FIELD OF THE INVENTION
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
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 cryopreservation and storage of sperm have facilitated widespread distribution and commercialization of sperm intended for artificial insemination or in vitro fertilization.
Further improvements in mammalian sperm collection, 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 containing 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 accelerating 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.
A variety of methods are available for selecting cells; however, the selection and subsequent processing of sperm presents unique challenges because sperm are incapable 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 betveen collection and use. As most of the available selection methods involve physical stresses and take time, selected sperm are typically somewhat 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.
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 insemination 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 105-106/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.
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
The present invention provides a method of cryopreserving sperm that have been selected for a specific characteristic. The method is particularly useful for cryopreserving 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.
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.
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
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 demonstrated 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.
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 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.
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.
The term “sex-type” refers to the type of sex chromosome present in the sperm (i.e., the X or Y chromosome).
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.
As used with reference to sperm, the term “cryoprotectant” refers to a molecule that protects sperm during a freeze-thaw cycle, promoting survival and retention of fertilizing capacity.
The term “dilution effect” refers to the rapid decline in motility and/or viability of sperm when highly diluted.
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 dictates 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.
The term “sorting” is used herein to describe a selection method carried out using a fluorescence-activated cell sorter (FACS).
The term “extender” refers to any medium that tends to preserve sperm viability. The term “extension” refers to the dilution of sperm with extender.
The term “initial extender” refers to a medium used to extend sperm prior to the isolation step of the method of this invention.
The term “final extender” refers to a medium used to extend sperm prior to the freezing step of the method of this invention.
An “organic substance” in an extender described herein is any organic substance that tends to reduce cold shock and preserve fertility of sperm.
An “energy source” in an extender described herein is any substance or substrate that sperm can utilize for cell maintenance and/or motility.
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 particles (i.e., osmoles) dissolved in 1 kg of water.
The invention provides a method of cryopreserving selected sperm includes the following steps:
(1) obtaining a selected sperm sample;
(2) cooling the selected sperm sample;
(3) isolating sperm from the selected sperm sample;
(4) adding final extender to the isolated sperm to produce a suspension of sperm; and
(5) freezing the suspension of sperm.
Obtaining a Selected Sperm Sample
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 invention. 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). Preferably, the selected sperm sample contains mammalian sperm. Human sperm, bovine, equine, porcine, ovine, elk, and bison sperm are particularly preferred.
Generally, the selected sperm sample contains normal, 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.
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.
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 discussed 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 selection 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.
Flow cytometry is a preferred method for separating 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-chromosome 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® flow cytometer produced by Cytomation, 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.
The selection method used to obtain the selected sperm sample is preferably one that preserves sperm viability. Because of the relative fragility of sperm, normal flow cytometry techniques should generally be modified for sorting 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.
Modifications that enhance viability are described the patent publications discussed above. For instance, procedures 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.
The selected sperm sample can contain a variety of components besides sperm and will often contain components added to protect the sperm during the selection process. 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).
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.
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 distinguish 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.
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. Suitable extenders commonly comprise one or more of the following additional components: a cryoprotectant, a component 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 substance that facilitates sperm capacitation, and one or more antibiotics.
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 concentration draws water out of the cells, which increases intracellular electrolyte concentration. Exemplary cryoprotectants 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.
The extender useful in the invention optionally includes one or more components that help maintain osmolality and provide buffering capacity. In preferred embodiments 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.
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, Tris[hydroxymethyl]aminomethane, and TES (N-Tris [Hydroxymethyl]methyl-2-aminoethanesulfonic acid), and monosodium glutamate buffers; milk; HEPES-buffered medium; and any combination thereof. The component employed to help maintain osmolality and provide 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.
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.
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.
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 useful in the extender include glucose, fructose, and/or mannose.
One or more antioxidants can optionally be included in the extender to provide additional protection against cold shock. Exemplary antioxidants include butylated 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.
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.
Finally, the extender preferably includes an antibiotic, 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 obtaining and handling the sperm sample, and the specific microorganism(s) to be targeted. Exemplary antibiotics include tylosin, gentamicin, lincomycin, spectinomycin, linco-spectin (a combination of lincomycin and spectinomycin), penicillin, 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.
Exemplary extenders are discussed in greater detail below and in the examples.
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 6×105 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.
