US 3687806 A
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United States Patent Office 3,687,806 Patented Aug. 29, 1972 3,687,806 METHOD FOR CONTROLLING SEX OF MAMMALIAN OFFSPRING Gustaaf J. van den Bovenkamp, Mill Valley, Califi, assignor to Bio-Controls, Inc. Filed Nov. 4, 1969, Ser. No. 873,787 Int. Cl. A61k 27/00 US. Cl. 1951.8 6 Claims ABSTRACT OF THE DISCLOSURE An immunological method for controlling the sex of mammaliam offspring, making use of a sperm fraction, containing a surplus of sex chromosomes of a single type (i.e., X-chromosomes or Y-chromosomes) and of a blood serum containing sperm antibodies, each antibody being selectively reactive by six chromosome with sperm type. Sperm fraction is introduced into the body of a mammal in sufficient quantity to produce antibodies in the blood stream. A blood serum is then taken from the mammal, the blood coagulated and the blood serum containing the antibodies isolated. The blood serum and sperm fraction are then mixed in proportions to effect inactivation and agglutination of between 80 and 100% of the antibodies in reactive excess over its sperm type is unaffected. The agglutinate and any remaining sperm is then precipitated with the supernatant portion containing said unaffected antibodies. These unaffected antibodies are selectively reactive to agglutinate and inactive sperm of only one sex chromosome type.
In one application of the invention, the antibodies reac tive with either the X- or Y-chromosomes may be added to semen to agglutinate and inactivate the sperm containing that type of chromosome before insemination. Alternatively, the antibodies may be introduced into the female prior to copulation (e.g., in a vaginal jelly or a vaccine) to provide the possibility of a sex selection at conception.
CROSS-REFERENCE TO RELATED APPLICATIONS Reference is made to the following applications: Ser. No. 443,473, filed by Bhairab Chandra Bhattacharya on Mar. 29, 1965, now abandoned and Ser. No. 814,906, filed by Wallace Shrimpton on Apr. 10, 1969, now copending.
Ser. Nos. 443,473 and 814,906 disclose two different methods for separating sperm into a fraction containing a surplus of sperm having either X-chromosomes (hereinafter called X-sperm) or Y-chromosomes (hereinafter called Y-sperm) BACKGROUND OF THE INVENTION As discussed in the aforementioned applications, the sex of offspring is controlled by the chromosomes of the particular spermatozoan or sperm cell which fertilizes the egg. More specifically, some spermatozoa (hereinafter called sperm) are genetypically known to contain X- while others contain Y-chromosomes which carry maleproducing genes. As further disclosed therein, X-sperm are somewhat more dense than the Y-sperm. This difference in density makes possible the separation of sperm in the ejaculate of a mammalian male to obtain sperm fractions containing a surplus of either X- or Y-sperm. Separation techniques utilizing this density differential are suitable for use with all mammals including human beings and other primates, cattle, swine (i.e., hogs and pigs), sheep, rabbits, cats, goats, horses, donkeys and buffalo. In general, the method of separation is to apply a buoyant force to the separation to cause the more buoyant sperm to attain a different level in a separation medium than the less buoyant sperm, the buoyant force being either positive or negative.
In the separation technique disclosed in the aforementioned abandoned application, Ser. No. 443,473, the sperm is separated by sedimentation in a system wherein gravity acts as an negative buoyant force. More specifically, the application discloses feeding fresh sperm to the top of a separation medium and recovering the lighter top fraction (Y-sperm) or heavier bottom fraction (X-sperm) before the bulk of the sperm reached the bottom of the column. This method is particularly effective in the recovery of X- sperm.
An alternative method of separating by applying a buoyant force based on the differences in density is disclosed in the aforementioned application, Ser. No. 814,- 906, wherein sperm is introduced into a central portion of separation medium containing a density gradient. The lighter fraction (Y-sperm) is buoyed in the direction of the upper portion of the column while the heavier fraction (X-sperm) settles to the lower portion of the column. This method produces a particular effective separation of Y-sperm.
