JPS6155975B2 - - Google Patents

Description

[Detailed description of the invention]

In order to control the sex of mammalian offspring, the present invention has developed an X
The present invention relates to a method and apparatus for separating chromosomal spermatozoa from Y-chromosomal spermatozoa that have other densities and potentials and generate male sex. The sex of the child is controlled by the chromosomes of the specific sperm cell that fertilizes the egg. Sperm with the X chromosome, which is responsible for female sex, are somewhat more concentrated than sperm with the Y chromosome, which is responsible for male sex. In addition,
It was determined that spermatozoa carrying an X chromosome and spermatozoa carrying a Y chromosome have different cell surface potentials. These differences in density and potential separate sperm into fractions that essentially contain either sperm with an X chromosome (hereinafter referred to as X sperm) or sperm with a Y chromosome (hereinafter referred to as Y sperm). It becomes possible to do so. Separation techniques using these density and potential differences can be applied to all mammals including humans and other animals, such as cows, pigs, sheep, rabbits, cats, dogs, goats, horses, donkeys, and water buffaloes. According to previously practiced methods, separation by density imparts buoyancy to the sperm, allowing more buoyant sperm to reach a different level in the separation medium than less buoyant sperm. The difference in potential causes Conventionally, it has been difficult to separate sperm cells from Y sperm cells.
Although a variety of electrophoresis cells have been used at potentials of 200 mV to 10 VDC to separate male and female sperm, the results are particularly poor when the separated cell concentration is too low to achieve a successful pregnancy. It was a disappointment. It was found that using higher currents decreased sperm viability while increasing separation purity. These problems have been substantially solved in the present invention. In the present invention, the sperm fraction containing substantially unequal populations of X and Y cells obtained in the first separation by countercurrent precipitation of thermal convection is transferred to a convection column held between the electrodes of an electrophoresis cell. By placing cells inside the electrode, cells of different charges are selectively attracted to their respective electrodes more efficiently than in the past. In the past, the presence of foreign particles in the medium interfered with the buoyancy or sedimentation velocity of the spermatozoa and their movement by direct current power, in addition to their ability to fertilize after separation. The medium disclosed in US Pat. No. 3,816,249 virtually eliminates this problem and also promotes cell hyperactivity control and extends sperm lifespan. The use of this medium prevents small differences in the density and surface potential of male and female spermatozoa from neutralizing the high metabolic activity of sperm cells, as well as cold addition that renders spermatozoa immobile. In the present invention, galvanic force is
used in combination with positive and negative buoyancy. The galvanic force referred to here is a biochemical term indicating a unidirectional force for moving charges obtained by direct current. In the first part of the method, the medium is kept at low temperature under the influence of countercurrent heat convection in a vertical settling column, and the sperm cells are separated according to their density. After this separation step, the Y chromosome and
The light and heavy fractions, each containing an unbalanced population of spermatozoa dominated by chromosomal spermatozoa, are treated separately in a convective galvanization device. Each fraction is injected into the central convection column between the two side tubes of the electrophoresis cell with positive and negative electrodes. While applying a direct current potential to the sperm cells, the sperm are circulated in the column similar to the circulation previously achieved with countercurrent thermal convection. Controlled temperature, ionic strength and PH conditions of the medium, and convective circulation combined with voltage
Separation of spermatozoa is achieved with a purity of 92% or higher. This is because when the female sperm obtained by the method of this invention was injected into cows and 9 of the cows were killed after a gestation period of 50 to 60 days, all of them had female fetuses. Estimated (U.S. Patent No.
