United States Patent
`Johnson
`
`[:9]
`
`[54] METHOD TO PRESELECT THE SEX OF
`OFFSPRING
`
`[75]
`
`Inventor:
`
`Lawrence A. Johnson, Silver Spring.
`Md.
`
`[13] Assignee:
`
`The United States of America as
`represented by the Secretary of
`Agriculture, Washington, DC.
`
`[21] Appl. No.: 692.958
`
`[22] Filed:
`
`Apr.26, 1991
`
`Related U.S. Application Data
`Continuation of Ser. No. 349,669, May 10. I989. aban-
`cloned.
`
`[63]
`
`Im. C1,: ............................................ .. A61K35/52
`[51]
`[52] us. CI. .................................... .. 424/561; 436/63;
`436/172; 435/2
`(531 Field of Search ................. .. 436/63. H2; 424/561
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4.:/i9'.i'S Lang ...................................42-W105
`4.083.951’
`4.191349 M1930 Bryant ............................... ..42-t.fl05
`
`FOREIGN PATENT DOCUMENTS
`
`2145112
`
`3/1935 United Kingdom .
`OTHER PUBLICATIONS
`
`Culling. Handbook of Histopathlological & Histochemi~
`cal Techniques, Butterworth & Co.. London, 1974, p.
`192.
`-
`I... A. Johnson, “Flow Sorting of Intact X and ‘Y
`Chromosome-Bearing Mammalian Sperrnatozoa,“ Cy-
`tometry. Suppl. 2, p. 66 Abstract (1938).
`J. C. Conover and R. B. L. Gwatkin, "Pre—Loading of
`Mouse Oocytes with DNA—Specific Fluorochrome
`(Hoechst 33342) Permits Rapid Detection of Sper-
`rn—Oocyte Fusion." J. Reprod. Fert. 32: 631-690 (1988).
`L. A. Johnson and R. N. Clarke, "Flow Sorting of X
`and Y Chromosome-Bearing Mammalian Sperm: Acti-
`vation and Pronuclear Development of Sorted Bull,
`
`llllllllllllll||I||||lll||ll|||lI|||||||l||||||l|||ll|l||||l|||||l||||l|||l
`usoos l35759A
`
`[11] Patent Number:
`
`5,135,759
`
`[45] Date of Patent:
`
`Aug. 4, 1992
`
`Boar, and Ram Sperm Microinjected into Hamster Oo-
`cytes," Gamete Res. 21: (1988).
`L. A. Johnson et al., "Flow Cytometry of X and Y
`Chromosome-Bearing Sperm for DNA Using an Irn-
`proved Preparation Method and Staining with Hoechst
`33342." Garnete Res. 17: 203-212 (1987).
`R. E. Hinkley et a1., “Rapid Visual Detection of Sper-
`m—Egg Fusion Using the DNA—Specific Fluorochrome
`Hoechst 33342,“ Dev. Biol. 113: 148-154 (1986).
`L. A. Johnson and D. Pin]-tel, "Modification of a
`Laser—Based Flow Cytometer for High—Re-solution
`DNA Analysis of Mammalian Spermatozoa," Cytome-
`try 1: 268-2‘.-'3 (1936).
`L. A. Johnson, “Gender Preselection in Farm Ani-
`mals,“ Research for Tomorrow, 1936 Yearbook of Ag-
`riculture. pp. 73-77 (1936).
`W. M. Grogan et al.. "DNA Analysis and Sorting of
`Viable Mouse Testis Cells,“ J. Histochem. Cytochem.
`29: 'i‘38—'.?46 (1981).
`M. R. Lolten. “Separation of Viable T and B Lympho-
`cytes Using a Cytoche-mica] Stain, Hoechst 33342," J.
`Histochem. Cytochem. 23: 36—39 (I930).
`
`Primary Exarm'ner——Douglas W. Robinson
`Assistant Examr'ner—Jean C. Witz
`Attorney. Agent. or Firm—M. Howard Silverstein; John
`D. Fado: Curtis P. Ribando
`
`[57]
`
`ABSTRACT
`
`intact X and Y chromosome—bearing sperm populations
`of rabbits and swine were separated according to DNA
`content using a flow cytometer/cell sorter. Sperm via-
`bility was maintained by special staining techniques and
`by sorting and collecting the sperm in nutrient media.
