US3894529A - Method and means for controlling the sex of mammalian offspring and product therefor - Google Patents

Method and means for controlling the sex of mammalian offspring and product therefor Download PDF

Info

Publication number
US3894529A
US3894529A US814906A US81490669A US3894529A US 3894529 A US3894529 A US 3894529A US 814906 A US814906 A US 814906A US 81490669 A US81490669 A US 81490669A US 3894529 A US3894529 A US 3894529A
Authority
US
United States
Prior art keywords
sperm
density
separation
medium
media
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US814906A
Other languages
English (en)
Inventor
Wallace Shrimpton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bio Controls Inc
Original Assignee
Bio Controls Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bio Controls Inc filed Critical Bio Controls Inc
Priority to US814906A priority Critical patent/US3894529A/en
Priority to ES378413A priority patent/ES378413A1/es
Priority to AT327770A priority patent/AT326402B/de
Priority to GB1702670A priority patent/GB1309781A/en
Priority to BR218158/70A priority patent/BR7018158D0/pt
Priority to TR16657A priority patent/TR16657A/xx
Priority to JP3023970A priority patent/JPS5536291B1/ja
Priority to CH534270A priority patent/CH563164A5/xx
Application granted granted Critical
Publication of US3894529A publication Critical patent/US3894529A/en
Priority to US06/179,044 priority patent/US4327177A/en
Priority to US06/179,045 priority patent/US4474875A/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes

Definitions

  • ABSTRACT A method of controlling the sex of mammalian offspring by separating spermatozoa into fractions having the desired sex characteristics and artificially inseminating the female to produce offspring of the desired sex.
  • the sperm is separated by applying a buoyant force or forces to a mixture of sperm in nutrient media so that separation occurs according to density of the sperm.
  • the nutrient media is controlled as to density characteristics and can have a uniform density gradient from top to bottom so that buoyant forces within such media are selectively applied to sperm of differing density to effect separation of the sperm and to hold sperm fractions of different density in suspended relation within the nutrient media.
  • Substantially pure sperm fractions (having the desired male or female sex characteristics) are isolated at the top or at the bottom of a separation column for use in artificially inseminating the female. Under certain circumstances, separation of the sperm into fractions is enhanced by the application of gas pressure (positive or negative) above the mixture of sperm and nutrient media in the column.
  • X-sperm When a sperm containing X chromosomes (hereinafter called X-sperm) combines with the egg (which contains X chromosomes), female offspring results.
  • Y-sperm When a sperm containing the Y chromosomes (hereinafter called Y-sperm) combines with the egg, male offspring results.
  • the sperm population in an ejaculate of a mammalian male contains both X-sperm and Y-sperm.
  • separation of these sperm in X and Y components has not been satisfactorily achieved. It is evident, however, that a satisfactory procedure for separating the two kinds of sperm, to isolate substantially pure X and Y-sperm fractions, would permit a choice or selection of the ultimate sex of the offspring.
  • This invention relates generally to a method and means for controlling the sex of mammalian offspring, and to compositions useful in providing offspring of one sex. More particularly, the invention relates to a method and means for separating spermatozoa containing X chromosomes from those containing Y chromosomes, to obtain substantially pure fractions of spermatozoa containing either X chromosomes or Y chromosomes.
  • the present invention is predicated on my discovery that the two sperm genotypes of mammals (X and Y) may be separated according to density characteristics by application of buoyant forces within a liquid separation medium to cause more buoyant sperm to attain a different level in the separation medium than less buoyant sperm.
  • buoyant force is used herein to include both positive buoyant forces which cause the sperm to rise or float in the medium and negative buoyant forces which cause the sperm to fall or sediment in the medium.
  • a separation medium or media arranged as a substantially vertical column and having a uniform density gradient from a lightest density at the top to a heaviest density at the bottom.
  • the separation medium is in the form of layers or zones of compatible liquids, each of slightly different density, to similarly provide a separation media which varies from a lightest density at the top to a heaviest density at the bottom of a column. Separation is achieved by introducing a sperm population to the separation medium at a point intermediate the ends of the column, in media equivalent in density to that of the point of introduction, so that the sperm are separated according to density by the simultaneous application of positive and negative buoyancy.
  • the velocity of rise or fall of the particle varies with the hydrodynamic efficiency which is related to the size and shape of the particle, the difference in density between the medium and the particle and the characteristics of the medium as a Newtonian solution at the temperature of separation.
  • the velocity of rise or fall in a particle class is closely related to Stokes law, particles in a liquid of uniform density tend to rise or fall in conformity with the factors outlined above.
  • the separation liquid varies in density, or has a density gradient, one can expect the velocity of rise or fall to reduce to zero at the point where the density of the particle equals the density of the liquid.
  • sperm fractions or populations at the top and bottom of the column will, when inseminated, produce offspring of different sex.
  • top and bottom fractions have been found to be related to sex genotypes, that is, the top fraction has been found to contain substantially all Y-sperm whereas the bottom fraction has been found to contain substantially all X- sperm.
  • the present invention has utility wherever it is desired to control the sex of mammalian offspring. It is of extreme practical and commercial importance in the field of animal husbandry, for example, in permitting the breeder or farmer to have a choice in selecting the sex of animal offspring.
  • the dairy farmer can elect to obtain only female offpsring from superior animals in the herd while obtaining male calves from the remainder of the herd, thereby increasing both his commercial return from the sale of calves and the genetic quality of his herd while also increasing milk production from improved cows.
  • the present invention therefore has important application and potential in solving the food problems in the hungry world by making possible:
  • a principal object of the present invention is to provide a truly successful method for controlling the sex of mammalian offspring.
  • a further object of the present invention is to provide a method for separating the X-sperm and Y-sperm in the ejaculate of a mammalian male, to obtain substantially pure fractions containing either X-sperm or Y- sperm, useful in artificial insemination of the female to obtain the desired sex.
  • a further object of the invention is to provide novel compositions containing either substantially pure X- sperm or substantially pure Y-sperm, capable of producing mammalian offspring of the desired sex.
  • a further object of the invention is to provide novel means for carrying out the sperm separation method of the present invention.
  • a further object of the invention is to provide a novel method and means of the above character which makes possible the separation of substantially pure fractions of X-sperm and Y-sperm, capable of producing normal fertilization.
  • FIG. I is a flow sheet illustrating the method of the present invention.
  • FIGS. 2, 3 and 4 are schematic representations of a particular step in the processing, at various points in time.
  • FIG. 5 is a graphical representation corresponding in point of time to the schematic representation of FIG. 4.
  • FIG. 6 is a schematic representation of one system of apparatus useful in carrying out the method of the present invention.
  • FIG. 7 is a fragmentary view in section and elevation of another embodiment of apparatus useful in carrying out the method of the present invention.
  • FIG. 8 is an enlarged detail view in section and elevation of a portion of the apparatus shown in FIG. 7.
  • FIG. 9 is a view in horizontal section along the lines 9-9 of FIG. 8.
  • FIG. I0 is a schematic representation of a further embodiment of apparatus useful in carrying out the invention.
  • FIG. 1 is a flow sheetillustrating the method of the present invention.
  • step I fresh sperm is collected from the male, containing approximately equal amounts of X-sperm and Y-sperm.
  • This sperm is mixed with a nutrient medium in step 12, following which the mixture of sperm and medium is gradually cooled in step 13.
  • the cooled mixture of sperm and medium is next subjected to processing in steps 16 and 17 to effect separation of the sperm according to density. This latter processing is carried out in the presence of a separation media which has been previously prepared to provide predetermined desired density characteristics. as in step 14.
  • the processing in step 14 provides the separation media with a uniform density gradient sufficient to span the range of individual sperm densities. Following preparation of the separation media, it is cooled to a temperature to immobilize the sperm, in step IS. The intermixed sperm and nutrient medium are also cooled in step 13 to insure substantial immobilization of the sperm, which is then introduced to the separation media in step 16. In the case of a separation media having a uniform density gradient, the sperm is introduced at an intermediate point of the density gradient. Thereafter the sperm is subjected to the buoyant forces within the separation media, in step 17, to cause separation of the sperm according to the individual sperm density.
  • the desired sperm fractions achieve a suspended state of separation in the separation media adjacent the top and bottom of the column, thereby facilitating separation of a sperm fraction of desired sex characteristics in step 18.
  • the separated sperm fraction (X-sperm and Y-sperm) can then be employed to inseminate the female, as represented by step 19, to obtain the desired offspring.
  • the present invention is suitable for use with all mammals.
  • mammals Of particular interest are cattle, swine (i.e., hogs and pigs), human beings and other primates, sheep rabbits, cats, dogs, goats, horses, donkeys and buffalo.
  • the sperm fraction separated in step 18 is inseminated in step 19 into the female of the species from which the sperm is taken.
  • horse may be crossed with ass and zebra as may wolf with dog.
  • the present invention is predicated on determination that there is a difference between the average or mean density of the X-sperm and Y-sperm of mammalian species, the X-sperm being generally more dense than the Y-sperm.
  • this difference in average density between sperm containing the X- and Y- chromosomes is believed to be about 0.5 percent at 0 C.
  • the difference appears to be greater than in bulls.
  • the medium selected for use in steps 16 and 17 must have a density sufficiently close to that of the sperm so that the slight difference in density between the X-sperm and the Y-sperm will result in the separation of at least part of the sperm into separate fractions containing predominantly X-sperm or Y- sperm.
  • density a density sufficiently close to that of the sperm so that the slight difference in density between the X-sperm and the Y-sperm will result in the separation of at least part of the sperm into separate fractions containing predominantly X-sperm or Y- sperm.
  • viscosity must also be appropriate for controlling the separation.
  • the medium selected for use in steps 16 and 17 must have a density sufficiently close to that of the sperm so that the slight difference in density between the X-sperm and the Y-sperm will result in the separation of at least part of the sperm into separate fractions containing predominantly X-sperm or Y- sperm.
  • viscosity must also be
  • the medium must not impair the viability or the fertility rate of the sperm, that is, the medium must not harm or destroy the sperm. To the contrary, the medium must provide nutrients to keep the sperm alive.
  • the medium must also have a suitable pH (i.e., within the range from 6.0 to 8.0) to permit it to act as a buffer and to avoid toxic effects or impairment to the fertility of the sperm.
  • the final consideration is that the medium should have the characteristics of a normal body fluid, and, in particular, its osmotic pressure should be within the range from about 277 to 280 milliosmos to avoid any possibility of harmful compression or expansion of the sperm.
  • a highly satisfactory nutrient medium for carrying out the separation technique of the present invention is derived from whole mammalian milk and its components.
  • Control of density for example, in preparing a separation column having a uniform density gradient, can be readily obtained through use of the components of commercially available types of cow's milk.
  • three media are initially prepared having average densities, respectfully, of about 1.025, 1.035, and 1.044.
  • the average density of the lightest media is achieved by using a milk product known as half-and-half (meaning that it contains approximately equal portions of homogenized milk and separated cream, mixed with ordinary homogenized milk).
  • half-and-half meaning that it contains approximately equal portions of homogenized milk and separated cream, mixed with ordinary homogenized milk.
  • An intermediate density (e.g., from about 1.034 to about 1.038) is similarly obtained by mixing homogenized milk with small amounts of dialyzed distilled nonfat milk.
  • a relatively dense media of average density 1.044 is prepared by mixing a larger proportion of dialyzed distilled nonfat milk with homogenized milk.
  • Antibiotics to protect the sperm introduced to the medium can also be employed.
  • Fresh mammalian milk such as cows milk and its components have a suitable viscosity, density and pH (about 6.4 to 6.8) for use in carrying out my separation process. Cows milk is also a normal body fluid, making it appropriate for use generally as a medium. Since mammalian milk is approximately isotonic with the blood of the animal from which it is drawn.
  • osmotic pressure or osmolality is also more easily adjusted to a value compatible with that of the sperm (i.e., between about 276 and 300 milliosmos).
  • a value compatible with that of the sperm i.e., between about 276 and 300 milliosmos.
  • specific desired values of density, viscosity, osmolality, and pH can easily be obtained during the operations to prepare the separation media for use in the sperm separation steps.
  • Satisfactory nutrient liquids are also derived from other mammalian sources (e.g., human milk) or derivatives of mammalian milk (e.g., milk powder).
  • Nutrient liquids based on egg yolk, dextrose, coconut cream (derived from green coconuts), tomato juice, glucose, fructose, lecithin, amino acids, living body fluids and extracts, tissue extracts (e.g., liver extract), and mixtures of these, are also satisfactory.
  • Egg yolk particularly includes glucose, fructose, and amino acids which provide nutrients and assist in fertility. When used in conjunction with glycine, desired values for density, viscosity, osmolality and pH can be obtained.
  • the glycine (preferably an aqueous solution of 2 to 5 percent glycine) serves to buffer the pH and to depress the freezing point of the egg yolk in the medium.
  • the ratio of glycine to egg yolk depends principally upon the initial density, viscosity, and osmolality of the egg yolk. The ratio can be varied, of course. as may be necessary in preparing a uniform density gradient. Since the glycine solution has a lower viscosity than the egg yolk, more will be required where a viscosity depressant is also needed. Generally, based on a 4 percent glycine solution, one part of glycine will be required for each one to four parts of egg yolk, depending on density requirements.
  • the separation method of the present invention is best carried out in conjunction with a separation media having a uniform density gradient. While various procedures are known for the preparation of density gradient columns, one particularly satisfactory procedure is carried out in connection with apparatus as generally represented at 20 in Figure.
  • apparatus as generally represented at 20 in Figure.
  • the medium of lightest average density e.g., 1.025
  • the media with an intermediate average density e.g., 1.035
  • the two containers, 2l, 22, are suspended on a pulley arrangement which functions to raise the container 22 at the same rate that it lowers the container 21.
  • the separate con tainers are suspended from the ends of a chain 23 reeved about the rotary supports 24 and the drive pul ley 25.