3976197, page 8, lines 22-26 and US Feature No. 4067965
(See page 8, lines 22-26). According to the invention, a countercurrent of thermal convection is created in the precipitation column, in which part of the medium moves upwards with a velocity V and the other part moves downwards with a velocity V. X sperm and Y sperm suspended in the medium move at speeds Vx and Vy, respectively. This velocity is determined by the velocity V of the medium, the direction of movement of the medium, and the density
It is influenced by gravity which exerts different forces on sperm and Y sperm. As a result of these factors, Y spermatozoa, which are less dense than X spermatozoa, rise faster in the upwardly moving portion of the medium and settle more slowly in the downwardly moving portion of the medium. As a result of these circulations over time, Y spermatozoa collect near the neck of the sedimentation column, while X spermatozoa collect near the bottom. Convection helps and facilitates the separation of the two types of sperm. In the second part of the invention, the debris of a medium with an unbalanced population containing either X sperm or Y sperm cells obtained from thermal convection countercurrent precipitation is determined by the difference in surface potential of X sperm cells or Y sperm cells. It is further separated and concentrated using convective galvanization. It is generally recognized that both X sperm and Y sperm are neutral (PH7.0) and negatively charged, and therefore both migrate toward the anode due to electrophoresis. However, male or Y spermatozoa have more negative charges in their heads than tails and are attracted to the anode head first, while female or X sperms have more negative charges in their tails than in their heads. It was found that the tail was attracted to the anode first.
Therefore, active or motile spermatozoa were found to swim in different directions during such electrophoresis. Y sperm swim toward the anode and move forward at a speed equal to the swimming speed plus the electrophoretic speed. On the contrary, the X sperm swims toward the cathode, and the forward motion of electrophoresis and the forward motion of swimming are opposite to each other. Since immotile spermatozoa, such as spermatozoa at low temperatures (eg, 3-5°C), have virtually no swimming speed, their movement is dominated by electrophoretic speed. However, it has been found that the surface charges of X and Y spermatozoa can differ according to the type of buffer used and the pH of the buffer. Therefore, the separation of X and Y spermatozoa depends on the regulation of the surface charge of the respective X and Y spermatids, and this charge depends on the temperature, current and voltage used in the electrophoresis cell as well as the pH of the buffer solution, Varies according to ionic strength and divalent ion concentration. Although greater separation rates in the electrophoresis cell can be expected if the sperm cells are motile, it is important to avoid the cells immediately after ejaculation in order to prevent them from adsorbing substances from the surrounding fluid or generating metabolic byproducts. It has been found that immobilizing sperm cells is necessary to perform the separation with maximum efficiency. Adsorption of substances or generation of by-products substantially alters the pheno-typical differences that enable separation by density and potential. The use of particulate-free media as disclosed in US Pat. No. 3,816,249 is important for controlling sperm cell metabolism and hyperactivity to take advantage of phenotypic differences in density and potential. Furthermore, the use of an unbalanced population of solids in the convective galvanization process, i.e., the use of a fraction of the medium containing substantially more sperm cells of one type than the other, can be achieved by electrical separation and Important for enrichment results. The thermal convection countercurrent precipitation step described above provides the unbalanced population conditions necessary for most effective separation in this electroseparation step. Countercurrent precipitation by thermal convection is the preferred method for creating an unbalanced sperm population for use in the convective galvanization process of the present invention. Various other methods for preliminarily separating X and Y sperm can also be used in combination with the convective galvanization process, with varying degrees of effectiveness. The thermal convection countercurrent precipitation step has been found to be the most preferred of the various initial separation methods and the most preferred in combination with the convective galvanization step. The convective circulation generated during convective galvanization may be either forced convective circulation or thermal convective circulation. Hereinafter, it should be understood that thermal convection and forced convection can be used interchangeably. It is also possible to combine a countercurrent precipitation column with thermal convection and a galvanization cell with forced convection in a single device and to carry out both steps in the same column. In this embodiment, precipitation by thermal convection is carried out until the desired separation by density is achieved, and then DC power is applied to the medium in the same column without transfer to other equipment. Thermal convection can optionally continue during galvanization. Although any ionic buffer solution known in the art can be used in the convective galvanization method of the present invention, the medium disclosed in US Pat. No. 3,816,249 is preferred. This medium has a pH of about 6-8 and typically about 250
It has an osmolality of ~350mos/Kg. Preferably a PH of about 6.8-7.0 and about 300 mos/
Use a medium with a permeability of Kg. In convective galvanization, the use of a slightly acidic medium was found to be most preferred as it accentuates the surface potential between the X and Y spermatozoa. The apparatus of the present invention for use in convective galvanization includes an electrophoresis cell having a central convection column and two side tubes interconnected and each containing an anode and a cathode. It should be noted that conventionally, electrophoretic or galvanic separation of X and Y spermatozoa was performed, but convective galvanization, which utilizes convective circulation of sperm cells, was not previously used during this separation. Should. In another embodiment of the invention, the apparatus for countercurrent precipitation by thermal convection has means adjacent to the precipitation column for providing the necessary temperature difference between the two parts of the medium therein. It may further include means for determining the extent to which the two sperm populations have accumulated at different heights within the sedimentation column during or after thermal convection. This is achieved by several different means. A small fraction of the medium was removed to contain X sperm and Y spermatozoa.