`The sorted sperm were surgically inseminated into the
`uteri of rabbits or swine. Of the offspring born from
`does inseminated with the sorted population of X-bear-
`ing sperm. 94% were females. Of ofispring born from
`does inseminated with sorted Y-bearing sperm from the
`same ejaculate. 81% were males.
`
`26 Claims, No Drawings
`
`Exhibit No. 1010
`
`PGR of U.S. Patent 8,933,395
`
`

`
`1
`
`METHOD TO PRESELECT THE SEX OF
`OFFSPRING
`
`5,135,'lS9
`
`This application is a continuation of application Ser.
`No. 07/349,669. filed May 10. I989. now abandoned.
`
`5
`
`BACKGROUND OF THE INVENTION
`l. Field of the Invention
`"I"his invention relates to a method of preselecting the 10
`sex of offspring by sorting sperm into X and Y chromo-
`some-bearing sperm based on differences in DNA con-
`tent.
`
`2. Description of the Prior Art
`Gender of animal offspring is important to livestock '5
`producers. Because the dairy farmer has little use for
`most bull calves, the use of sexed semen to produce only
`females would make "milk production more efficient.
`Swine fanners would produce pork more efficiently if
`they were able to market only female swine, because
`females grow faster than males.
`In beef cattle and sheep breeds. the male grows at a
`faster rate than the female and hence is preferred for
`meat production.
`In addition. the ability to specify male or female off-
`spring should shorten the time required for genetic
`improvements. since desirable traits are often associated
`with one or the other parent. Planning the sex of cattle
`offspring is already practiced on a limited basis. This
`procedure consists of removing embryos from the cow,
`identifying their potential gender. and re-implanting
`only those of the desired gender. However, an ability to
`separate sperm into male-producing and female-produo
`ing groups before they are used for artificial insemina-
`tion could enhance the overall value of offspring pro-
`duced by embryo transfer.
`Every living being has a set of paired chromosomes.
`which carry all the genetic material necessary to main-
`tain life and also to propagate new life.
`All but one pair of chromosomes are called auto-
`sornes and carry genes for all the characteristics of the
`body. such as skin, hair and eye color. mature size. and
`body characteristics. The remaining pair are called sex
`chromosomes. They carry the genetic material
`that
`specifies gender. One sex chromosome is called X. the
`other Y.
`A sperm from the male or an egg from the female
`contains one of each pair of autosomes; in addition. in
`mammals the egg always contains an X chromosome,
`while the sperm always carries either an X or Y chro-
`mosome.
`
`When a sperm and egg unite and the sperm carries the
`Y chromosome. the offspring is male (XY); however. if
`the sperm carries an X chromosome when it unites with 55
`the egg. the resulting offspring is female (XX).
`The only established and measurable difference be-
`tween X and Y sperm that is known and has been
`proved to be scientifically valid is their difference in
`deoxyribonucleic acid (DNA) content. The X chromo-
`some is larger and contains slightly more DNA than
`does the Y chromosome. The difference in total DNA
`between X-bearing sperm and Y-bearing sperm is 3.4%
`in boar, 3.8% in bull. and 4.2% in ram sperm.
`The amount of DNA in a spenn cell. as in most nor- 65
`mal body cells. is stable. Therefore, the DNA content of
`individual sperm can be monitored and Md to differen-
`tiate )t- and Y-bearing sperm.
`
`2
`Since the difference in DNA mass in the sex chromo-
`somes of most mammals is the only scientifically vali-
`dated. measurable difference between X- and Y-bearing
`sperm. the chromosomal constitution [Moruzzi. J. Re-
`prod. Fertil. 57: 319 (l9'i'9)] and/or measurement of
`DNA mass [Pinkel et al. (1). Science 218: 904 (1982);
`Pinkel et al. (2). Cytometry 3:
`I (1982); Johnson and
`Pinkel, Cytometry 7: 263 (1986); Johnson et al. (I).
