Thermal convection counter streaming sedimentation and forced convection galvanization method for controlling the sex of mammalian offspring

• US RE32,350 E
• Filed: 04/29/1982
• Issued: 02/10/1987
• Est. Priority Date: 11/22/1974
• Status: Expired due to Term

• Current Assignee: Bhattacharya Bhairab C and Bhattacharya Manju
• Original Assignee: Bhairab C. Bhattacharya, Manju Bhattacharya
• Inventors: Bhattacharya, Bhairab C.
• Primary Examiner(s): Williams, Howard S.
• Application Number:
  US06/373,143
• Time in Patent Office: 1,748 Days
• Field of Search: 204/180 R
• US Class Current: 435/2
• CPC Class Codes: A01K 67/02 Breeding vertebrates
• CPC Class Codes: A61K 35/52 Sperm; Prostate; Seminal fluid; Leydig cells of testes
• CPC Class Codes: B03B 13/02 using optical effects
• CPC Class Codes: B03B 5/28 by sink-float separation
• CPC Class Codes: G01N 27/44721 by optical means

Abstract

A method and apparatus for controlling the sex of mammalian offspring by separation of the X-chromosome female producing sperm and Y-chromosome male producing sperm according to their different characteristics of density of the respective cells and electric potential on the respective cell surfaces. Separation is accomplished by first producing at thermal convection counter stream within a sedimentation column containing a universal medium with sperm suspended therein and allowing the two sperm populations to settle into different fractions according to different densities. Subsequently, the fractions are further separated and concentrated utilizing convection galvanization. The positive and negative geotaxis applied to the sperm during thermal convection sedimentation in combination with galvanic forces applied during the convection galvanization facilitate a more efficient separation than previously obtained. This is due to the fact that a greater degree of separation of X and Y sperm is achieved by subjecting an unbalanced population of sperm cells, i.e., one predominating in X or Y cells, to convection galvanization. Thermal convection counter streaming sedimentation has been found to be a preferred method for attaining this unbalanced sperm population. The apparatus used to accomplish the above separation includes means for producing a temperature differential between axial and peripheral portions of the medium contained in the sedimentation column, thus creating the necessary thermal convection counter stream, as well as an electrophoreses cell comprising a convection column disposed between the two electrodes of the cell. Alternatively, the sedimentation apparatus and the convection galvanization apparatus may be combined. Additionally, the apparatus may comprise a laser capable of scanning the length of the thermal convection sedimentation column as well as laser detecting means to determine the distribution of sperm produced within the medium therein.

Claims

1. A method of separating sperm cells of differing densities and electrical potentials from semen comprising the steps of:

mixing semen with a liquid suspending medium;

immobilizing the sperm by cooling the mixture;

applying both positive and negative buoyant forces to the sperm whereby more dense sperm attain a different level in the liquid medium than less dense sperm;

applying galvanic force to .Iadd.a portion of .Iaddend.said medium .Iadd.containing an unbalanced sperm population as a result of the preceding step .Iaddend.while circulating said medium by convection so as to separate sperm having different net electrical cell surface potentials; and

withdrawing a fraction of the medium containing the desired sperm.

2. A method as recited in claim 1 wherein said circulation by convection is produced by causing a forced convection circulation of said medium.

3. A method as recited in claim 1 wherein said circulation by convection is produced by causing a thermal convection circulation in said medium.

4. A method as recited in claim 1 wherein said more dense sperm are X-chromosome containing sperm, and said less dense sperm are Y-chromosome containing sperm, and said sperm of different electric cell surface potentials correspond to those sperm of a different chromosomal type.

5. A method as recited in claim 1 further comprising the step of allowing the sperm to further separate by gravitational sedimentation after the liquid medium is at rest.

6. A method as recited in claim 1 further comprising the step of applying the positive and negative buoyant forces by creating a temperature differential between portions of the mixture of sperm and medium in order to circulate the sperm by thermally produced convection counter stream.

7. A method as recited in claim 1 further comprising the step of centrifuging the fraction withdrawn to further purity the desired sperm.

8. A method as recited in claim 1 wherein said medium comprises a mixture of glycine, alpha-aminopropionic acid and egg yolk in amounts effective in aqueous solution to extend the life of said sperm.

9. A method as recited in claim 1 further comprising the step of determining the ratio of X chromosome sperm to Y chromosome sperm present in said withdrawn fraction of medium by subjecting said fraction of medium to a B-body test.

10. A method as recited in claim 1 further comprising the steps of:

staining any Y chromosome containing sperm cells present in said withdrawn fraction of medium with a compound selected from the group consisting of quinacrine HCl and quinacrine-mustard; and

determining the proportion of sperm cells contained in said withdrawn fraction of medium which are stained relative to those sperm cells which are not.

11. A method as recited in claim 10 further comprising the step of treating sperm cells contained in said withdrawn fraction of medium with an enzyme capable of enhancing the penetration of said staining compound into the sperm cells.

12. A method as recited in claim 11 wherein said enzyme is papaya protease.

13. A method of separating mammalian sperm according to phenotypical differences related to normal and abnormal genotypes comprising the steps of:

mixing fresh semen with a liquid suspending medium;

separating the mixture by thermal convection counter streaming sedimentation according to different sperm densities into three fractions, a first fraction containing predominantely X-sperm of a normal genotype, a second fraction containing predominately Y-sperm of a normal genotype, and a third fraction containing both X and Y-sperm carrying defective genes; and

subjecting a desired fraction of said medium to convection galvanization thus further separating and concentrating all X-sperm of a normal genotype and all Y-sperm of normal genotype.

14. A method as recited in claim 13 wherein said sperm are human sperm.

15. A method as recited in claim 13 further comprising the steps of:

mixing at least one separated fraction with a solution of from 5 percent to 20 percent glycerol; and

freezing the mixture in liquid nitrogen.

16. A method of separating sperm cells of differing cell surface electrical potentials from semen comprising the steps of:

mixing the semen with a liquid suspending medium;

applying galvanic force to said medium while subjecting it to convection circulation whereby sperm of one net cell surface electrical potential attain a different position within said medium than sperm of a differing net cell surface electrical potential; and

withdrawing a fraction of the medium containing the desired sperm type.

17. A method as recited in claim 16 further comprising the step of subjecting said mixture of semen and liquid suspending medium to a separation process which creates an unbalanced X-chromosome/Y-chromosome population ratio within said liquid suspending medium prior to the application of said galvanic force.

18. A method as recited in claim 16 wherein said convection circulation is produced by causing forced convection circulation of the medium.

19. A method as recited in claim 16 wherein said convection circulation is produced by causing a thermal convection circulation within said medium.

20. A method as recited in claim 16 further comprising the step of determining the ratio of X chromosome sperm to Y chromosome sperm present in said withdrawn fraction of medium by subjecting said fraction of medium to a B-body test.

21. A method of controlling the sex of mammalian offspring comprising the steps of:

mixing fresh sperm with a liquid suspending medium;

subjecting said mixture to thermal convection counter streaming sedimentation to separate according to differing densities sperm having X-chromosomes from sperm having Y-chromosomes;

removing a desired fraction of said mixture from said sedimentation column;

subjecting the desired fraction of said mixture to forced convection galvanization to further separate and concentrate according to different cell electrical potentials substantially all sperm having X-chromosomes from all sperm having Y-chromosomes; and

introducing the desired fraction into a female whereby conception occurs and offspring of the desired sex are produced.