Sperm location and concentration are determined by measuring density or by placing a number of small liquid hydrometers into the sedimentation column. Alternatively, conductivity may be measured at several points within the column. In a preferred embodiment, the means for determining the position and concentration of the precipitation layer comprises a laser and laser sensing means in combination with means for scanning a laser beam over the length of the precipitation column. With this method, changes in the opacity of a medium for specific wavelengths can be measured without physically disturbing the contents of the precipitation column. This also facilitates recording changes in particle distribution, location and concentration of separated layers of X and Y spermatozoa. A preferred embodiment of the apparatus used to carry out the countercurrent precipitation step by thermal convection, which is the first half of the method of the present invention, is shown in FIG. A sedimentation column 1 containing a medium 3 in which X spermatozoa and Y spermatozoa are suspended is surrounded by a water sheath 5 into which a water flow 7 is applied by a pump 9.
is washed away. Water is drawn out from the water tank 11 after its temperature is adjusted by the temperature control means 13. This may include cooling elements with precise automatic temperature control. Inside the medium 3 is a second water jacket 15 which is coaxial with the precipitation column 1 and has an inlet 17 and an outlet 19 . Second water flow 2
1 flows through the outer tube 15, is pumped from the water tank 24 by the pump 22, and is temperature controlled by another temperature control means 26. The temperature of water flow 7 is equal to the temperature of water flow 2
If the temperature is the same as that in step 1, the entire medium in the precipitation column will have a uniform temperature. If there is a temperature difference between two streams of water, thermal convection occurs in the medium. Of course, other fluid heat exchange media can be used instead of water. An inlet 23 is provided for introducing sperm cells into the medium in the sedimentation column, and an outlet 25 is provided for removing and collecting a fraction of spermatozoa of substantially one chromosome type after sedimentation into a container 27. In order to measure the progress and location and concentration of sperm cell precipitation within the medium, means are provided for scanning the length of the precipitation column and measuring the comparative opacity at each location. These means are described in U.S. Patent No. 3,976,197.
Details are given in the issue. The above-described device can effectively generate countercurrent flow due to thermal convection of the present method and can effectively determine the location and concentration of separated X sperm cells and Y sperm cells. A preferred embodiment of a convective galvanization apparatus for carrying out the second half of the method is shown in FIG. The unbalanced sperm population fractions obtained in the thermal convection countercurrent precipitation step are further separated and concentrated by this device. Although a method using forced convection will be described, in the present invention, thermal convection may also be used as described above. A forced convection electrophoresis cell 71 generally has an interconnected central convection column 73, a side tube 75 containing an anode 79, and a side tube 77 containing a cathode 81. The two side tubes are connected to the convection column at their opposite positions. The convection column 73 consists of an upper part 83 and a lower part 85. The upper part is substantially narrower than the lower part, and in this embodiment has an upper diameter of 1 cm and a lower diameter of 1.5 cm. At the lower end of the convection column there is an inlet 87 connected via tube 91 to a peristaltic pump 89, heat exchange means 129 and tube 92. The pump is connected to a semen receiver 95 via a tube 93. The inlet of the semen receptacle leads to a tube 97 which connects at the upper end of the convection column 73 with its outlet 99. Semen receptacle 95 has an inlet 101 for supplying a semen mixture containing an unbalanced sperm population of X and Y sperm. Heat exchange means 129 is provided in close proximity to inlet 87. The temperature of the medium flowing through the convection cell is controlled to keep the sperm immobilized and at a low metabolic rate. A power supply means 103 is provided together with the electrophoresis cell 71. It includes a rectifier 105 that plugs into a DC power source or a standard AC outlet, and the DC output is controlled by a potentiometer 107 to obtain the desired voltage. The voltage is voltmeter 10
