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

• This invention relates to an improved method of preserving collected mammalian semen and separating the sperm contained therein into X and Y components so that a female of the species may be artificially inseminated with separated sperm to produce offspring of a desi red and predetermined sex .
• Th is invention is particularly useful for preserving cattle semen and separating it so that sperm bearing only X ch romosomes can be sepa rated and used to produce dairy herds and sperm bearing only Y ch romosomes can be used to produce beef herds .
• This invention also has usage in breeding horses and other animais .
• Some physical cha racteristics of sperm that are of potential use in separating sperm cells are: higher density or packing of the Y ch romosome, greater DNA content of the X ch romosome, slightly larger size and heavier weight of sperm contain i ng the X ch romosome; generally h igher velocity of movement of the Y ch romosome containing sperm; and an appa rent dense negative electrical su rface charge on the X ch romosome .
• the methods of separation of sperm utilizing these physical properties have, for the most part, been on ly partially successfu l , with separation percentages ranging f rom 60° o to 80%.
• SUBSTITUTE SHEET very great need for a method of sperm separation which is virtually 100% accu rate and which can be done very rapidly. Furthermore, in order to accomplish separation , it is necessary to maintain or get the semen which has been collected in a fluid non- coagulated condition before the separation of the sperm can be accomplished .
• a method of separating viable sperms cells according to whether they carry an X chromosome or a Y chromosome is described.
• the separated batches of cells are suitable for use in artificial insemination of humans, cows and other livestock animais so that offspring of predetermined sex are obtained.
• the method of separation depends on basic differences in X and Y sperm cells. For example, each X sperm has a greater DNA content then a corresponding Y sperm since the X chromosome has a greater DNA content than a comparable Y chromosome. Hence, it is postulated that the X sperm should incorporate greater absolute amounts of certain dyes and other labels then Y sperm. On the other hand, it is believed that the Y chromosome of Y sperm is more dense or tightly packed than a corresponding X chromosome.
• Semen which contains sperm is collected from a mammal and treated by adding an anti-coagulant, such as mammal saliva, to prevent coagulation and hasten liquefaction. Any anticoagulant that does not affect viability will work. In particular, human saliva has been found very satisfactory for this purpose and is readily available.
• the semen is then diluted with a diluting solution and stored in a water bath at substantially the body temperature of the mammal or the sperm cells are centrifuged out of the semen fluid and diluted in a suitable saline solution.
• a cell membrane diffusion material such as sperm-free human semen
• sperm-free human semen can be added to the mammalian semen so that a fluorescent dye can penetrate the cell membrane of the sperm cell and become localized around the chromosomes therein. The dye will penetrate the cell membranes of the sperm cell of primates and vole semen without prior preparation.
• SUBSTITUTE SHEET The selection of dye depends on the method of detection used to select the X and Y sperm. If a scanning procedu re is used wherein any bright spots of fluorescent dye would be detected, such as that which it is postulated characterize the Y chromosome because of its higher density than an ideal dye is quinicrine or quinicrine mustard .
• a more suitable dye has been found to be acridine, acridtne orange and derivatives thereof such as ethidium, bromide, mith ramycin or any combination thereof .
• Another suitable dye is DAPI (4, 6-diamidino-2-phenylindole) .
• the semen is put into a detection chamber.
• the semen is flowed through the chamber so that ideally only one sperm ceil at a time is irradiated with ultraviolet light.
• the light causes the dye to fluorescence.
• a detection devise in the chamber senses the degree of fluorescence and provides an output signal in response to the sensed cell which varies according to the degree of fluorescence.
• the stream then passes through a fluidic amplifier and the stream is switched in response to each sensed cel l in the detection chamber to direct the sensed cell to one of two outlet ports .
• the detection device is programmed to scanning the cell for bright spots the Y sperm will trigger the system presumably because of the higher density of the dye localized around the denser Y ch romosome. If instead the greater absolute degree of fluorescence is being detected , the X sperm will trigger the system presumably because the X sperm contains more DNA; and, hence more dye is localized a round it. I n any way, the X and Y ch romosomes can be effectively separated into separate containers for use in artificial insemination of female animals in order to produce the desi red sexed offspring .
• Semen is first collected from a mammal in any manner which is known to one skilled in the art. For example, with cattle and horses, teaser animals or electrical stimulation may be used. The various methods of collecting semen from animals is explained in conventional vetenarian reference texts.
• the collected semen is placed in a clean, sterile container and may be treated with an enzyme solution to hasten liquefaction.