  • the entire unit is powered by the motor 26 through the drive take-off 27 and worm gear arrangement 28.
  • the illustrated apparatus serves to simultaneously introduce the liquids from the containers 21 and 22 to a mixing chamber 29, at controlled rates of flow determined by the hydrostatic or liquid head for the media in each container as respects the mixing chamber.
  • the flow of the less dense nutrient media in container 2] begins at a maximum and decreases to a minimum, as represented by the dotted line 30.
  • the flow of the denser nutrient medium in container 22 begins at a minimum and increases to a maximum, as represented by the dotted line 31.
  • the total flow at any time through the mixing chamber 29 remains constant.
  • the mixture discharged from the bottom of the mixing chamber 29 will therefore have a linear density gradient ranging from the density of the lightest media in container 2] to that of the intermediate media in container 22.
  • a desired intermixing of the media introduced to the mixing chamber 29 is accomplished by conventional agitation means, for example, as illustrated in FIG. 6.
  • a media having the described linear density gradient passes from the mixing chamber 29 through a surge coil 32 to a vented manifold 33, whence it is discharged in equal proportionate amounts to the bottom of the various separation columns, represented at 34.
  • a medium of relatively heavier average density e.g., 1.044
  • the heavier medium in container 35 is used to adjust the height of the density gradient columns within the separation devices 34.
  • the separation media is introduced to form substantially uniform density gradient columns within each of the separation devices 34 and thereafter is gradually cooled to a temperature which will immobilize without harming the sperm.
  • this cooling is accomplished by a circulatory refrigeration apparatus including the refrigeration unit 38, the cooling tank 39 surrounding the separations devices, and a body of cooling water 40 containing glycerol which is circulated between the refrigeration unit and a tank by the inlet and outlet conduits 4] and 42, respectively.
  • cooling of the density gradient columns within the separation devices from room temperatures to a temperature below about 1 C. is accomplished in a very short period of time, ranging from a few minutes up to a few hours, at the most.
  • sperm Prior to initiating the separation, fresh sperm is collected and intermixed with a nutrient medium of the same type and corresponding in density to an average of median density of the media forming the density gradient column (e.g.. about L028 in the system described).
  • the sperm sample is also pre-cooled to a temperature below IT. and thereafter. as illustrated in FIGS. 2 and 6, is introduced to the separation column at an intermediate point of the density gradient column.
  • the density of the sperm plus extender sample should correspond closely to the density of the separation media at the point of introduction.
  • a small amount of the nutrient medium for the sperm can be initially introduced to the separation column, preferably containing a small amount of a coloring ingredient so its relative density with respect to that of the column can be readily determined by the movement of the colored segment within the column.
  • a small amount of the heavier medium in the container 35 can be introduced through the manifold 36 and valving means 37 to the bottom of the density gradient column in question.
  • a small amount of the separation media originally present can be discharged through the stopcocks 43 positioned at the bottom of each of the separation columns 34.
  • the important consideration is that the density of the separation medium opposite the point of sperm introduction (represented at 44 in FIGS. 2 and be approximately equal to that of the nutrient medium containing the introduced sperm.
  • each of the separation columns 34 is provided with inlet valving means 45 operable by the valve controls 46.
  • the valve controls 46 are then operated to introduce the sperm through the filling lines 49 to the midpoints of the density gradient columns contained within the separation devices 34.
  • sperm populations shown in zones 51 and 52 These isolated substantially pure fractions are represented by the sperm populations shown in zones 51 and 52. As a practical matter, I have found that the sperm capable of being isolated in either of these zones generally. comprise less than lO percent of the total sperm population. On the other hand. since the ejaculate of a mammalian male normally contains many millions of sperm (e.g., a portion of the ejaculate will average 400 million sperm for a typical male bull), the number of sperm present in one or more of the isolated fractions (i.e., 20 to million sperm) is sufficiently large under normal circumstances to permit insemination and conception of offspring of the desired sex.
  • the intermixed sperm contained within the intermediate portion of the column, represented at 53 in FIG. 4, is not lost, but remains available for use by animal breeders and the like to effect normal artificial insemination wherever sex predetermination or control is not desired.
  • FIGS. 2 to 4 represent different points in time.
  • FIG. 2 illustrates the point of introducing the sperm to the columns.
  • FIG. 3 represents a point in time where the sperm are still in motion. clue to the continuing effect of the buoyant forces within the separation media (positive and negative) upon the individual sperm.
  • FIG. 4 represents a final equilibrium condition wherein the individual sperm have achieved a state of suspension within the density gradient of the separation medium, according to individual densities.
  • the equilibrium state may not be achieved and the separation of pure sperm fractions may not result.
  • the medium itself may tend to separate. That is, the inorganic ions and other heavy particles in the colloid medium may separate after long periods of time. Moreover, the particles of the medium may tend to salt out" or precipitate from the fluid if substantially more than 24 hours are used.
  • the median density of the separation medium (i.e., at the midpoint) should be close to the median density of the sperm of the mammal from which the sperm is taken.
  • the density range is about 1.01 to about 1.19 grams per cubic centimeter, with the median density falling in the range from about 1.028 to about 1.036.
  • a density gradient column for use in separating bull and human sperm should range from about 1.010 to about 1.150 grams per cubic centimeter, with a median density of the order of 1.028.
  • the median density of the density gradient column should closely approximate the average density of the sperm sample.
  • the range of densities in the density gradient column should be sufficient to insure that a full range of rise and fall of the sperm is permitted, particularly as respects the lightest Y-sperm and the heaviest X-sperm at the extreme density ranges of the sperm sample.
  • the viscosity of the separation medium should also fall within a predetermined viscosity range for satisfactory separation.
  • the viscosity of the separation medium is related to the density. For humans, rabbits and cattle, viscosity should be between 2 to 9 centipoise measured at C.
  • Table 1 sets forth the range of densities and viscosities for various species of mammals, and also indicates a medium density and viscosity for satisfactory separation in a density gradient column. All measurements taken in Table l are at 0C. with the density in grams per cc and viscosity in centipoise.
  • osmotic pressure of the medium may vary between a lower limit of about 276 and an upper limit of about 300 milliosmos, as a practical matter I have found that best results are obtained when the osmotic pressure of the medium is about 280 milliosmos.
  • a temperature between about 5C. and about 2C. is required. Below this temperature range, the physical properties of the medium are changed to such an extent that the desired rise and fall of the sperm to achieve a desired state of suspended separation will not result. At higher temperatures, the sperm tend to swim or move on their own through the medium so that the buoyant forces within the separation medium fail to separate the sperm as substantially inert particles. Accordingly. a preferred temperature for use of a density gradient column prepared from whole mammalian milk is 08C.
  • step 19 particular care is required to avoid vibration and resultant intermixing and contamination of adjacent fractions.
  • Another factor to be considered is the avoidance of visible light.
  • Light affects the fertility of the sperm. Fertility can also be affected by extremely high or low pH of the medium (outside the range 6.0 to 6.8 for most species), age of the sperm. and number of motile sperm.
  • the use of a density gradient column results in a suspended state of separation of the sperm according to sperm density.
  • the desired substantially pure sperm fractions should be immediately separated from the top or bottom of the Column and preserved for artificial insemination.
  • the Y-sperm fraction at the top of the column is done by draining the entire column, with the Y-sperm fraction being the last to be recovered.
  • the Y-sperm fraction may be initially removed from the top of the column with a spe cial apparatus for this purpose, for example, a special burette fitted with a Pasteur pipette.
  • the separation apparatus comprises a cylindrical burette or column 60, suitably provided with apertures 62 along its length to facilitate the separation of closely spaced liquid fractions.
  • Each of the apertures 62 is in communication with a separating or fractionating valve mechanism 64 which controls the discharge of a sperm fraction into the receiving vials 66 mounted on the column by means of the bottom supports 67 and clips 68.
  • each aperture 62 is closed by a spring biased valve member 70 operable by a pull rod 72.
  • the pull rod can be actuated through the pulls 74 to move outwardly, thereby discharging a sperm fraction through the discharge spout 76 into the vial 66. Since individual valving mechanisms 64 and vials 66 are spaced along the length of the column with respect to each aperture 62, the illustrated mechanism makes possible the separation of minute fractions along the entire length of the column. Alternatively, of course, a larger specimen may be withdrawn from a substantially larger portion of the column by operation of a single valving mechanism 64 at the bottom of a desired column length. The vials 66 can thus be made sufficiently large to accommodate the volume of the several fractions, as may be necessary.
  • the individual valving mechanisms 64 are constructed to be operated in automatic or semi-automatic fashion by means of the cam actuated rod and actuator mechanisms 76, 78.
  • the latter are adapted to engage between the actuators 74 of the pull rods and the body of the valve mechanisms 64, to bias the valve members 70 outwardly.
  • each of the valve actuators 76 and 78 are provided with spring biased cam followers 79 which cooperate with the rotating cams 80 and 82 at the top and bottom of the column. respectively.
  • the cam actuators can be rotated at a predetermined rate to effect a desired sequential operation of the valving mechanism 64 by suitable drive means (e.g., a variable speed electric motor) at the bottom of the column and similar means at the top (not shown).
  • suitable drive means e.g., a variable speed electric motor
  • the number and positioning of the valve actuators 78 with respect to the camming mechanisms can be predetermined to facilitate fractional separation of sperm samples from the interior of the column 60, as may be appropriate to a particular separation technique
  • FIG. 7 illustrates the mounting of the separating column 60 within a refrigeration enclosure.
  • cooling of the columns in this embodiment is accomplished through use of a dry gaseous atmosphere, for example, dry cold air at a temperature below about lC.
  • Refrigerated gas from the cooling operation may be supplied by any appropri ate means, as represented by the refrigeration chamber 86 and impeller 88, which supply refrigerated gas to the cooling chamber 90 through the conduit 92. Cooling gas discharged from the outlet 94 may be recovered for recycling or discharged to the atmosphere, depending upon the particular cooling system employed.
  • the separating column 60 is employed in the separation process in similar fashion to the processing described with respect to columns 34, the valving mechanisms 96 and 98 being the counterparts of the manifolds 33 and 36 in the apparatus of FIG. 6. As illustrated, the valving mechanisms 96, 98 and the associated conduits 100, 102 are in direct communication with the bottom opening of the columns 60 (i.e., through chamber 104).
  • the columns 60 are prepared in the previous manner by introducing a separation media of predetermined desired density characteristics. Each column is then cooled by suitable application of the refrigeration unit and circulating system 86, 88 to obtain a desired temperature of the separation media.
  • the sperm samples to be separated are likewise prepared by intermixing with a nutrient medium in a manner previously de scribed. Assuming the use of density gradient columns, the vertical position of the separation media or columns can be adjusted by the technique previously described.
  • a sperm sample after being first gradually cooled to immobilize the sperm, is then injected through the conduit 49 and valving mechanism 45 at the point of average or median density to initiate the sperm separation through action of the buoyant forces within the density gradient column.
  • the camming mechanisms for the valve operators 78 are energized to initiate withdrawal of sperm fractions.
  • the sperm fractions are withdrawn from the top of the column in descending fashion, to obtain substantially pure fractions of the lightest sperm (Y-sperm).
  • the central portion of the column 60 is then discharged in a single operation through a timed operation of a particular valving mechanism (e.g., 106 in FIG.
  • the separation processing is facilitated by use of a gas under positive pressure (i.e., 0.1 to ID psi) in the head space above the separation media in the column 60.
  • a gas under positive pressure i.e., 0.1 to ID psi
  • gaseous pressure which may be exerted by a cold dry air or an inert gas such as N or CO supplements the hydrostatic head serving to discharge the sperm fractions into the vials 66.
  • gas under pressure is particularly useful, for example, in facilitating discharge of the intermediate sperm fractions through the valving mechanism 106.
  • a milk media can be appropriately prepared to a desired density gradient by subjecting commercially available homogenized milk to the substantial centrifugal forces possible with modern ultracentrifuges.
  • Such devices which rotate at speeds in excess of 1,000 revolutions per minute, generate forces enormously greater than gravity.
  • the result of such centrifugation is to distribute the molecules in solution in the centrifuge cell in such way that the density is higher the greater the distance from the center of the rotor.
  • a milk media with a desired density gradient can be prepared in relatively short time with ordinary laboratory centrifuges, without any change in the osmolality of the milk media which remains substantially constant throughout the density gradient.