Electric wire 11 measured at 9 and connected to the anode and cathode
The current flowing into the electrophoresis cell through 3 and 115 is detected by an ammeter 111. When the separation of X sperm cells and Y sperm cells is completed to a desired degree, the desired fraction is taken out from outlets (valves) 117 and 119 communicating with anode 79 and cathode 81, respectively. The fraction of the medium removed from the outlet 117 of the side tube containing the anode 79 contains substantially entirely Y sperm cells attracted to the anode by the negative surface potential. Cathode 8
The portion of the medium taken out from the outlet 119 of the side tube containing 1 contains predominantly Y sperm drawn to the cathode by the positive surface potential. That is, the sperm head has no cytoplasm and is not involved in osmotic exchange of positive or negative ions. That is, osmotic conversion occurs between the sperm tail and the middle part. Therefore, if the medium surrounding the spermatozoon contributes to positive ion conversion, the X spermatozoon neutralizes the cathode-charged tail, making the entire X spermatozoa positively charged. As a result, the X sperm is attracted to the cathode. On the other hand, Y spermatozoa remain negatively charged. 121 and 1 of the side tube containing the electrodes of the electrophoresis cell
The section designated 23 is slightly less than 1 cm in diameter in this embodiment, and the sections designated 125 and 127 are wider than said sections, for example 1.5 cm in diameter. The degree of compression in the horizontal portions 121 and 123 of the side tubes compared to the lower part 85, with a slight compression in the upper part, helps to generate convective circulation within the lower part 85 and
and promotes the pulling of Y spermatids into the lateral tubes 75 and 77. As another specific example of the present invention, a device combining a precipitation device and a galvanization device is shown in FIG. All the elements described above with respect to the thermal convection countercurrent device are exactly the same in FIG. The precipitation column 1 connects two bent glass tubes 141 and 143 at different vertical levels. These glass tubes have an anode 145 and a cathode 147, and outlets 149 and 151, respectively.
DC power is provided by means similar to that illustrated at 103 in FIG. Precipitation column 1 performs the function of convection column 73 in FIG. The preferred medium used in the precipitation column in this method is the medium disclosed in US Pat. No. 3,816,249, as described above. This medium is glycine, α
- Contains a mixture of aminopropionic acid and egg yolk in an effective amount in an aqueous solution to extend the life of semen.
A preferred composition is, by weight, about 0.01-1.0% glycine, about 0.01-1.0% alpha-aminopropionic acid, about 0.1-2.0% sodium chloride, potassium chloride or calcium chloride, about 30-55% egg yolk and about
It is an aqueous solution containing 30-70% water, having a pH of about 6.0-8.0, and passed through a filter with a pore size of 0.2μ. The permeability of this medium may be approximately 250-350 mos/Kg. Although this medium is preferred, other particulate-free media of appropriate composition and appropriate pH and ionic conductivity concentrations for the galvanization step of the method may also be practiced. Fresh sperm containing equal amounts of X and Y sperm are collected from males and immediately mixed with medium at 22°C. The sperm mixture is then diluted to 30 million cells per ml and checked microscopically for its quality. After gradually lowering the temperature of the sperm cells to 15° C., they are introduced into a precipitation column, such as column 1 in FIG. The outer water stream 7 (FIG. 1) is kept at 3.5°C during operation, and the water stream 21 inside the outer jacket tube 15 is brought to 10°C for one and a half hours, after which the water flow is kept at 30°C by simply stopping the water flow.