Cooling the Selected Sperm Sample
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 conditions, the sperm are able to adapt to thermal effects. Example 2, among others, describes conditions for cooling bovine sperm, and determining suitable conditions for cooling sperm of other species is within the level of skill in the art.
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. Cooiing 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 Sample
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.
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 centrifugation. 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 850×g, 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).
Final Extension of Isolated Sperm Cells
After isolation, the sperm are pooled, if desired, and extended with final extender to an appropriate concentration for freezing. The concentration of sperm after the final extension and prior to freezing is preferably in the range of about 1×106/ml to about 300×106/ml, more preferably about 10×106/ml to about 50×106/ml, and most preferably about 10×106/ml to about 20×106/ml.
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 embodiments, 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.
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 Tris[hydroxymethyl]aminomethane (buffer); egg yolk (organic substance); fructose (energy source); tylosin, gentamicin, and linco-spectin (antibiotics). The typical approximate concentrations of these components after addition of the final extender to the isolated sperm are:
|Components of Egg Yolk-Tris Extender |
| ||Glycerol: ||4-8% ||vol/vol |
| ||Citric Acid: ||55-75 ||mM |
| ||Tris [hydroxymethyl]aminomethane: ||190-210 ||mM |
| ||Egg yolk: ||5-25% ||vol/vol |
| ||Fructose: ||45-65 ||mM |
| ||Tylosin: ||25-100 ||μg/ml |
| ||Gentamicin: ||200-300 ||μg/ml |
| ||Linco-spectin: ||100-400 ||μg/ml* |
| || |
| || |
In a variation of this embodiment particularly suitable 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 Tris[hydroxymethyl]aminomethane, about 20% (vol/vol) egg yolk, about 56 mM fructose, about 50 μg/ml tylosin, about 250 μg/ml gentamicin, and about 150/300 μg/ml linco-spectin (i.e., 150 μg/ml lincomycin and 300 μg/ml spectinomycin), in deionized water.
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 typical 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 ||μg/ml |
| ||Gentamicin: ||200-300 ||μg/ml |
| ||Linco-spectin: ||100-400 ||μg/mL* |
| || |
| || |
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 μg/ml tylosin, about 250 μg/ml gentamicin, and about 250/300 μg/ml linco-spectin.
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 concentrations of these components after addition of the final extender to the isolated sperm are:
|Components of Egg Yolk TES-Tris Extender |
| ||Glycerol: ||3-7% ||vol/vol |
| ||Tris [hydroxymethyl-methyl]- ||140-170 ||mM |
| ||2-aminoethanesulfonic acid: |
| ||Tris [hydroxymethyl]aminomethane: ||60-80 ||mM |
| ||Egg yolk: ||5-25% ||vol/vol |
| ||Fructose: ||5-12 ||mM |
| ||Tylosin: ||50-150 ||μg/ml |
| ||Gentamicin: ||400-600 ||μg/ml |
| ||Linco-spectin: ||200-700 ||μg/mL* |
| || |
| || |
Exemplary, preferred concentrations for freezing bovine sperm are about 5% (vol/vol) glycerol, about 158 mM Tris[hydroxymethy-methyl]-2-aminoethanesulfonic acid, about 72 mM Tris[hydroxymethyl]aminomethane, about 20% (vol/vol) egg yolk, about 8 mM fructose, about 100 μg/ml tylosin, about 500 μg/ml gentamicin, and about 300/600 μg/ml linco-spectin.
In yet another alternative embodiment of the invention, a Milk extender is employed. After the addition of the extender, the sperm suspension comprises glycerol (cryoprotectant); heated homogenized milk (organic substance); fructose (energy source); and tylosin, gentamicin, and linco-spectin (antibiotics). The typical approximate concentrations of these components after addition of the final extender to the isolated sperm are:
|Components of Milk Extender |
| ||Homogenized Milk ||90% ||(vol/vol) |
| ||Glycerol: ||3-7% ||(vol/vol) |
| ||Fructose: ||1.25% ||(wt/vol) |
| ||Tylosin: ||50 ||μg/ml |
| ||Gentamicin: ||250 ||μg/ml |
| ||Linco-spectin: ||250/300 ||μg/ml |
| || |
| || |
Exemplary preferred concentrations for freezing bovine sperm are about 90% milk, about 10% (vol/vol) glycerol, about 1.25% fructose (wt/vol?), about 50 μg/ml tylosin, about 250 μg/ml gentamicin, and about 250/300 μg/ml linco-spectin.