`Gum. Res. 16: I (1987); Johnson et al. (2), Gem. Res. 1?;
`203 0937)] are the only verifiable means other than
`fertility for determining the sex-producing capability of
`a population of sperm. The literature describes many
`physical, biochemical. and functional methods that
`have purportedly sexed sperm [Amann and Seidel.
`"Prospects for Setting Mammalian Sperm," Colorado
`Assoc. Univ. Press. Boulder 0982)]; several of these
`methods have been tested for relative DNA content
`[Pinltel et al., J. Anim. Sci. 60: i303 (1985); Johnson (I).
`Theriogenoloy 29: 265 0988)}. However. no method
`has been proven in controlled experiments to actually
`affect the sex ratio of offspring.
`Previous studies have demonstrated that the differ-
`ence in DNA content between X and Y chromosome-
`bearing sperm can be repeatedly measured and the
`spenn sex ratio of a sample of semen predicted [Johnson
`and Pinkel, supra; Johnson et al. (1), supra; Johnson et
`al. (2). supra; Johnson (I). supra; Johnson (2). Cytome-
`try. Suppl. 2: 66 (Abstract) (l9B3)]. Verifiable separa-
`tion by soning of X and Y sperm based on DNA con-
`tent has been accomplished with the vole [Pinkel et al.
`(1), supra; Johnson. In “Beltsville Symposia in Agricul-
`tural Research X,“ P. C. Augustine, H. D. Danforth, &
`M. R. Bakst (eds). Martinus Nijltoff, Boston, pp.
`121-134 (1986)) and the chinchilla [(Johnson et al. (1).
`supra]. However, preparation procedures damaged
`DNA viability. The sorting of sperm nuclei from sev-
`eral mamrnaiian (bull, boar, ram, vole, chincilla} species
`into separate X and Y chromosome-bearing populations
`at purities ranging from 92 to 99% has been accom-
`plished [Johnson and Clarke, Gam. Res. 21: 335 (l98B}].
`Nuclear decondensation and pronuciear development
`was demonstrated in hamster eggs that had been mi-
`croinjected with sorted X- or Y-bearing bull, boar, or
`ram sperm [Johnson and Clarke, supra].
`SUMMARY OF THE INVENTION
`
`It is an object of this invention to provide a method
`for sorting mammalian sperm into X and Y chromo-
`some fractions based on DNA content.
`It
`is a further object of this invention to teach a
`method of staining the DNA of mammalian sperm
`while maintaining viability of the sperm.
`It is a further object of this invention to provide a
`sheath fluid adapted to be used in a cell-sorting appara-
`tus while maintaining viability of sperm cells.
`It is a further object of this invention to provide in
`collecting fluid capable of maintaining the viability of
`sorted sperm cells.
`Other objects and advantages of this invention will
`become readily apparent from the ensuing description.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`I have now demonstrated the separation. by flow
`sorting. of‘ intact. viable X and Y chromosome-bearing
`rabbit and swine sperm populations based on relative
`DNA content; surgical insemination of the sorted spenn
`into does; and the subsequent birth of sexed offspring
`
`

`
`5,135,759
`
`IS
`
`25
`
`55
`
`3
`with a phenotypic sex ratio consistent with predictions
`based on the relative DNA content of the sorted sperm
`populations.
`A flow cytometer measures the amount offluorescent
`light given off when the sperm. previously treated with
`it fluorescent dye. pass through a laser beam. The dye
`binds to the DNA. The fluorescent light is collected by
`an optical lens assembly; the signal is transported to a
`photomultiple lube, amplied. and analyzed by com-
`puter. Because the X chromosome contains more DNA
`than the Y chromosome. the female sperm (X) takes up
`more dye and gives off more fluorescent light than the
`male sperm (Y).
`For small differences in DNA to be detected between
`X and Y. the sperm must pass single file through the
`laser beam. which measures the DNA content of indi-
`vidual sperm.
`In orthogonal flow cytometry. a suspension of single
`cells stained with a fluorochrome is made to flow in a
`narrow stream intersecting an excitation source (laser 20
`bearn). As single cells pass through the beam, optical
`detectors collect the emitted light, convert the light to
`electrical signals, and the electrical signals are analyzed
`by a multichannel analyzer. The data are displayed as
`multi- or single-parameter histograms. using number of
`cells and fluorescence per cell as the coordinates.