Cool down to 3.5℃ in minutes. The above temperatures are merely representative. In fact, the method can be carried out at any temperature at which the activity of the sperm cells is low enough not to interfere with the precipitation step. During periods when there is a temperature difference between the center and the outer part of the medium in the precipitation column, a countercurrent due to thermal convection occurs, as schematically shown at 55 in FIG. Creates positive buoyancy. Precipitation due to gravity is a negative buoyant force and continues even when there is no temperature difference and no movement of the medium. 1/1 after introducing the sperm mixture into the medium in the precipitation column until a satisfactory separation is achieved.
It takes 2-8 hours. During this period, U.S. Pat.
The distribution of spermatozoa in the column measured over time using the laser scanning system detailed in No. 3976197 is shown at 57, 59, 61 and 63 in FIG. When convective separation is stopped, the concentration of cells due to sedimentation continues to pull both light and heavy sperm towards the bottom. By using the chart recorder 51 connected to the laser detection means 33, the distribution of spermatozoa can be recorded and observed as time passes during the separation period. When the distribution is deemed suitable, outlet 25 is opened and the fluid drips into receiver 27 at a rate of about 20 drops per minute. The first fraction collected from the sedimentation column contains heavy X sperm, the next fraction contains many Y sperm and a few X sperm, and the last fraction contains virtually all Y sperm. At this point, the fraction containing light Y sperm and the fraction containing heavy X sperm can be concentrated and purified by centrifugation, and the fractions of X sperm and Y sperm may be used for insemination. However, it is desirable to further concentrate and purify each fraction by convective galvanization as described below. After removing the desired fraction containing an unbalanced population of X or Y spermatozoa from the sedimentation device, preferably about
This fraction, on the order of 50 c.c., was incubated at 3-5°C for 150
Mix with cc fresh medium. This mixture is added to flask 95 of FIG. 3 and pumped at very low speed by pump 89 through inlet 87 into the convection column.
The electrophoresis cell having side tubes 75 and 77 with convection column 73 has heat exchange means 129 and pump 89.
pre-filled with fresh medium maintained at the desired temperature by When the semen-containing medium enters the column through the inlet 87, a flow in the opposite direction is created in the wide section 85 of the convection column by the action of the narrow section 83;
Therefore, convection circulation as shown by the arrows occurs at the bottom of the convection column 73. Due to this convection, the filled spermatozoa pass through the side tubes 75 and 77 containing the anode or cathode.
carried near the opening. A rectifier 105 and a potentiometer 107 are used to supply current at a desired voltage. The voltage used is approximately 1 to 5V and the current is several 100mA.
However, it is preferably about 2 to 4 V and about 100 to 400 mA depending on the ion concentration of the medium. The lower limit of useful voltage is the voltage required to cause the sperm cell to move substantially toward each electrode in an efficient amount of time, and the upper limit of voltage is the voltage that permanently affects sperm cell motility and fertility. It is a voltage that does not give The circulating flow contains relatively low concentrations of sperm (e.g. per cc
15 million pieces). Due to the low concentration, the sperm do not aggregate and move in their own directions relative to the electric field. By applying voltage to electrodes 79 and 81, sperm cells circulating in the convection column are exposed to DC power. These DC powers are enhanced by the use of media with appropriate PH and ion concentration. PH is approx.
It has been found that the potential difference on the surface of each cell of X sperm and Y sperm can be emphasized by using a medium of 6.0 to 8.0. As mentioned above, the ionic concentration or permeability of the medium is also important in this method;
Compositions with a permeability of 400 mos/Kg or higher may be used, but values of 250 to 350 mos/Kg are preferred;
A value of approximately 300 mos/Kg was found to be useful in the above specific example. During galvanization with forced convection, the hydrogen and oxygen exiting each electrode accumulates in the necks of side tubes 75 and 77, where the gas easily escapes or mixes with the circulation flow. This is a unique advantage over conventional devices where nascent hydrogen and oxygen damage the sperm. Gentle circulation of the medium within the electrophoresis cell is maintained until the desired degree of separation is reached, which in the above embodiment is approximately 30 minutes for each fraction processed. Circulation is then stopped and the electrodes are de-energized. Male and female media debris exit 117
and 119. The amount of medium removed is carefully controlled so that a substantial amount of medium that was circulating in the central convection column is not removed. Fluid is then forced back into the flask through the central convection column. Alternatively, fluid from the center of the column is drawn into the flask, but in this case all separated sperm populations are drawn towards the electrodes through the folds in side tubes 75 and 77. It is necessary to confirm. As a result, when the medium is withdrawn from the central column, 121
The medium flowing from sections 123 and 123 does not contain a substantial amount of separated sperm and can mix with the medium withdrawn from the central column. The debris in the lower parts 125 and 127 of the side pipes is then removed from the outlets 117 and 11.