Variations of the latter two mentioned applications have been used to form a surplus sperm fraction (X- sperm or Y-sperm). For example, see E. Schilling, Separation of Bull Sperm by Sedimentation and Centrifugation and the Sex of the Born Calves, Zeitschrift Fur Saugertierkunde, vol. 31. No. 4, pp. 314-323 (1966). Although this article describes the use of a different separation medium than that in the above applications, the principle of applying a buoyant force in a separation medium is followed.
A SUMMARY OF THE INVENTION AND OBJECTS This invention relates generally to an immunological method for controlling sex of mammalian offspring, and to compositions useful in providing offspring of one sex. More particularly, the invention relates to an immunological method for successfully isolating a substantially pure antibody composition selectively reactive with either X- sperm or Y-sperm and to the use of such antibody composition to permit conception by sperm containing chromosomes of the opposite type.
According to the invention, fresh sperm is introduced as an antigen into the body of a mammal in sufficient quantities to produce antibodies in the blood stream. A blood sample is then taken from the mammal, the blood coagulated and the blood serum containing the antibodies is isolated to produce an agglutination reaction with sperm of the same sex chromosome type as the antigen which ties up or inactivates the sperm. Such serum is mixed with a sperm fraction containing a surplus of one sex chromosome type (e.g., X-sperm) in proportions carefully selected to effect an agglutination and inactivation of substantially all of the antibodies reactive with the X-sperm, while a portion of the opposite antibodies are in reactive excess over the Y-sperm. Therefore, a substantial portion of the Y-sperm reactive antibodies remain free of agglutination. The agglutinate is then precipitated from the mixture and the supernatant containing a serum of the unaffected antibodies reactive only with Y-sperm is separated. The separated supernatant antibody serum which would inactivate Y-sperm but not X-sperm could be used to select the ultimate sex of offspring through use of various techniques: for example, by introduction of the serum into the vaginal cavity before copulation by various techniques (e.g., in a vaginal jelly or as a vaccine). In another technique, fresh sperm could be inoculated with this serum to cause inactivation of the Y-sperm before insemination to induce conception of only the X-type. It is understood that a serum containing only X-sperm reactive antibodies may be produced by a technique starting with a sperm fraction containing a surplus of Y-sperm (instead of X-sperm).
In general, it is an object of the present invention to provide a. successful method of controlling the sex of mammalian offspring.
Another object of the present invention is to provide a method for isolating antibodies in a form capable of inactivating sperm containing sex chromosomes of only one type.
Another object of the present invention is to provide a method for isolating antibodies capable of being used in procedures for artificial insemination of the female, to obtain offspring or the desired type.
A further object of the invention is to provide novel compositions containing antibodies reactive to inactivate and agglutinate substantially only either the X-sperm or the Y-sperm, thereby enabling use of such compositions to induce conception by a sperm of the opposite type on contact of said serum with a mammalian ejaculate prior to insemination to thereby produce offspring of the desired sex.
A still further object of the invention is to provide novel compositions of the above character which can be positively tested prior to use to insure their utility as antiserums with respect to sperm of particular sex characteristics.
A still further object of the invention is to provide a method for obtaining an upgrading in the effectiveness of sex determination as compared with conventional techniques starting with an X- or Y-sperm surplus fraction.
Additional objects and advantages of the invention will appear from the following description in which the preferred embodiments have been set forth in detail in conjunction with the accompanying drawing.
BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a flow sheet illustrating the method of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to the flow sheet of FIG. 1, fresh sperm ejaculate or sperm preserved in a nutrient medium such as fresh mammalian milk is introduced into the body of a mammal, in step 11, as an antigen, the purpose being to induce antibody production in the blood of such mammal. In accordance with known procedures, the antigen sperm fraction can be injected in successive doses to induce antigenic stimulation, leading to the highest possible levels of antibody formation. In due course a blood sample is removed from the mammal, in step 12, and the blood allowed to coagulate, as in step 13. The coagulation of the blood (a phenomenon related to clotting of the plasma) causes the non-coagulable blood serum to separate from the blood cells and coagulated plasma, as represented in step 14. The blood serum separated in step 14 is of primary importance to the method of the present invention for it contains the antibodies developed as a result of introduction of the sperm as an antigen. While the precise mechanism by which the antibodies are formed in the blood stream of the mammal is not known or fully understood, it is evident that the antibodies are finally present in the blood serum as separated in step 14. In a preferred practice of the invention, the blood serum separated in step 14 is inactivated by heating in step 15, viz, at a temperature of 56 C. for a period of about onehalf hour. The blood serum containing antibodies of the desired type is now in a form suitable for carrying out antibody reactions in accordance with the present invention.