`In order to use an orthogonal flow cytrometric sys-
`tem to differentiate between X- and Y-bearing sperm
`DNA. a beveled sample injection tip and a second fluo-
`rescence detector in the forward position is required
`[Johnson and Pinkel. supra]. This paper is herein incor-
`porated by reference. The modified system allows one
`to control the orientation of the flat ovoid sperm head
`as it passes the laser beam. Elimination of the unoriented
`sperm by electronic gating enhances precision. Typi-
`cally. 30% of sperm nuclei (without tails) are properly
`oriented as they pass the laser beam.
`In the modified Epics V flow cytometer/cell sorter.
`hydrodynamic forces exerted on the flat. ovoid mam-
`malian sperm nuclei orient the nuclei in the plane ofthe 40
`sample stream as they exit the beveled injection tip.
`Fluorescent signals are collected simultaneously by 90
`and 0 degree optical detectors, from the edge and flat
`side ofthe sperm nucleus, respectively. For sorting, the
`sample stream is broken into uniform droplets by an
`ultrasonic transducer. Droplets containing single sperm
`of the appropriate fluorescence intensity are given a
`charge and electrostatically deflected into collection
`vessels. The collected sperm nuclei then can be used for
`microinjection into eggs. Since the spenn nuclei have
`no tails, they cannot be used for normal insemination.
`Accurate measurement of mammalian sperm DNA
`content using flow cytometry and cell sorting is dill"-
`cult because the sperm nucleus is highly condensed and
`flat in shape. which makes stoichiometric staining diffi-
`cult and causes stained nuclei to have a high index of
`refraction. These factors contribute to emission of fluo-
`re-scence preferentially from the edge or thin plane of
`the sperm nucleus. In most flow cytometers and sorters,
`the direction of sample flow is orthogonal to the direc- 60
`tion of propagation of the laser beam and the optical
`axes of the fluorescence detection. Consequently. fluo-
`rescence measurement is most accurate when the spenn
`fluorescence is excited and measured on an axis perpen-
`dicular to the plane of the sperm head [I-‘inltel et al. (2),
`supra]. At relatively low sample flow rates, hydrody-
`namics are used to orient tailless sperm so that DNA
`content can be measured precisly on 60 to 80% of the
`
`4
`Sperrn passing in front of the laser beam. The modified
`Epics V system used in this study can measure the DNA
`content oftailless spenn from most species at the rate of
`50 to 150 sperm per second [Johnson and Pinltel. supra].
`Intact sperm (with tails). whether viable or nonvia-
`ble. cannot be oriented as effectively as tailless sperm
`nuclei [Johnson (2). supra]. However. a 9'0-degree de-
`tector can be used to select the population of properly
`oriented intact sperm to be measured by the 0 degree
`detector. Since no hydrodynamic orientation is at-
`tempted, thc sample flow rate can be much higher,
`which compensates somewhat for the fact that only 15
`to 20% ofintact spenn pass through the laser beam with
`proper orientation. In this invention, the overall flow
`rate was approximately 2500 intact sperm per second.
`The intact X- and Y-bearing sperm fractions were
`sorted simultaneously from the population of input
`sperm at a rate of 80-90 sperm of each type per second.
`It is, of course, of critical importance to maintain high
`viability of the intact sperm during the sorting process
`and during storage after sorting but prior to insemina-
`tion.
`
`Of the factors involved in maintaining sperm viabil-
`ity, the method of staining. the sheath fluid. and the
`collecting fluid have been found to be especially impor-
`tant.
`
`A nontoxic DNA stain must be selected. A preferred
`stain is Hoechst bisbenzimide H 33342 fluorochrotne
`(Calbiochem-Behring Co., La Jolla, Calif). To our
`knowledge. this fluorochrome is the only DNA binding
`dye that is nontoxic to sperm. Concentration of the
`fluorochrome must be minimal to avoid toxicity, and
`yet be sufficient to stain sperm uniformly and to detect
`the small differences in the DNA of)( and Y sperm with
`minimal variation. A suitable concentration was found
`to be S pg/ml. but this may be varied from 4 to 5 pg/ml.
`The sperm must be incubated with stain at sufficient
`temperature and time for staining to take place, but
`under mild enough conditions to preserve viability.