drawn from 9. When using the apparatus shown in FIG. 4, the method can be carried out in the same manner as described above except for the step of transferring the sperm cells from the precipitation apparatus to the galvanization apparatus. After the countercurrent precipitation by thermal convection is complete, the circulation is slowed and the electrodes 145 and 147 of FIG.
Apply DC power to. A population rich in Y spermatozoa gathers near the nuchal part of the sedimentation column, and since these spermatozoa have a negative surface potential, they move toward the anode 145 in the side tube 141.
being drawn inside. On the contrary, the population rich in X sperm gathers near the bottom, and since these sperm have a positive surface potential, they are drawn into the side tube 143 toward the cathode 147. The desired fraction is then collected at outlets 149 and 151.
Each is extracted from When the separated fractions are processed as desired, the following method is preferably used. Fractions from outlets 117 and 119 are diluted with an equal volume of medium (containing approximately 20% glycerol) and ml
The volume should preferably be such that the number of sperm cells per unit is 60 million. The mixture is then held at 5-8° C. for 4-6 hours to equilibrate the cells and glycerol. This material is then placed in 1 ml ampoules, sealed, labeled as male or female or a mixture of fractions from the central convection column, frozen and stored in liquid nitrogen. The purity of the male or female fraction obtained may be tested serologically by raising antibodies or checked by B-body or F-body tests. Fractions from the anode and cathode of the precipitation column or convective galvanization device are centrifuged repeatedly with fresh media to concentrate and purify the different sperm types. The precipitate in the heavy part and the supernatant in the light part are then centrifuged repeatedly and washed to obtain the purest male and female forms, respectively. Thereafter, the serological tests described in column 3, lines 11-43 and column 5, lines 5-55 of US Pat. No. 3,692,897 are performed. Alternatively, the purity of separated and/or unseparated semen may be checked with a B-body test. This test is based on the finding that human and primate Y spermatozoa tend to fluoresce at specific brightnesses when stained with quinacrine-HCL or quinacrine-mustard using simple, conventionally accepted staining techniques. This method identifies Y sperm by observing stained sperm. Attempts have been made in the past to extend this technique to distinguish between male and female sperm in humans and non-primate livestock, but many failures have been reported.
Surprisingly, a satisfactory technique for staining Y spermatozoa in other species has been found by this method. As a result of using different enzymes at different concentrations, temperatures, pH, etc., papaya protease (commercially available from Gigma Chemicals, USA) is suitable for artificially performing similar functions on the cell membranes of spermatozoa of other domestic animals. I understood. The above method is carried out as follows: approximately 1 ml of semen or media mixture (containing approximately 20-50 million cells) is washed with saline and centrifuged three times for 15 minutes at 2500 g. good. This centrifugation and washing are optional. In this example, the precipitate is diluted with 1 ml of fresh saline, and 3 ml of this suspension is mixed with 5 mg of protease and digested for about 10 minutes at room temperature. next,
One drop of 0.005% quinacrine-mustard is added to one drop of the digestion mixture, placed on a slide and immediately mounted on the microscope. so that the dye enters the internal structure of the sperm
After 40 minutes, the outer membrane is digested by protease, which accelerates staining. Observations are made with a Leitz Ortholux microscope using a KP-490 Eciter Filter with two heat barriers HP430 and HP460 at a transmission wavelength of 530 nanometers. Human sperm treated with this method may be heavily stained without identifying Y sperm. However, Y-sperm from cows and horses has excellent transparency and exhibits unique bright spots (B-body);
Sperm undergoes dull and diffuse staining. Untreated human, bovine and horse semen as well as treated bovine semen were checked for B-bodies as is customary in the practice of this invention to check product purity. The results of 523 experiments are listed in Table 1. In the table the results of the B-Body test are compared with the results of the biological test.