Further referring to the flow sheet of FIG. 1, at step 16, a sperm fraction containing a surplus of either X-sperm or Y-sperm is isolated by techniques known or available in the art. For example, surplus X- or Y-sperm fractions can be isolated in accordance with the aforementioned abandoned application Ser. No. 443,473, copending application Ser. No. 814,906, or the aforementioned Schilling article. While sedimentation is specifically described therein as a procedure for separating a sperm fraction containing a surplus of X- or Y-sperm, other procedures may also be used. For example, employing forces greater than gravity in a centrifuge or forces to achieve positive buoyancy, as well as sedimentation in columns containing medium of carefully controlled density. In general, the sperm fraction so isolated is maintained and preserved within a nutrient medium such as fresh mammalian milk or other body fluids and nutrient liquids, as therein disclosed.
Although for purposes of the following description the sperm fraction will be assumed to contain a surplus of X-sperm, it will be understood that the technique may be followed using a Y-sperm surplus fraction to obtain a final product of antibodies of the opposite type.
As further illustrated in the fiow sheet of FIG. 1, in step 17, the blood serum containing sperm antibodies is mixed with the X-surplus sperm fraction. In step 17, the ratio of antibodies to sperm is extremely important. In an optimum ratio, there would be just enough X-sperm antibodies to agglutinate and tie up or inactivate substantially all the X-sperm. On the other hand, since X- sperm is present in excess of Y-sperm, there would be an insufficient number of Y-sperm to agglutinate all the Y- sperm reactive antibodies. In step 18, the agglutinate and any unreacted sperm can then be precipitated suitably by centrifugation. The supernatant serum, containing antibodies only reactive with Y-sperm, is then separated from the precipitate as in step 19. If desired, the blood serum in this form may be separated and held as an intermediate product capable of controlling the sex of offspring. This product containing antibodies reactive to agglutinate sperm of only the Y-type may be introduced into the female prior to copulation by conventional means (e.g., a vaginal jelly or a vaccine) to provide the possibility of sex selection at conception. The antibodies would selectively inactivate the Y-sperm while having no effect on the X- sperm. As above explained, the process could employ a Y-surplus sperm (instead of X-surplus sperm) to produce substantially all X-sperm reactive antibodies.
In an alternative to directly introducing the product of step 14 into the female, the product could be used for artificial insemination to produce offspring of the desired sex. For example, if the product contained antibodies reactive with only the Y-sperm, a mammalian ejaculate could be mixed with the product in proportions to agglutihate all of the Y-sperm. This agglutinate could be precipitated and the resulting supernatant would contain a substantially pure sperm fraction of X-sperm which could be used for artificial insemination.
It is apparent that starting with a surplus X-sperm (or Y-sperm) fraction produced in a conventional method (e.g., based on sperm density differentials) and following the technique of the invention may yield a product (containing substantially all X- or Y-sperm reactive antibodies) which is capable of controlling the sex of offspring in substantially all cases. This result is in marked contrast to other known attempts to control sex which are limited in their effectiveness to the extent of surplusage of X- or Y-sperm in a sperm fraction attainable by conventional methods. For example, direct artificial insemination with the sperm fraction would produce offspring in the same sex percentage as in the initial sperm fraction. The ability of the technique of the invention to upgrade the effectiveness of controlling sex is extremely important since to date a completely successful sperm separation by conventional means has not been demonstrated. 'Even if the antibody product of the invention were to contain less than 100% purity (e.g. or X-sperm reactive antibodies), this could ultimately produce a higher sex predictability than available in any other known tecl1- nique. Such a product would have great value to animal breeders or parents desiring a child of a particular sex.