`lncubation for 1 hr at 35° C. was found to beacceptable,
`but ranges of 30" to 39' C. would also be effective.
`Incubation time has to be adjusted according to temper-
`ature; that is. 1.5 hr for 30' C.; 1 hr for 39‘ C.
`Sheath fluid used in sorting cells must be electrically
`conductive and isotonic. A concentration of IO mM
`phosphate buffered saline provided the necessary elec-
`trical properties, and 0.1% bovine serum albumin was
`added to enhance sperm viability by providing protein
`support for metabolism and viscosity for the sperm. The
`sheath fluid must be free of sugars and excess salts.
`Dilution of sperm as occurs in sorting tends to reduce
`viability of the cells. To overcome this problem, sperm
`were collected in test egg yolk extender [Graham et al..
`J. Dairy Sci. 55: 372 0972)] modified by adjusting the
`pH and adding a surfactant. Details of the composition
`of the extender are shown in Example 1. The surfactant
`is believed to enhance capacitation of the sperm prior to
`fertilization.
`
`To confirm the DNA content and predict the sex of
`the offspring of surgically inseminated X or Y sorted
`sperm fractions, an aliquot of the sorted spenn was
`sonicated to remove the tails, stained. and the nuclei
`was reanalyzed for DNA content to predict the propor-
`tion of X and Y sperm.
`Although the detailed description which follows uses
`the sorting of rabbit sperm as an example of this inven-
`tion,
`it
`is expected that the sperm of most mammals
`could be effectively sorted by following these proce-
`
`

`
`5.l35,'.+'59
`
`6
`kindling rate of near 80% and litter size of about six
`from does of this age and breed. However, the kindling
`rate across the three treatment groups averaged 28%.
`with an average litter size of 3.9. The cause ofthe appar-
`ent high rate of embryonic death is though to be due to .
`the fluorochrome binding to the DNA and/or to the
`effect of the laser beam exciting the DNA bound fluoro-
`chrome. Earlier work has shown that sorted vole sperm
`nuclei that were microinjected into hamster eggs exhib-
`ited chromosome breakage in the developing sperm
`pronucleus [Libbus et al.. Mut. Res. 182: 265 0987)].
`Those sperm had been sonicated, stained, sorted, and
`microinjected,
`ll somewhat more rigorous treatment
`than the staining and sorting used in this study.
`I have demonstrated that DNA can be used as a dif-
`ferentiating marker between X- and Y-bearing sperm,
`that DNA can be used to accurately predict the sex of
`offspring from separated X- and Y-bearing sperm popu-
`lations. and that flow sorting is an effective means for
`separating viable X- and Y-bearing sperm populations
`suitable for production of offspring.
`The following examples are intended only to further
`illustrate the invention and are not intended to limit the
`scope of the invention. which is defined by the claims.
`EXAMPLE I
`
`Semen was collected from mixed breed mature bucks
`by use of an artificial vagina. Sperm concentration was
`detennined with a hemocytometer. The semen was
`diluted with Tris bufTer, pH 6.9, to a concentration of
`10X 105 per ml. Bisbenzimide I-I 33342 lluorochrome
`was added at a concentration of5 p.g/ml. The samples
`were incubated for 1 hr at 35° C. Intact sperm were
`sorted on a modified EPICS V flow cytometer/cell
`sorter. The stained intact spenn were excited in the
`ultraviolet (UV; 361 and 364 um) lines of a 5-watt 90-5
`lnnova Argon-ion laser operating at 200 mW. Data
`were collected as 256-channel histograms. Sheath fluid
`was 10 mM phosphate-buffered saline (PBS) containing
`0.1% bovine serum albumin (BSA). Sperm were sorted
`into a test egg yolk extender.
`The composition of the extender was N-tris(hydrox-.
`ymethyl)-methyl-2-amino ethane sulfonic acid. 2.16 g;
`tris hydroxymethyl arninomethane, 0.51 g; dextrose. 0.1
`g; streptomycin sulfate. 0.13 g; penicillin (3. 0.08 g; egg
`yolk,
`I2.5 ml; Equex STM (Nova Chemical Sales,
`Scituato, Mass), 0.5%; and distilled water, 50 ml. This
`
`5
`dures. Those skilled in the art will recognize that minor
`modifications may be made in the procedure without
`departing from the spirit and scope of the invention.