  • the range of the density gradient can also be varied by using mixtures of half-and-half and homogenized milk to vary the starting fat content. Control over the density gradient can additionally be obtained by variations in the speed and time of centrifugation.
  • FIG. 10 illustrates interconnected vessels 110 and 112 which may be simultaneously discharged by valve means 114 and I16.
  • the dense liquid contained in vessel I10 is introduced into the bottom of the vessel I12 containing the less dense liquid, where the two liquids are mixed by a simple propeller agitator H8.
  • the resulting mixture flows out of the vessel I12 through a tube I22 that leads to the bottom of the density gradient column being formed in the column 120.
  • the processing of the present invention makes possible the separation of sperm fractions of varying degrees of purity, that is, fractions containing varying amounts of either X-sperm or Y-sperm. As represented in FIG.
  • the zone 52 in FIG. 4 contains substantially pure Y-sperm.
  • the fraction contained in zone 51 contains substantially pure X-sperm.
  • Fractions effective to carry out the purposes of the present invention should have at least 70 to 80 percent of either the X-sperm or the Y-sperm (according to the selection) to overcome the -50 ratio existing in nature.
  • at least to percent of the sperm should be of the desired sex, if the expense involved in carrying out the method is not to be prohibitive.
  • each of the fractions represented by the sperm in zones 51 and 52 comprises essentially sperm having only the desired chromosomes in a carrier.
  • one suitable procedure involves means for measuring density, such as a plurality of containers containing solutions of known varying density (e.g., CuSO into which droplets of media may be introduced to determine density.
  • a plurality of small hydrometers might be placed in the separation column to determine different density levels within the column, this latter embodiment would avoid the necessity of taking small fractions and testing each one.
  • determination of the density in vari- LII ous zones within the separation column may be determined without draining by using monochromatic light or radiant energy of various frequencies that measure the opacity of the mixture.
  • a light source may be placed on one side of the column and a photoelectric cell on the other side (connected to an amplifier and a recorder) to measure the opacity of the mixture to determine where the separated layers of sperm are located.
  • Location of desired density layers or the desired fractionating point between the desired layers may also be determined by measuring the conductivity at various points in the column.
  • multiple electrodes are placed in the column to determine the change in conductivity of the contents of the column at various points along its length.
  • the conductivity of the separated sperm fractions is different from that of the supporting medium.
  • determining the location of the separated sperm fractions include a micro-densitometer, which measures density using polarized light, and mi croscope techniques which measure the scattering of polarized light.
  • a gelatin tube may replace the burette forming the separation column.
  • the tube and its contents are gradually cooled to a temperature well below freezing, for example 20C., and the separated sperm fractions frozen in situ.
  • the frozen tube may then be cut into appropriate fractions as desired. Storage of the separated sperm fractions for relatively long periods is then possible.
  • the apparent density of the sperm being separated within the density gradient column is decreased, with the apparent density of the Y-sperm at the top end of the tube decreasing to a greater degree than the apparent density of X-sperm at the bottom end of the tube. While the physiological factors underlying this decrease in apparent density is not clearly understood, it is postulated that the spermatozoa have an elastic exterior which is therefore responsive to the negative pressure in the space above the column. By way of illustration, the individual spermatozoa may be compared in the initial state to tiny balloons carrying tiny weights so that the resultant effect of the buoyant forces on the sperm is close to zero (i.e., neither positive nor negative).
  • the sperm will tend to move up and down in the media in response to any change in the gas pressure above the media surface.
  • the explanation for this phenomenon is that the separation media is relatively incompressible and readily transmits to the compressible sperm the force of the changing gas pressure, thus causing variations in the volume of the sperm and consequently its apparent density. Consequently, in circumstances where the positive buoyancy is less than desired. so that the Y-sperm do not rise as rapidly as desired in the upper portion of the density gradient column, imposing a negative pressure above the column serves to increase the positive buoyancy and thereby the separation effects of the separation media upon the Y-sperm. In practice.
  • separation efficiencies can be increased as much as 30 to 50 percent by application of a vacuum of about 10 to 20 inches of mercury in the head space above the density gradient column.
  • the normal procedure is to apply the vacuum gradually, for example. reaching a vacuum equivalent to 15 inches of mercury in a period of about minutes. Best results are obtained when the vacuum pump is energized shortly after the introduction of the sperm sample to the separation column, with the vacuum being drawn continuously until the end of the separation process. To protect the separated sperm. the vacuum is reduced to zero over a period of at least two minutes. prior to separation of the desired sperm fractions.
  • a separation media in the form ofa density gradient column can be prepared from commercially available cows milk and its components by ini tially heating to 90F. cooling to room temperature (e.g., 72F), filtering all the starting materials (i.e., homogenized commercial milk. half-and-half. distilled nonfat and low-fat milk) through sterilized glass wool at approximately room temperature. After cooling, each component can be treated with suitable antibiotics (e.g.. potassium. penicillin g. and streptomycin sulfate solution).
  • suitable antibiotics e.g. potassium. penicillin g. and streptomycin sulfate solution.
  • the value of density can be increased without substantial change or alteration in the osmotic pressure by dialysis to remove various organic and inorganic salts and without reduction in protein content or other impairment of the milk.
  • the milk is redistilled with vacuum to remove the excess water. thereby achieving the higher density. 1
  • dialysis in laboratory dialysis equipment e.g., Oxford Multiple Dialyser
  • redistillation at 58C. for about 3 to 10 hours will produce the desired effect.
  • the following are representative of typical densities and osmotic pressures ob tained with the indicated starting materials. after preparation:
  • sperm for separation To prepare sperm for separation, freshly collected sperm are mixed with a mixture of homogenized milk and half-and-half of known density (e.g., 1.0295) in proportions to provide a population of about 200 to 300 million sperm per cc of media, the total sample providing at least 400 to 600 million sperm for the separation processing.
  • the intermixed sperm and nutrient media are then placed in a cold room and gradually cooled to a temperature close to 0C. (i.e.. 08C.).
  • the sperm sample is introduced to the midpoint" of the density gradient column by first determining the location within the column of the zone of average or median density corresponding to that of the sperm sample undergoing separation (e.g., 1.028). This point is easily located by injecting a small amount of color into a separate portion of the medium of density, say, 1.028, and introducing this small amount of colored medium through the inlet valve 45 to a central portion of the column. Thereafter the 1.028 density strata (as determined by the color) is vertically adjusted within the column to a point opposite the inlet valve 45 (FIG. 6).
  • the density gradient column Prior to initiating separation, the density gradient column is cooled to the desired separation temperature (0.8C.) and the sperm sample introduced through the filling spout 48 and tube 49 to the midpoint of the density gradient column. Separation of the sperm according to density thereafter occurs through the buoyant effects of the density gradient medium, causing the less dense sperm to rise and the more dense sperm to fall within the density gradient column. After a sufficient period of time to achieve equilibrium (e.g., about 2% hours), a sperm fraction is separated from the top or bottom of the column (according to selection) to obtain nutrient media containing a sperm fraction comprising from 4 to 8 percent of the total sperm population in the sample introduced.
  • the desired separation temperature 0.8C.
  • the sperm fraction is separated from the bottom of the column in any of the manners previously described. Where Y-sperm are desired. the sperm fraction is similarly separated from the top of the column. In practice, the separated fraction is counted for the number of sperm present therein, and the number plotted against the fraction.
  • Sperm samples containing approximately 480 million sperm were introduced to the individual separation columns through the side tubes 49 and valve mechanisms 45, using a hypodermic syringe 47 cooled to a sperm immobilizing temperature of 0.8C.
  • Sperm was introduced to the column at a relatively slow rate, not exceeding about 1 cc per minute. To insure that all the sperm were introduced to the columns. a small amount of nutrient medium was additionally pushed through the side tubes 49 thereby clearing the side tubes of sperm. Following the introduction of the sperm. which was done under infra-red light conditions. the refrigeration tank and separation columns were covered to exclude light during the separation processing.
  • sperm fractions were removed from the top of the columns by means of pipettes equipped with water pumps (hereinbefore described) following which the more dense sperm fraction was removed from the bottoms of the column. (as represented at 34), through the stopcocks 43 at the bottom of the columns.
  • the portions removed from the top and bottom of density gradient columns were maintained at a volume of approximately cc intermixed sperm fractions were also removed from central portions of the column. All of the sperm samples were then checked for sperm count. using a standard procedure with a haemacytometer.
  • the separated fractions of relatively pure sperm were subsequently mixed with a small quantity (0.l65 cc) of percent glycerol in homogenized milk. and the mixture held 15 minutes at 1 to 8C. After a holding period of approximately 15 minutes, an additional amount of glycerol (0.165 cc) was added and the mixture held at 1 to 8C. for an additional 15 minute period, following which a third addition of glycerol (0.165cc) was added to the mixture.
  • Samples were selected for insemination from the sperm population at the bottom of the tube representing less than 4 /2 percent of the total population. Collected samples of sperm were then deep frozen using standard procedures as normally practiced in the artificial insemination industry.
  • sperm samples prepared as above were removed from cold storage and used in artificial insemination of female mammals.
  • cows found to be in heat were inseminated with l to 2 ccs containing at least 10 to million sperm (before freezing), again using procedures standard in the artificial insemination industry.
  • Sperm purity as determined by laboratory testing was in excess of 70 percent with the procedure described.
  • At least part of said separation medium being substantially equivalent in density to the density of said mixture and having a uniform density gradient extending from a lightest density at the top to a heaviest density at the bottom; applying buoyant forces to the sperm introduced to said separation medium tending to separate the sperm at levels ofsuspension within the separation medium according to individual sperm density; separating a portion of the separation medium of known density containing a suspended sperm fraction of equivalent density and desired predetermined sex characteristics; and artificially inseminating a female with the separated sperm fraction whereby conception occurs to produce offspring of the desired sex.
  • said separation media contains at least one member of the group consisting of whole mammalian milk, cream, nonfat milk, egg yolk, dextrose, coconut cream, tomato juice, glucose, fructose, sugar alcohols, lecithin, amino acids, living body fluids and tissue extracts.
  • said sperm is a member of the group consisting of primates, cattles, pigs. sheep, rabbit, buffalo, goat and horse sperm.
  • a method of controlling the sex of mammalian offspring the steps of mixing fresh sperm with a nutrient medium; cooling the mixture of sperm and medium to a low temperature to immobilize the sperm; introducing the cooled mixture of sperm and medium to a separation medium in the form of a separate body of nutrient medium, at least part of said separation medium being substantially equivalent in density to the density of said mixture and wherein the separation medium is subjected to a positive gas pressure to thereby increase the apparent density of the sperm relative to the separation media; applying buoyant forces to the sperm introduced to said separation medium tending to separate the sperm at levels of suspension within the separation medium according to individual sperm density', separating a portion of the separation medium of known density containing a suspended sperm fraction of equivalent density and desired predetermined sex characteristics; and artificially inseminating a female with the separated sperm fraction whereby conception occurs to produce offspring of the desired sex.
  • a separation media from nutrient liquids, said separation media having predetermined density characteristics; cooling the separation media to a temperature between about 5 C. to about +2 C.; independently collecting fresh sperm and mixing the same with a nutrient medium of the same general type as said separation media; gradually lowering the temperature of the resultant mixture of sperm and nutrient medium to a temperature between about 5C.
  • said separation media comprises a mixture of at least two of said nutrient liquids, said mixture of nutrient liquids being characterized by a uniform density gradient extending from a lightest density of at least 1.010 grams per cc to a heaviest density no greater than about 1.150 grams per cc, measured at 0C, continuing the application of said buoyant forces to the sperm in said separation media until such time as the sperm achieves a state of relative suspension in said separation media; separating a portion of said separation media containing a suspended sperm fraction of equivalent density and desired predetermined X- or Y- sperm characteristics; and then inseminating a female with the separated sperm fraction to achieve conception and offspring of the desired sex.
  • the nutrient liquids employed in preparing said separation media comprise at least one member of the group consisting of whole mammalian milk, cream, nonfat milk, egg yolk, dextrose, coconut cream, tomato juice, glucose, fructose, sugar alcohols, lecithin, amino acids, living body fluids, tissue extracts, and mixtures thereof.
  • a separation media comprising a density controlled mixture of nutrient liquids at least two of which are selected from the group consisting of whole mammalian milk, cream,

