Selected Sperm Sample, typically, by
`reducing the temperature at a controlled rate. Cooling too
`rapidly can cause cold shock, which can result in a loss of
`membrane integrity and cell function. The Severity of the
`effects of cold Shock vary from Species to species and
`depend on factorS Such as the rate of cooling and the
`temperature range. Under Suitable controlled cooling con
`ditions, the Sperm are able to adapt to thermal effects.
`Example 2, among others, describes conditions for cooling
`bovine Sperm, and determining Suitable conditions for cool
`ing Sperm of other Species is within the level of Skill in the
`art.
`0057. In a preferred embodiment of the invention, the
`Selected Sperm Sample is cooled typically from about 22
`Celsius, to about 5 Celsius, and cooling is generally carried
`out over a period of about 60 minutes to about 24 hours,
`preferably over a period of about 90 minutes to about 240
`minutes, and most preferably over a period of about 90
`minutes to about 120 minutes. Cooing can be accomplished
`by any convenient method. including simply placing the
`Selected Sperm Sample in a 5 Celsius environment.
`Isolation of Sperm Cells from the Selected Sperm
`0058
`Sample
`0059. After initial extension of the selected sperm
`Sample, Sperm are isolated from the Sample using any
`Sufficiently gentle isolation method that provides at least
`about 50% recovery of sperm, more preferably about 75% to
`about 90% recovery of sperm, and most preferably about
`80% to about 90% recovery of sperm. During the isolation
`Step, the cooled Sperm should generally be kept cold, i.e.,
`between about 1 and about 8 Celsius, and preferably close
`to 4 or 5 Celsius.
`0060 Any of a variety of methods suitable for recovering
`cells from a Suspension can be used to isolate the Sperm,
`including for example, filtration, Sedimentation, and cen
`
`Exhibit 1035
`Select Sires, et al. v. ABS Global
`
`

`

`US 2003/O157475 A1
`
`Aug. 21, 2003
`
`trifugation. In an exemplary, preferred embodiment, the
`Selected Sperm Sample is aliquoted into 50 ml tubes at
`Volumes not exceeding about 27 ml, and preferably between
`about 20 to about 27 ml. Centrifugation is carried out at
`about 4 Celsius, at about 850xg, for about 20 minutes.
`Preferably, the centrifugation Step provides at least about
`50% to about 90% recovery of sperm, more preferably about
`60% to about 90% recovery of sperm, and most preferably
`about 70% to about 90% recovery of sperm. After isolation,
`the Supernatant is removed and the pellet is Suspended by
`Vortexing gently or repeated aspiration at 4 Celsius. The
`Sperm concentration is then typically determined (e.g., using
`a hemacytometer).
`0061 Final Extension of Isolated Sperm Cells
`0.062. After isolation, the sperm are pooled, if desired,
`and extended with final extender to an appropriate concen
`tration for freezing. The concentration of Sperm after the
`final eXtension and prior to freezing is preferably in the
`range of about 1x10"/ml to about 300x10"/ml, more pref
`erably about 10x10/ml to about 50x10/ml, and most
`preferably about 10x10/ml to about 20x10/ml.
`0.063. The description of the initial extender above also
`applies to the final extender, which can be the same as or
`different from the initial extender. In particular embodi
`ments, the composition of the Sperm Sample extended with
`the final extender is Substantially similar to (if not the same
`as) the composition of the Sperm Sample after addition of the
`initial extender.
`0064.
`In a preferred embodiment of the invention, an egg
`yolk-Tris extender is used. After the addition of the extender,
`the Sperm Suspension comprises glycerol (cryoprotectant);
`citric acid and Trishydroxymethylaminomethane (buffer);
`egg yolk (organic Substance); fructose (energy Source);
`tylosin, gentamicin, and linco-Spectin (antibiotics). The typi
`cal approximate concentrations of these components after
`addition of the final extender to the isolated Sperm are:
`
`Components of Egg Yolk-Tris Extender
`4-8% wolf vol
`SS-75 nM
`190-210 mM
`5-25% vol?vol
`45-65 mM
`25-100 tug/ml
`200-300 tug/ml
`100-400 tug/ml
`
`Glycerol:
`Citric Acid:
`Tris hydroxymethylaminomethane:
`Egg yolk:
`Fructose:
`Tylosin:
`Gentamicin:
`Linco-spectin:
`
`* 100-400 tug/ml lincomycin and 100-400 tug/ml spectinomycin
`
`0065. In a variation of this embodiment particularly suit
`able for freezing bovine Sperm, the concentrations of these
`components after addition of the final extender to the
`isolated sperm are about 6% (vol/vol) glycerol, about 65
`mM citric acid, about 200 mM Trishydroxymethylami
`nomethane, about 20% (vol/vol) egg yolk, about 56 mM
`fructose, about 50 lug/ml tylosin, about 250 lug/ml gentami
`cin, and about 150/300 ug/ml linco-spectin (i.e., 150 lug/ml
`lincomycin and 300 ug/ml spectinomycin), in deionized
`Water.
`0.066. In an alternative embodiment, an egg yolk-citrate
`extender is employed. After the addition of the extender, the
`
`Sperm Suspension comprises glycerol (cryoprotectant);
`Sodium citrate (buffer); egg yolk (organic Substance);
`tylosin, gentamicin, and linco-Spectin (antibiotics). The typi
`cal approximate concentrations of these components after
`addition of the final extender to the isolated Sperm are:
`
`Components of Egg Yolk-Citrate Extender
`Glycerol:
`4-8% vol?vol
`Sodium Citrate:
`60-80 mM
`Egg yolk:
`5-25% vol?vol
`Tylosin:
`25-100 tug/ml
`Gentamicin:
`200-300 tug/ml
`Linco-spectin:
`100-400 tug/mL*
`
`*100–400 tug/ml lincomycin and 100-400 tug/ml spectinomycin
`
`0067 Exemplary, preferred concentrations for freezing
`bovine sperm are about 7% (vol/vol)glycerol, about 72 mM
`sodium citrate, about 20% (vol/vol) egg yolk, about 50
`tug/ml tylosin, about 250 lig/ml gentamicin, and about 250/
`300 ug/ml linco-Spectin.
`0068. In another alternative embodiment, an egg yolk
`TES-Tris (“EY TEST) extender is employed. After the
`addition of the extender, the Sperm Suspension comprises
`glycerol (cryoprotectant), egg yolk and heated milk, e.g.,
`homogenized milk containing 1.25% fructose with 10%
`glycerol (organic Substances); tylosin, gentamicin, and
`linco-Spectin (antibiotics). The typical approximate concen
`trations of these components after addition of the final
`extender to the isolated Sperm are:
`
`Components