`Rabbit semen was collected. diluted, and stained with
`a lluorochrome dye. Sperm were sorted in a modified
`Epics V flow cytometer/cell sorter.
`After being sorted. sperm were surgically insemi-
`nated into the uteri of rabbits.
`The results obtained by surgical insemination of does
`with sorted intact sperm are presented in Table 1. Re-
`covery of ova 4-0 hr post-insemination indicated that
`stained sorted sperm, as well as unstained unsorted
`sperm, were capable of fertilizing rabbit ova in vivo.
`Inseminations were also made to determine the com-
`
`parability of predicted sex of offspring to phenotypic 15
`sex. As the data in Table 11 indicate. the predictability of
`the phenotypic sex based on DNA analysis of the sepa-
`rated intact sperm was very high. Reanalysis of the
`sorted Y population used for insemination indicated that
`31% of the spent: were Y-bearing. The sex ratio of 20
`offspring from these inseminations was identical to that
`predicted. These values were significantly different
`from theoretical 50:50 sex rates (P<0.003)._ Reanalysis
`of the sorted X-bearing sperm population used for in-
`semination indicated that 86% were X-bearing and 14%
`were Y-bearing sperm. The phenotypic sex of the off-
`spring from these inseminations was 94% female. which
`was different from the theoretical 50:50 (P -(0.0003).
`Inseminations were made with sorted X and Y popu-
`lations that were recombined (recombined X and Y
`group) immediately before insemination. The assump-
`tion was made that the proportions of X and Y in the
`recombined samples were equal (50:50). The pheno-
`typic sex resulting from the inserninations was 57%
`female and 43% male (Table II} and was not signifi-
`cantly different from the theoretical (50:50) sex ratio
`(p=o.4o).
`
`ABLE I
`Fertilizing Capacity of Flow-Sorted Rabbit
`Sgrmatozoa Alter Intrauterine Insemination of Docs
`Number of
`Ovulation
`Eggs
`Does
`Treatment
`Points
`Recovered
`Inseminaled
`of Sperm
`I6
`9
`2
`Unsorted
`59
`£6
`6‘
`Sorted
`‘One doe accounted for ‘l' recovered and T unferliltwd eggs.
`
`Eggs
`Fertilized
`9
`39
`
`- T
`
`TABLE II
`Predicted and Actual Sex Ratios of Offspring After Intrauterine
`Insemination of Soned X and Y Chrcmosorne-Bearing Rabbit 551111
`Total
`Percentage and Numbers of Offspring
`No. of
`Predicted
`Actual
`9!:
`9!:
`Young
`%
`%
`Born Males
`Fernales Ma|es('N')
`FemaIea(N)
`1]
`ll
`19
`81 (17)
`I9 (4)
`16
`I4
`36
`6 ll)
`94 (I5)
`I4
`50
`50
`43 (d)
`57 (B)
`
`I3 T —
`
`—
`
`47 (24;
`
`53 (27)
`
`Number of Docs
`lnserninnted Kindling
`16
`5
`14
`3
`I?
`5
`_
`47
`
`Treatment
`oifiperrn
`Sorted Y
`Sorted X
`llot:ombi.ned
`x and Y
`Total
`
`The phenotypic sex ratio of offspring born of dots in-
`seminated with either sorted X-bearing or sorted Y-
`bearing sperm was different
`[P<0.£K)02 for X and
`P<0.C0l for Y) from the theoretical (50:50) sex ratio
`expected from untreated semen.
`Embryonic mortality was significant in the does in-
`seminated with sorted intact sperm. With a reasonably
`high fertilization rate (Table I). one would expect a
`
`mixture was centrifuged, and only the supernatant was
`65 used. The sorted sperm were concentrated by incubat-
`ing at room temperature for 1 hr, after which the more
`dilute fraction was removed and the remainder was
`used for insemination 1 to 4 hr later.