It can be seen from Table 3 above that the combination of countercurrent precipitation with convection and convective galvanization substantially improves product purity over countercurrent precipitation with convection alone. Table 3 shows only the purity of the female, and the female is the case where there is no galvanic voltage (sample No. 2) - 40.9% is male sperm and 59.1% is female sperm. When galvanic voltage is introduced (No. 3), 30.3% of the sperm become male and 69.7% become female, improving the purity of the female by about 10%. Another method for identifying male sperm in humans and primates is the F-body test. This method is based on the fact that one arm of the Y chromosome in cells emits light when exposed to quinacrine detergent, and is a method for identifying Y sperm by observing the emitted arm of the Y chromosome. Human semen that has not been treated with this method is
It was found that approximately half of the individuals emitted light when treated using the body staining method. Although biological tests are not possible in humans, the obvious conclusion is that the luminescent population is synonymous with a luminescent Y chromosome, and therefore luminescent cells are Y chromosome-bearing, ie, androgenetic cells. The reproducibility of the F-body test has been demonstrated and is accepted as a standard technique for identifying male spermatozoa in human semen. By using countercurrent precipitation by thermal convection, convective galvanization, or a combination thereof, the heaviest and Light sperm or sperm with abnormal phenotypic characteristics can be avoided. This method can reduce Klinefelter's syndrome, Turner's syndrome and autosomal defects due to chromosome non-disjunction or translocations, which can lead to abnormal phenotypic characteristics of sperm with regard to cell surface potential or defective heavy or light sperm. This is possible by excluding. As mentioned above, the cell surface potential difference is directly related to the chromosomal structure of each cell, and therefore abnormal chromosomes can be easily detected and eliminated by carefully sorting spermatozoa based on such potential differences. In this method, the combination of countercurrent precipitation by thermal convection and convective galvanization is useful in controlling the sex of mammalian offspring. Counter-current precipitation, convective galvanization or a combination thereof are of great practical and economic importance in order to meet the needs of breeding cattle, horses, pigs, etc., especially female breeding. This method allows selection of the sex of the animal's offspring. For example, by obtaining only female offspring, it is possible to raise only milk-producing cows, or conversely, it is also possible to raise only male cows. With respect to human birth, the method and apparatus allow parents to choose or control the sex of their children if they desire a child of a particular sex, thus giving them the opportunity to reduce the number of children they have.
The sperm cells obtained by this method are highly fertile and will generally be successful in artificial insemination. Even if both parents carry the defective gene, this method allows them to exclude defective spermatozoa with the phenotypic characteristics of density and surface potential, giving them a chance of producing a normal child.

[Brief explanation of the drawing]

FIG. 1 is a sectional view showing a specific example of a countercurrent precipitation apparatus using thermal convection according to the present invention. FIG. 2 is a schematic explanatory diagram for explaining the distribution of spermatozoa over time in the countercurrent sedimentation device. FIG. 3 is a partially cutaway view showing a specific example of the convection galvanization device of the present invention. FIG. 4 is a sectional view showing a specific example of an apparatus combining countercurrent precipitation and convective galvanization according to the present invention. 1... Precipitation column, 5, 15... Outer tube, 23...
Sperm inlet, 71... Electrophoresis cell, 75, 77... Side tube, 79,145... Anode, 81,147... Cathode,
95...Sperm receptacle.