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Cell Biology (AREA)
  • Reproductive Health (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Dairy Products (AREA)
US814906A 1969-04-10 1969-04-10 Method and means for controlling the sex of mammalian offspring and product therefor Expired - Lifetime US3894529A (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
US814906A US3894529A (en) 1969-04-10 1969-04-10 Method and means for controlling the sex of mammalian offspring and product therefor
AT327770A AT326402B (de) 1969-04-10 1970-04-09 Verfahren und vorrichtung zum fraktionieren von sperma
GB1702670A GB1309781A (en) 1969-04-10 1970-04-09 Method for fractionating sperm
ES378413A ES378413A1 (es) 1969-04-10 1970-04-09 Metodo y aparato para regular el sexo de la descendencia enlos mamiferos mediante fraccionamiento del esperma.
BR218158/70A BR7018158D0 (pt) 1969-04-10 1970-04-10 Processo para a separacao de espermatozoides de mamiferos composicoes espermatozoidicas por ele obtidas bem como aparelho usado neste processo
TR16657A TR16657A (tr) 1969-04-10 1970-04-10 Memelilerin yavrularinin cinsiyetinin kontrolune mahsus usul ve tertibat
JP3023970A JPS5536291B1 (fr) 1969-04-10 1970-04-10
CH534270A CH563164A5 (fr) 1969-04-10 1970-04-10
US06/179,044 US4327177A (en) 1969-04-10 1980-08-18 Method and means for controlling the sex of mammalian offspring and product therefor
US06/179,045 US4474875A (en) 1969-04-10 1980-08-18 Method and means for controlling the sex of mammalian offspring and product therefor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US814906A US3894529A (en) 1969-04-10 1969-04-10 Method and means for controlling the sex of mammalian offspring and product therefor