• Cattle semen for example is highly viscous and will not flow in a stream as necessary for separation unless an anti-coagulant is added and a dilutant is also added.
• one of the best enzyme solutions and one of the most physiologically compatible is mammalian saliva, and particularly human saliva.
• about 1 ml of human saliva is added to each quantity of about 2.0 ml to about 3.5 ml of semen. If the quantity of human saliva added is significantly less than 1 ml, the reaction will be too slow. However, an amount larger than 1 ml will have no adverse effect, but is unnecessary.
• semen diluting solution contains egg yolk, glycerol, glucose and citrate. Preparation of these solutions is set forth in Fertility and Sterility by Berman and Sasada, published in 1966.
• One such anti-coagulant is alpha-amylase.
• the semen is then stored in a water bath at approximately the body temperature of the animal. This is between about 35°C and about 38.5°C, but is usually about 37 C. If the temperature is substantially above or below this range, sperm mobility will decrease and the survival rate of the sperm will decrease below acceptable limits. When it is time to selectively identify and separate the X and
• a cell membrane diffusion material must be added to the collected semen so that the dye can penetrate the cell. The dye will penetrate in primate and vole semen without further treatment.
• DMSO dimethylsuifoxide
• disulfomethoxide SUBSTITUTE ET membrane diffusion material which has been found satisfactory. It has been found that the addition of about 0.05 ml of DMSO to a quantity of about 2.0 ml to about 3.5 ml of semen provides sufficient penetration of the cell membrane by the dye.
• sperm-free human semen Another cell membrane diffusion material which has been found quite satisfactory is sperm-free human semen .
• the sperm - free human semen is added in a quantity of about 0. 5 ml human semen to a quantity of about 2.0 mi to about 3.5 mi of mammal semen . If either DMSO or human semen is added in quantities substantially below the indicated ranges , the diffusion of the dye will be very slow whereas if the amounts added are substantially above the indicated ranges, the DMSO and human semen will have a toxic effect and kill substantial numbers of sperm .
• the dyes which has been found most satisfactory are taken from the group consisting of quinicrine, quinicrine hydrochloride, quinicrine dihydrochloride and quinicrine mustard .
• quinicrine hydrochloride or quinicrine dihydrochloride is used , it is diluted in a sol ution of ion-free distilled water to a strength in the range of about 0.05° o to about 5.0° .
• the most satisfactory range has been found to be between about 0.5° o and about 1 .0° o in sol ution .
• quinicrine mustard When quinicrine mustard is used , it is diluted with ion- free distilled water to a solution having a strength in the range of about 0.001°o to about 0.01° o whereas the optimum range is about 0.005°o solution . If the solutions are substantially below the indicated range in concentration , the dye will ta ke too long to stain , whereas if the solutions are substantially above the indicated ranges in concentration , the dye will have a toxic effect and kill substantial numbers of the sperm . The dye solution is put in contact for a period of at least several minutes , but usually for a period of at least 15 minutes to assu re good dye penetration . Confirmation of staining is obtained by separating a small part of the specimen and checking for staining under a fluorescent microscope.
• the difference in fluorescence of X and Y chromosome bearing cells entrained in a stream of diluted semen is detected and the resultant different signals which are produced as a result of this detection are used to control a fluidic amplifier located downstream to switch the X sperm to one outlet port and the Y sperm to another outlet port so they can be separated and collected.
• the semen is fed in a very narrow stream so that the sperm cells in the semen move essentially single file past a laser light beam which is provided with an ultraviolet filter.
• the laser projects a very narrow beam in which a pattern of illumination of the beam, when it strikes the sperm cell, appears as a thin line of light transverse to the stream of cells.
• Electrical photoresponsive pick-up elements are arranged around the outside of the detection chamber through which the stream passes to detect any scattering of light due to the ultraviolet light striking a fluorescent Y chromosome.
• the excitation wavelength of the laser beam is in the range of 457 nm to 488 nm.
• the emission wavelength from the fluorescent Y sperm will be around 351 nm.
• the fluid amplifier includes a transducer which is responsive to any scattering of light due to fluorescence of a Y sperm which it is postulated will create a turbulance in the stream to cause a "wall attachment effect" as is well known in fluidics.
• any X chromosome it is postulated will pass along the stream uninterrupted and out a first outlet port whereas the Y chromosomes will be diverted due to reaction of the transducer to the scattered light causing them to be diverted and pass through a second outlet port.
• the semen can be separated into X and Y sperm and separately collected for

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• 1982
  • 1982-10-05 WO PCT/US1982/001478 patent/WO1984001265A1/en not_active Application Discontinuation
  • 1982-10-05 EP EP19830900092 patent/EP0120854A4/de not_active Withdrawn

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