`
`

`
`7
`
`EXAMPLE 2
`
`5,135,759
`
`Mature New Zealand White does were injected with
`ISO international units of human chorionic gonadotro-
`pin (HCG) to induce ovulation. which was expected to
`occur I0 hr later. Seven hours after treatment with
`HCG. the docs were surgically prepared by injection
`with Ketamine hydrochloride containing acepromazine
`and anesthetized under halothane and oxygen. The
`uterus was exposed by midline incision, and I00 pl of
`sorted or unsorted sperm was placed into the lumen of
`the anterior tip of each uterine horn through a 21-gauge
`needle. Standard management practices were used in
`caring for the rabbits. These does were sacrificed 4-0 hr
`post-insemination; uteri were flushed and recovered
`eggs evaluated. All fertilized eggs recovered were clas-
`sified as morula. The results of these experiments are
`shown in Table I.
`
`EXAMPLE 3
`
`Table II shows the results of inseminations made into
`the tip of the uterine horn: the number of does that
`kindled and the phenotypic sex of the offspring com-
`pared to the predicted sex. Predicted sex of offspring
`was based on reanalysis of sorted intact sperm to deter-
`mine relative DNA content. For reanalysis. the sorted
`sperm was sonicated for 10 sec and centrifuged at
`I5.0{}0 g, the supernatant was discarded, and the pellet
`was resuspended in 9 pM bisbenzimide H 33342. Pheno-
`typic sex of the offspring was determined soon after
`birth and confirmed at later ages up to 10 weeks. Re-
`eombined X and Y is the sorted X and Y sperm popula-
`tions recombined immediately before insemination.
`EXAMPLE 4
`
`Using the methods of Examples 1. 2, and 3, viable
`swine sperm was sorted into viable X and Y chromo-
`some-bearing populations. Two litters (18 pigs) from
`surgically inseminated boar semen produced 83% fe-
`males from X-sorted sperm and 67% males from Y-
`sorted sperm.
`It is understood that the foregoing detailed descrip-
`tion is given mainly by way of illustration and that
`modification and variation may be made therein with-
`out departure from the spirit and scope ofthe invention.
`1 claim:
`1. A method for sorting intact. viable. mammalian
`sperm into X- and Y-chromosome—bearing populations
`based on DNA content, the method comprising:
`in) staining intact. viable sperm collected from a male
`mammal with a fluorescent dye capable of selec-
`tively staining DNA in living cells by incubating
`the sperm with the dye at a temperature in the
`range ofabout 30'—39‘ C. for a period of time suffi-
`ciently long for staining to take place uniformly but
`sufficiently short to preserve viability of the sperm;
`b) passing the sperm into an electrically conductive
`and isotonic viability-supporting sheath fluid to
`form a suspension of sperm which are caused to
`flow singly in a stream of sheath fluid;
`c) passing the sheath fluid containing the sperm be-
`fore an excitation light source causing the stained
`DNA to fluoresce;
`_
`d} passing the sheath fluid containing the sperm
`through both a means for detecting the fluores-
`cence of the stained DNA and also a cell sorting
`means, the means for detecting fluorescence having
`at least two detectors arranged such that a first
`
`8
`detector determines the orientation of sperm on the
`basis of magnitude of fluorescence and controls a
`second detector to measure the DNA content of
`sperm on the basis of magnitude of fluorescence of
`those sperm that have been determined to be in a
`preselected orientation;
`e) selecting by said cell sorting means the sperm hav-
`ing a DNA content corresponding to a desired
`chromosome which will produce a desired gender
`of olTspring. and separating the selected sperm
`from nonselected sperm; and
`f) collecting the selected sperm in a viability-support
`ing collecting fluid.
`2. The method of claim 1, wherein said mammal is a
`rabbit.
`3. The method of claim 1, wherein said mammal is a
`swine.
`4. The method of claim 1, wherein said mammal is a
`bovine.
`5. The method of claim 1, wherein said dye is bisben-
`zimide H333-12 fluorochrorne.
`6. The method of claim 1. wherein said incubation is
`at a temperature of about 39° C. for a period of about 1
`hr.
`7. The method of claim 1. wherein said incubation is
`at a temperature of about 35" C. for a period of about 1
`hr.
`8. The method of claim 1. wherein said incubation is
`at a temperature of about 30° C. for about 1.5 hr.