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US05564807 Division 1975-04-03

Publications (1)

Publication Number Publication Date
US3894529A true US3894529A (en) 1975-07-15

Family

ID=25216316

Family Applications (1)

Application Number Title Priority Date Filing Date
US814906A Expired - Lifetime US3894529A (en) 1969-04-10 1969-04-10 Method and means for controlling the sex of mammalian offspring and product therefor

Country Status (8)

Country Link
US (1) US3894529A (fr)
JP (1) JPS5536291B1 (fr)
AT (1) AT326402B (fr)
BR (1) BR7018158D0 (fr)
CH (1) CH563164A5 (fr)
ES (1) ES378413A1 (fr)
GB (1) GB1309781A (fr)
TR (1) TR16657A (fr)

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4067965A (en) * 1974-11-22 1978-01-10 Bhattacharya Bhairab C Thermal convection counter streaming sedimentation method for controlling the sex of mammalian offspring
US4092229A (en) * 1975-12-17 1978-05-30 Bhattacharya Bhairab C Thermal convection counter streaming sedimentation and forced convection galvanization method for controlling the sex of mammalian offspring
US4605558A (en) * 1984-04-20 1986-08-12 Wallace Shrimpton Process for cell separation
USRE32350E (en) * 1974-11-22 1987-02-10 Bhairab C. Bhattacharya Thermal convection counter streaming sedimentation and forced convection galvanization method for controlling the sex of mammalian offspring
US4999283A (en) * 1986-01-10 1991-03-12 University Of Kentucky Research Foundation Method for x and y spermatozoa separation
US5660997A (en) * 1987-04-08 1997-08-26 Cytogam, Inc. Methods for determining antibodies specific for sex associated sperm membrane proteins
US5908380A (en) * 1998-01-12 1999-06-01 Zavos; Panayiotis M. Compartmentalized Zavos sperm swim-up column
WO1999033956A1 (fr) 1997-12-31 1999-07-08 Xy, Inc. Insemination de mammiferes specifique au sexe, avec nombre reduit de cellules spermatiques
US6149867A (en) * 1997-12-31 2000-11-21 Xy, Inc. Sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US20040132001A1 (en) * 2000-11-29 2004-07-08 Seidel George E System for in-vitro fertilization with spermatozoa separated into x-chromosome and y-chromosome bearing populations
WO2004072220A2 (fr) * 2003-02-17 2004-08-26 Fundacão De Amparo A Pesquisa Do Estado De São Paulo Procede de selection du sexe d'un spermatozoide mammaire et procede de controle de la qualite de doses de sperme congele avec sexe
US20050130115A1 (en) * 2003-10-31 2005-06-16 Abs Global,Inc. Method for altering the gender ratio of offspring in mammals by manipulation of spermatozoa
US7169548B2 (en) 2002-09-13 2007-01-30 Xy, Inc. Sperm cell processing and preservation systems
EP1769067A2 (fr) * 2004-07-22 2007-04-04 Monsanto Technology LLC Procede pour enrichir une population de spermatozoides
US7208265B1 (en) 1999-11-24 2007-04-24 Xy, Inc. Method of cryopreserving selected sperm cells
US7221453B2 (en) 1997-01-31 2007-05-22 Xy, Inc. Optical apparatus
EP1917974A1 (fr) 1998-07-30 2008-05-07 XY, Inc. Système d'insémination équine artificielle non chirurgicale
US7371517B2 (en) 2000-05-09 2008-05-13 Xy, Inc. High purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
US7618770B2 (en) 2005-07-29 2009-11-17 Xy, Inc. Methods and apparatus for reducing protein content in sperm cell extenders
US7713687B2 (en) 2000-11-29 2010-05-11 Xy, Inc. System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations
US7723116B2 (en) 2003-05-15 2010-05-25 Xy, Inc. Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
US7758811B2 (en) 2003-03-28 2010-07-20 Inguran, Llc System for analyzing particles using multiple flow cytometry units
US7838210B2 (en) 2004-03-29 2010-11-23 Inguran, LLC. Sperm suspensions for sorting into X or Y chromosome-bearing enriched populations
US7855078B2 (en) 2002-08-15 2010-12-21 Xy, Llc High resolution flow cytometer
US8211629B2 (en) 2002-08-01 2012-07-03 Xy, Llc Low pressure sperm cell separation system
US8486618B2 (en) 2002-08-01 2013-07-16 Xy, Llc Heterogeneous inseminate system
CN102643780B (zh) * 2004-07-22 2015-09-02 英格朗公司 富集精细胞群的方法
US9879222B2 (en) 2007-12-14 2018-01-30 Mofa Group Llc Gender-specific separation of sperm cells and embryos
WO2019043656A1 (fr) 2017-09-01 2019-03-07 Genus Plc Procédés et systèmes d'évaluation et/ou de quantification de populations de spermatozoïdes à asymétrie sexuelle