One method of ascertaining the mixing proportions in step 17 is by a predetermination of the titer of the serum. To illustrate this technique, a brief discussion with assumed quantities is set forth. The serum is prepared from whole fresh bull sperm and so contains an equal number of X- and Y-sperm reactive antibodies. To simplify the discussion, each antibody will be assumed to be capable of agglutinating one sperm cell. This serum may be diluted in a series of serum-to-dilution ratios (e.g., 1:10, 1:50, and 1:100) and divided into given volumes (e.g., 10 ml. each). Fresh bull sperm (e.g., 1,000,000 sperm) is placed in each of the three tubes and agglutination is allowed to proceed. An agglutinate is then formed and precipitated by techniques described hereinafter. Then the supernatant for each dilution is separated and mixed with more fresh bull sperm. The first diluted volume in an ascending order of dilution that produces no visible agglutination is the titer of the solution. It is assumed that this titer contains a suflicient amount of anibodies in that test tube to just agglutinate all of the sperm (i.e., 500,000 X-sperm antibodies and 500,000 Y-sperm antibodies).
Once the titer is above-determined, a serum containing all Y-sperm reactive antibodies may be produced in the following manner. A sperm fraction containing 650,000 X-spenn and 350,000 Y-sperm is introduced into 13 ml. of the titered dilution (containing 650,000 X-antibodies and 650,000 Y antibodies). This volume of serum is selected since it is sufficient to agglutinate all the X- sperm with all the X-sperm antibodies. However, since there are 650,000 Y-sperm antibodies and only 350,000 Y-sperm, on agglutination 300,000 Y-sperm antibodies would remain in an unaffected form. By precipitating the agglutinated sperm, these 300,000 solely reactive with Y-sperm antibodies may be recovered as a supernatant product as represented at step 14. In an alternative approach, a substantially pure X-sperm reactive antibody may be produced by starting with a surplus Y-sperm fraction.
As a generalized example, illustrating the practice of the invention, serum containing antibodies selectively reactive with either X-sperm or Y-sperm can be prepared as follows:
In a procedure (called step A) for obtaining the blood serum, the bull serum is first suspended in a saline solution (0.9% NaCl in distilled H and washed three times by centrifuging at 2,000 r.p.m. for ten minutes, decanting the supernate and resuspending the precipitated sperm in saline. After adding antibiotics (e.g., penicillin, streptomyocin, etc.) in appropriate amount, the thricewashed sperm is injected subcutaneously or intra-abdominally into rabbits in successive injections, each injection involving the introduction of approximately 100,000,000- 200,000,000 sperm (approximately equal number of X- and Y-sperm). Following standard practice in hyperimmunization techniques, the rabbits are injected twice a week over a six-week period to thereby achieve a total injectional level of approximately 2,000,000,000 sperm. It should be noted that before initiation of each of the foregoing injection series, the experimental rabbits are tested for eventual spontaneous agglutination of bull sperm and any rabbits showing such agglutination are eliminated. At the end of the six-week period of injections, a blood serum is taken from each test rabbit by a procedure involving opening of a suitable blood vessel (e.g., an ear vein) from which the blood is caused to flow into a container. Blood specimens typically average four to five cc. The blood serum in inactivated to destroy substances in the sperm potentially harmful to sperm by heating at 56 C. for half and storing at --2 C. A blood serum is prepared in conventional manner by letting the blood stand for a sufficient period of time (e.g., overnight) to achieve coagulation. Clear, non-coagulable blood serum (e.g., approximately 3 cc.) is thereafter removed from the coagulate with a Pasteur pipette and placed in a small test tube or other suitable container.
The titer of the serum is then established in an agglutination series (step B) using bull sperm washed as described in step A. In accordance with this procedure, the blood serum is diluted with saline (0.9% NaCl in distilled H O) to achieve dilutions in the ratios of 1: 10, 1:50, and 1:100, etc. The washed sperm (1 ml. containing 10,000,- 000 sperm) is added to 1 ml. of each of these serum dilutions in a series of test tubes. The sperm and serum are mixed by sucking into a Pasteur pipette and releasing to insure thorough intermixing. The test tubes are placed in an incubator at 37 C. for twenty minutes. The test tube containing the mixture in which agglutination is just visible (the test tube next in number showing no visible agglutination) is the test tube selected as containing the dilution indicating titer. All test tubes having a greater dilution than the one containing the titer are discarded. Following this period of incubation, the agglutinate in the remaining test tubes is precipitated by centrifugation at 2,000 r.p.m. and the supernate again placed in a series of test tubes. The washed bull sperm is again added to this series of dilutions and incubated at 37C. for twenty minutes. Although the 1:10 dilution produced visible agglutination, the 1:50 and 1:100 produced none. Therefore, the 1:50 dilution is assumed to contain serum'in a concentration to just cause complete agglutination of all the antibodies in the serum by the 10,000,000 sperm.