Other extenders standardly used to freeze sperm can also be employed as the final extender in freezing selected sperm. A variety of extenders optimized for use in freezing sperm from various species have been described, and many are commercially available. Freezing extenders for equine sperm typically consist of milk, egg yolk, various sugars, electrolytes and a cryoprotectant. Exemplary freezing extenders are described by Squires, E. L., et al., Cooled and Frozen Stallion Semen Animal Reprod. and Biotechnology Laboratory, Bulletin No. 69, Chapter 8, “Seminal Extenders” pp. 49-51 (July, 1999).
Equilibration and Freezing of Sperm
Extension of the sperm sample produces a suspension of sperm, which is then transferred into containers for freezing. If the sperm are intended for use in fertilization, the cells are conveniently aliquoted into individual doses sufficient to achieve fertilization. The required dose can vary substantially from one species to the next and is either well-known (e.g., for cattle and horses) or can readily be determined. In the case of sex-selected bovine sperm, convenient doses range from about 1.0×106 sperm to about 3.0×106sperm.
Any suitable container can be employed for freezing, including, for example, an ampule, a vial, and a straw Sperm intended for AI are typically frozen in straws (e.g., 0.25 ml or 0.50 ml straws) designed for use with an insemination gun. Preferably, a bolus of extender is drawn into the straw, followed, in sequence, by air, sperm, air, and extender, so that the sperm are flanked on either side by an air space, which separates the sperm from a bolus of extender at either end of the straw.
Prior to freezing, the sperm are generally allowed to equilibrate at about 5° C. Preferably, the sperm are allowed to equilibrate for a period in the range of about 1 hour to about 18 hours, more preferably between about 3 hours and about 18 hours, and most preferably between about 3 hours and about 6 hours (see Example 2).
Following equilibration, any standard freezing method can be employed, provided the freezing rate is not too rapid (i.e., not in excess of about 0.5° C./minute). Preferably, the freezing rate is about 0.5° C./minute. In an exemplary, preferred embodiment, the sperm are placed in static liquid nitrogen vapor, and freezing is carried out in three distinct stages over a period of about 10 minutes. In the first stage of freezing, the sperm are cooled from about 5° C. to about −15° C. at a rate of about 40° C./minute to about 65° C./minute. In the second stage of freezing, the sperm are cooled from about −15° C. to about −60° C. at a rate of about 25° C./minute to about 35° C./minute. In the third stage, the sperm are plunged into liquid nitrogen at about −100° C.
Selected Sperm Samples
In addition to a freezing method, the invention provides a frozen sperm sample including sperm selected from a source sample for a particular characteristic. The sperm can be from any species, including any of those discussed above with reference to the freezing method. The invention encompasses frozen sperm selected for any characteristic by any suitable method, such as those described above. Preferred embodiments include frozen sex-selected human, bovine, equine, porcine, ovine, elk, or bison sperm. Sex-selection is preferably carried out using flow cytometry as described generally above.
Also within the scope of the invention is a container containing a frozen sperm sample according to the invention. The container can be formed from any material that does not react with the frozen sperm sample and can have any shape or other feature that facilitates use of the sample for the intended application. For samples intended for use in AI, for example, the container is conveniently a straw (e.g., 0.25 ml or 0.5 ml straw) designed for use with an insemination gun. The container is sealed in any manner suitable for preserving the sample at the intended storage temperature, which is typically below −80° Celsius. 0.25 ml straws can be sealed, for instance, with PVC powder, ultrasonically, or with a cotton-polyvinyl plug and/or a stainless steel ball (BB).
As the frozen sperm sample of the invention is typically thawed before use, the invention also provides a thawed, previously frozen, selected sperm sample and a container including such a thawed sample.
Methods of Using the Selected Sperm Sample
The frozen selected sperm sample of the invention is suitable for use in any method in which sperm are used. The sample can be thawed and used in any conventional fertilization method, such as artificial insemination or in vitro fertilization. Thawing is carried out in the same manner as for frozen, non-selected sperm. Briefly, the straw containing the frozen sperm is submerged in a water bath maintained at a temperature of about 35° C. to about 37° C. for a period of about 20 to about 30 seconds. After thawing, semen deposition (e.g., insenination) is carried out according to standard procedures, taking care to protect the sperm from environmental fluctuations.