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NO782997L (no) * 1978-09-01 1980-03-04 Kaare Fossum R fremgangsmaate og middel for aa oeke fertiliteten hos husdy
US4326026A (en) * 1980-06-02 1982-04-20 The Regents Of The University Of California Method for fractionating cells
WO2009002849A2 (fr) * 2007-06-22 2008-12-31 Millennium Medical Technologies, Inc. Concentrateur de fluide, fluides corporels concentrés autologues, et utilisation de ceux-ci

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3185623A (en) * 1960-05-31 1965-05-25 Smith Fred Preservation of animal semen

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3185623A (en) * 1960-05-31 1965-05-25 Smith Fred Preservation of animal semen

Cited By (68)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE32350E (en) * 1974-11-22 1987-02-10 Bhairab C. Bhattacharya Thermal convection counter streaming sedimentation and forced convection galvanization method for controlling the sex of mammalian offspring
US4067965A (en) * 1974-11-22 1978-01-10 Bhattacharya Bhairab C Thermal convection counter streaming sedimentation method for controlling the sex of mammalian offspring
US4092229A (en) * 1975-12-17 1978-05-30 Bhattacharya Bhairab C Thermal convection counter streaming sedimentation and forced convection galvanization method for controlling the sex of mammalian offspring
US4605558A (en) * 1984-04-20 1986-08-12 Wallace Shrimpton Process for cell separation
US4999283A (en) * 1986-01-10 1991-03-12 University Of Kentucky Research Foundation Method for x and y spermatozoa separation
US5660997A (en) * 1987-04-08 1997-08-26 Cytogam, Inc. Methods for determining antibodies specific for sex associated sperm membrane proteins
US7221453B2 (en) 1997-01-31 2007-05-22 Xy, Inc. Optical apparatus
US7929137B2 (en) 1997-01-31 2011-04-19 Xy, Llc Optical apparatus
US7586604B2 (en) 1997-01-31 2009-09-08 Xy, Inc. Optical apparatus
US6524860B1 (en) 1997-12-31 2003-02-25 Xy, Inc. Methods for improving sheath fluids and collection systems for sex-specific cytometer sorting of sperm
WO1999033956A1 (fr) 1997-12-31 1999-07-08 Xy, Inc. Insemination de mammiferes specifique au sexe, avec nombre reduit de cellules spermatiques
EP2341130A2 (fr) 1997-12-31 2011-07-06 Xy, Llc Insémination de mammifères spécifique au sexe, avec nombre reduit de cellules spermatique
US9365822B2 (en) 1997-12-31 2016-06-14 Xy, Llc System and method for sorting cells
US9422523B2 (en) 1997-12-31 2016-08-23 Xy, Llc System and method for sorting cells
US7629113B2 (en) 1997-12-31 2009-12-08 Xy, Inc Multiple sexed embryo production system for bovine mammals
US6149867A (en) * 1997-12-31 2000-11-21 Xy, Inc. Sheath fluids and collection systems for sex-specific cytometer sorting of sperm
US6372422B1 (en) 1997-12-31 2002-04-16 Colorado State University Through Its Agent Colorado State University Research Foundation And Xy, Inc. Multiple sexed embryo production system for mammals
US7195920B2 (en) 1997-12-31 2007-03-27 Xy, Inc. Collection systems for cytometer sorting of sperm
US5908380A (en) * 1998-01-12 1999-06-01 Zavos; Panayiotis M. Compartmentalized Zavos sperm swim-up column
US7772005B1 (en) 1998-07-30 2010-08-10 Xy, Llc Method of establishing an equine artificial insemination sample
EP1917974A1 (fr) 1998-07-30 2008-05-07 XY, Inc. Système d'insémination équine artificielle non chirurgicale
EP2283848A1 (fr) 1998-07-30 2011-02-16 Xy, Llc Système équin d'insémination artificielle non chirurgicale
US7208265B1 (en) 1999-11-24 2007-04-24 Xy, Inc. Method of cryopreserving selected sperm cells
US7820425B2 (en) 1999-11-24 2010-10-26 Xy, Llc Method of cryopreserving selected sperm cells
US10208345B2 (en) 2000-05-09 2019-02-19 Xy, Llc Method for producing high purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
US7371517B2 (en) 2000-05-09 2008-05-13 Xy, Inc. High purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
US9145590B2 (en) 2000-05-09 2015-09-29 Xy, Llc Methods and apparatus for high purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
US7094527B2 (en) 2000-11-29 2006-08-22 Xy, Inc. System for in-vitro fertilization with spermatozoa separated into X-chromosome and Y-chromosome bearing populations
US8137967B2 (en) 2000-11-29 2012-03-20 Xy, Llc In-vitro fertilization systems with spermatozoa separated into X-chromosome and Y-chromosome bearing populations
US20040132001A1 (en) * 2000-11-29 2004-07-08 Seidel George E System for in-vitro fertilization with spermatozoa separated into x-chromosome and y-chromosome bearing populations
US7713687B2 (en) 2000-11-29 2010-05-11 Xy, Inc. System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations
US7771921B2 (en) 2000-11-29 2010-08-10 Xy, Llc Separation systems of frozen-thawed spermatozoa into X-chromosome bearing and Y-chromosome bearing populations
US8652769B2 (en) 2000-11-29 2014-02-18 Xy, Llc Methods for separating frozen-thawed spermatozoa into X-chromosome bearing and Y-chromosome bearing populations
US9879221B2 (en) 2000-11-29 2018-01-30 Xy, Llc Method of in-vitro fertilization with spermatozoa separated into X-chromosome and Y-chromosome bearing populations
US8497063B2 (en) 2002-08-01 2013-07-30 Xy, Llc Sex selected equine embryo production system
US8486618B2 (en) 2002-08-01 2013-07-16 Xy, Llc Heterogeneous inseminate system
US8211629B2 (en) 2002-08-01 2012-07-03 Xy, Llc Low pressure sperm cell separation system
US7855078B2 (en) 2002-08-15 2010-12-21 Xy, Llc High resolution flow cytometer
US11261424B2 (en) 2002-09-13 2022-03-01 Xy, Llc Sperm cell processing systems
US11230695B2 (en) 2002-09-13 2022-01-25 Xy, Llc Sperm cell processing and preservation systems
US7169548B2 (en) 2002-09-13 2007-01-30 Xy, Inc. Sperm cell processing and preservation systems
WO2004072220A3 (fr) * 2003-02-17 2004-10-07 Fundacao De Amparo A Pesquisa Procede de selection du sexe d'un spermatozoide mammaire et procede de controle de la qualite de doses de sperme congele avec sexe
WO2004072220A2 (fr) * 2003-02-17 2004-08-26 Fundacão De Amparo A Pesquisa Do Estado De São Paulo Procede de selection du sexe d'un spermatozoide mammaire et procede de controle de la qualite de doses de sperme congele avec sexe
US8748183B2 (en) 2003-03-28 2014-06-10 Inguran, Llc Method and apparatus for calibrating a flow cytometer
US9377390B2 (en) 2003-03-28 2016-06-28 Inguran, Llc Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
US11718826B2 (en) 2003-03-28 2023-08-08 Inguran, Llc System and method for sorting particles
US11104880B2 (en) 2003-03-28 2021-08-31 Inguran, Llc Photo-damage system for sorting particles
US10100278B2 (en) 2003-03-28 2018-10-16 Inguran, Llc Multi-channel system and methods for sorting particles
US8664006B2 (en) 2003-03-28 2014-03-04 Inguran, Llc Flow cytometer apparatus and method
US8709817B2 (en) 2003-03-28 2014-04-29 Inguran, Llc Systems and methods for sorting particles
US8709825B2 (en) 2003-03-28 2014-04-29 Inguran, Llc Flow cytometer method and apparatus
US7943384B2 (en) 2003-03-28 2011-05-17 Inguran Llc Apparatus and methods for sorting particles
US9040304B2 (en) 2003-03-28 2015-05-26 Inguran, Llc Multi-channel system and methods for sorting particles
US7758811B2 (en) 2003-03-28 2010-07-20 Inguran, Llc System for analyzing particles using multiple flow cytometry units
US7799569B2 (en) 2003-03-28 2010-09-21 Inguran, Llc Process for evaluating staining conditions of cells for sorting
US7723116B2 (en) 2003-05-15 2010-05-25 Xy, Inc. Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
US20050130115A1 (en) * 2003-10-31 2005-06-16 Abs Global,Inc. Method for altering the gender ratio of offspring in mammals by manipulation of spermatozoa
US7838210B2 (en) 2004-03-29 2010-11-23 Inguran, LLC. Sperm suspensions for sorting into X or Y chromosome-bearing enriched populations
US7892725B2 (en) 2004-03-29 2011-02-22 Inguran, Llc Process for storing a sperm dispersion
EP2269617A1 (fr) * 2004-07-22 2011-01-05 Inguran, LLC Suspensions de sperme pour trier des populations enrichies contenant les chromosomes X ou Y
US20080039680A1 (en) * 2004-07-22 2008-02-14 Monsanto Technology Llc Process for Enriching a Population of Sperm Cells
CN102643780B (zh) * 2004-07-22 2015-09-02 英格朗公司 富集精细胞群的方法
EP1769067A2 (fr) * 2004-07-22 2007-04-04 Monsanto Technology LLC Procede pour enrichir une population de spermatozoides
US7833147B2 (en) * 2004-07-22 2010-11-16 Inguran, LLC. Process for enriching a population of sperm cells
US7618770B2 (en) 2005-07-29 2009-11-17 Xy, Inc. Methods and apparatus for reducing protein content in sperm cell extenders
US9879222B2 (en) 2007-12-14 2018-01-30 Mofa Group Llc Gender-specific separation of sperm cells and embryos
WO2019043656A1 (fr) 2017-09-01 2019-03-07 Genus Plc Procédés et systèmes d'évaluation et/ou de quantification de populations de spermatozoïdes à asymétrie sexuelle
US10961577B2 (en) 2017-09-01 2021-03-30 Genus Plc Methods and systems for assessing and/or quantifying sperm cell subpopulations bearing a specific genetic signature