`9. The method of claim 1. wherein said sheath fluid is
`phosphate-buffered saline solution.
`the solution also
`containing |J.l% bovine serum albumin to enhance
`spenrt viability.
`1|]. The method of claim 1, wherein said collecting
`fluid is modified test egg yolk extender.
`11. The method of claim 1. wherein said sperm are
`hydrodynamically oriented in the flow of sheath fluid
`prior to being passed before said light source.
`12. The method of claim 1, wherein said sperm are
`hydrodynamically oriented in the flow of sheath fluid
`by passing the fluid in a narrow stream through and out
`ofa bevelled injection tip prior to being passed before
`said light source.
`13. A method to preselect the sex of mammalian off-
`spring comprising:
`a) sorting sperm according to the method of claim 1;
`and
`
`b) inseminating a female mammal of the same species
`as the male mammal with the selected sperm in the
`collecting fluid.
`14. A method to preselect the sex of mammalian off-
`spring comprising:
`tt) sorting spenn according to the method of claim 1;
`and
`b) fertilizing an egg obtained from a female mammal
`of the same species as the male mammal with the
`selected sperm in the collecting fluid.
`15. The method of claim 1, further comprising elimi-
`nating sperm which are not properly oriented with an
`electronic gating system before sorting by said cell
`sorting means.
`16. The method of claim 1, wherein the flow of sperm
`through the cell sorting means is regulated by an ultra-
`sonic transducer.
`
`17. The method of claim 1, wherein said spenn are
`sorted on the basis of X- or Y-chromosome DNA con-
`tent with about 90% efficiency.
`
`

`
`5,135,759
`
`9
`18. The method of claim 1, wherein said sperm are
`hydrodynarnically oriented in the flow of sheath fluid
`and sperm which are not properly oriented are elimi-
`nated by an electronic gating system prior to being
`passed before said light source.
`19. A method to preselect the sex of mammalian off-
`spring comprising:
`in) staining intact, viable sperm collected from a male
`mammal with a fluorescent dye capable of selec-
`tively staining DNA in living cells by incubating to
`sperm with the dye at a temperature in the range of
`about 30°—39' C. for a period of time sufliciently
`long for staining to take place uniformly but suff-
`ciently short to preserve viability of the sperm;
`b) passing the spenn into an electrically conductive
`and isotonic viability-supporting sheath fluid to
`form a suspension of sperm which are caused to
`flow singly in a stream of sheath fluid;
`c) passing the sheath fluid containing the sperm be-
`fore an excitation light source causing the stained
`DNA to fluoresce;
`d) passing the sheath fluid containing the sperm
`through both a means for detecting the fluores-
`cence of the stained DNA and also a cell sorting
`means to measure the DNA content of the sperm
`on the basis of magnitude of fluorescence of the
`spenn;
`e) selecting by said cell sorting means the sperm hav-
`ing a DNA content corresponding to a desired
`chromosome which will produce the desired gen-
`
`10
`der of offspring, and separating the selected sperm
`from nonselected sperm; and
`1) collecting the selected sperm in a viability-support-
`ing collecting fluid.
`20. A method for preparing intact. viable. mammalian
`sperm for sorting into X- and Y-chromosome-beating
`populations based on DNA content. the method com-
`prising staining intact, viable sperm collected from a
`male mammal with a fluorescent dye capable of selec-
`tively staining DNA in living cells by incubating the
`sperm with the dye at a temperature in the range of
`about 30'—39° C. for a period of time sufficiently long
`for staining to take place unifonnly but sufficiently
`short to preserve viability of the sperm.
`2].. The method of claim 20, wherein said mammal is
`in swine.
`22. The method of claim 20, wherein said mammal is
`a bovine.
`23. The method of claim 20, wherein said dye is bis-
`benzimide I-£33342 fiuorochrorne.
`24. The method of claim 20, wherein said incubation
`is at a temperature of about 39° C. for a period of about
`1 hr.
`25. The method of claim 20. wherein said incubation
`is at a temperature of about 35‘ C. for a period of about
`I hr.
`26. The method of claim 21. wherein said incubation
`is at a temperature of about 30‘ C. for about 1.5 hr.
`I
`I
`U
`U
`I