Claims (1)

[Claims] 1. Mixing semen with a liquid medium; applying galvanic force while providing convective circulation to the medium in which the sperm are suspended, to differentiate sperm of one surface potential from sperm of other surface potentials. A method of separating sperm cells from semen, comprising: reaching different locations in the medium; removing a fraction of the medium containing the desired type of sperm. 2. A method according to claim 1, wherein the convective circulation is produced by forcing convection in the medium. 3. The method of claim 1, wherein said convective circulation is produced by causing thermal convection in said medium. 4. A patent claim comprising cooling the mixture to immobilize the spermatozoa and imparting positive and negative buoyancy to the spermatozoa, thereby causing high-density spermatozoa and low-density spermatozoa to reach different positions in the medium. The method described in item 1. 5. The method according to any one of claims 1 to 4, which comprises further purifying desired spermatozoa by centrifuging the fraction. 6. A method according to any one of claims 1 to 5, wherein the medium comprises glycine, alpha-aminopropionic acid and an amount of egg yolk effective in an aqueous solution to extend the lifespan of spermatozoa. 7. mixing the semen with a liquid medium, creating an unbalanced X-chromosome/Y-chromosome population ratio in this medium; applying galvanic forces while imparting convective circulation to the medium in which the sperm are suspended; It involves allowing spermatozoa of one surface potential to reach a different location in the medium than spermatozoa of another surface potential; removing a fraction of the medium containing the desired type of spermatozoa. ; A method for separating sperm cells from semen. 8. The method of claim 7, wherein said convective circulation is produced by causing controlled convection in said medium. 9. The method of claim 7, wherein said convective circulation is produced by causing thermal convection in said medium. 10 A claim comprising cooling the mixture to immobilize the sperm and imparting positive and negative buoyancy to the sperm, thereby causing high density sperm and low density sperm to reach different positions in the medium. The method described in item 7. 11. The method according to any one of claims 7 to 10, comprising centrifuging the fraction to further purify the desired sperm. 12. A method according to any one of claims 7 to 11, wherein the medium comprises glycine, alpha-aminopropionic acid, and an amount of egg yolk effective in water to prolong sperm lifespan. 13. An apparatus for separating sperm cells suspended in a medium; an electrophoresis cell having an electrode having a pair of electrodes; a convection column communicating with the electrophoresis cell and disposed between the electrodes; means for introducing into the cell a medium containing sperm cells having different surface potentials; and means provided adjacent to each electrode for removing from the electrophoresis cell a desired fraction of sperm cells having the same surface potential; The above device including. 14 a precipitation column; means for introducing into said precipitation column a medium in which sperm cells with different densities and different surface potentials are suspended; adjacent to the precipitation column;
means for applying a temperature difference between two portions of the medium therein to cause countercurrent flow by thermal convection; means for recovering from the precipitation column a fraction of the medium containing sperm cells separated by density; and means for transferring the fractions collected from the precipitation column to the electrophoresis cell;
14. The apparatus of claim 13 comprising: 15 The convection column includes an upper part and a lower part, and either the upper part or the lower part is narrower in diameter than the other part, and the part containing the electrodes of the electrophoresis cell is in communication with the convection column between the upper part and the lower part. 15. The device according to claim 13 or 14. 16. Apparatus according to any one of claims 13 to 15, further comprising means for creating convection currents within the convection column. 17. The convection column has an inlet at one end and an outlet at the other end, and the apparatus further includes a pump connected between the inlet and the outlet for creating forced convection of a medium in the convection column. Claim 13
17. The device according to any one of paragraphs 1 to 16. 18 A patent in which the means for creating convection currents within a convection column includes means for creating thermal convective circulation of the medium within the column by bringing a portion of the medium contained in the column to a substantially different temperature than other portions. Apparatus according to claim 16. 19. The apparatus of any one of claims 13 to 18, further comprising means for measuring changes in sperm cell distribution within the sedimentation column. 20. A device for separating sperm cells with different densities and different surface potentials suspended in a medium;
Precipitation column; means for introducing into said precipitation column a medium in which sperm cells with different density densities and different electrical potentials are suspended; provided adjacent to said precipitation column to create a temperature difference between the two parts of the medium in said column; means for generating; forming part of said precipitation column;
an electrophoresis cell comprising two side tubes communicating with the column at different heights and each containing an electrode;
and means for recovering a desired fraction of the medium from the side tube containing the precipitation column and electrodes. 21 Claim 20 further comprising means for measuring changes in sperm cell distribution within said sedimentation column.
Apparatus described in section.