Also Published As

Publication number Publication date
TR16657A (tr) 1973-03-01
ATA327770A (de) 1975-02-15
AT326402B (de) 1975-12-10
BR7018158D0 (pt) 1973-04-12
GB1309781A (en) 1973-03-14
CH563164A5 (fr) 1975-06-30
JPS5536291B1 (fr) 1980-09-19
ES378413A1 (es) 1973-08-16

Similar Documents

Publication Publication Date Title
US3894529A (en) Method and means for controlling the sex of mammalian offspring and product therefor
US4327177A (en) Method and means for controlling the sex of mammalian offspring and product therefor
US4474875A (en) Method and means for controlling the sex of mammalian offspring and product therefor
US4155831A (en) Thermal convection counter streaming sedimentation and forced convection galvanization method and apparatus for controlling the sex of mammalian offspring
US3976197A (en) Thermal convection counter streaming sedimentation method and apparatus for controlling the sex of mammalian offspring
Evers The milkfat globule membrane—compositional and structural changes post secretion by the mammary secretory cell
Lake et al. Preservation of fowl semen in liquid nitrogen: application to breeding programmes
USRE32350E (en) Thermal convection counter streaming sedimentation and forced convection galvanization method for controlling the sex of mammalian offspring
EP0159044A2 (fr) Procédé de séparation du sperme
Cristanelli et al. Effects of egg yolk and glycerol levels in lactose-EDTA-egg yolk extender and of freezing rate on the motility of frozen-thawed stallion spermatozoa
New et al. Opossum fetuses grown in culture
US3692897A (en) Immunological method and composition for controlling the sex of mammalian offspring
Baracaldo et al. Steps for freezing bovine semen: from semen collection to the liquid nitrogen tank
CA1069071A (fr) Methode de determination du sexe des descendants chez les mammiferes
Whittlestone 507. Variations in the fat content of milk throughout the milking process
Hervonen et al. The effect of hypoxia on the catecholamine content of human fetal abdominal paraganglia and adrenal medulla
Heinemann Milk
US5976389A (en) Method of semen filtration
WO2009123889A1 (fr) Dispositif pour optimiser la qualité du sperme
Wunder Gravitational Aspects of Growth as Demonstrated by Continual Gentrifugation of the Common Fruit Fly Larvae.
CA1088025A (fr) Methode de manipulation des sexes de la progeniture des mammiferes et appareil servant a cette fin
US3709361A (en) Device for separating living cells
Juma et al. Cocunut Water And OPTIXcell For Alpine Goat Semen Extension: Comparative Evaluation Of Pot-extension Semen Parameter With And Without Seminal Plasma
RU226773U1 (ru) Устройство для моделирования микрогравитации на мышах
EP0446509A1 (fr) Colonne filtrante pour le sperme