The procedure described in the aforementioned Schilling article is then employed to obtain an X-sperm surplus fraction. A portion of the above washed bull sperm is placed at the top of a column containing low fat milk and a buffer at an overall viscosity of two to three centipoises and a specific gravity between 1.026 and 1.030. The fraction of the sperm that reaches the bottom of the column after sixty minutes is separated. This bottom fraction presumably contains a surplus of X-sperm (since X- sperm is believed denser than Y-sperm).
To prepare the serum having antibodies reactive with only X-sperm (step C), 1 ml. of serum at the above-determined titer dilution (1 :50) is mixed with 1 ml. of the above-prepared sperm fraction (approximately 20,000,000 sperm) presumably containing a surplus of X-sperm (called the X-sperm fraction) and the mixture is incubated. The mixing and incubation condition are as above described. During incubation an agglutinate is formed. The mixture is then centrifuged and the supernatant decanted. If the titer is correct and the sperm fraction ac tually contains a substantial surplus of X-sperm, there should be sufiicient sperm to agglutinate all X-sperm reactive antibodies but only part of the Y-sperm reactive antibodies. Therefore, the supernatant serum should contain only Y-sperm reactive antibodies. This serum can be used to control the sex of offspring as disclosed herein.
To test whether the above technique is successful in producing substantially all Y-sperm reactive antibodies, the above supernatant is divided and placed into two series of three test tubes. To one series of the supernatant is added the same type and concentration of thrice-washed fresh bull sperm (labeled XY sperm) used to determine the titer in step A and to the other series is added the same type and concentration of washed X-sperm surplus fraction (labeled X-sperrn) prepared in step B. Table I summarizes the amounts added.
Table II shows the degree of agglutination seen in the above series in Table I at an incubation temperature of 37 C.
TABLE lL-DE G REE O F A G GL UTINATION 1a 2a 3a 1b 2b 3b After halt-hour Slight Slight None None None None. After one hour Marked Marked Moderate do do Do.
Tables I and II illustrate that the blood serum formed according to the invention in step C contains substantially all Y-reactive sperm antibodies. By reversing the starting sperm from X-surplus to Y-surplus sperm, all X-reactive sperm antibodies would be produced.
From the foregoing, it will be apparent that the immunological method of the present invention has utility to control the sex of mammalian offspring. It is especially important as a commercial technique 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. By way of illustration, the dairy farmer can elect to obtain only female offspring and thereby breed only milk-producing cows rather than bulls. Regarding human procreation, it provides parents with a simple, easily employed means to select or control the sex of otfspring to quickly satisfy the desire to have a child of a particular sex, thus providing the opportunity to reduce the total number of children.
1. In a method for producing an immunological product for controlling the sex of mammalian offspring the steps of forming a blood serum containing sperm antibodies, each antibody being selectively reactive by sex chromosome type with sperm, isolating a sperm fraction containing a surplus of one sex chromosome type, mixing said antibodies with said sperm fraction in proportions to effect an inactivation and agglutination of at least 80% of the antibodies reactive with said surplus sperm while that substantial portion of the opposite antibodies in reactive excess over its sperm type is unaffected.
2. A method as in claim 1 wherein said mixing effects inactivation and agglutination of at least 90% of the antibodies reactive with said surplus sperm type.
3. A method as in claim 1 wherein said mixing effects an inactivation and agglutination of substantially all the antibodies reactive with said surplus sperm type.
4. A method as in claim 1 wherein said agglutinate is precipitated and the unaffected antibodies are separated as a supernatant product.
5. A method as in claim 1 wherein said sperm fraction contains a surplus of X-sperm.
6. A method as in claim 1 wherein said sperm fraction contains a surplus of Y-sperm.
No references cited.
RICHARD L. HUFF, Primary Examiner US. Cl. X.R. 42485; l281