U.S. patent number RE32,350 [Application Number 06/373,143] was granted by the patent office on 1987-02-10 for thermal convection counter streaming sedimentation and forced convection galvanization method for controlling the sex of mammalian offspring.
This patent grant is currently assigned to Bhairab C. Bhattacharya, Manju Bhattacharya. Invention is credited to Bhairab C. Bhattacharya.
United States Patent |
RE32,350 |
Bhattacharya |
February 10, 1987 |
Thermal convection counter streaming sedimentation and forced
convection galvanization method for controlling the sex of
mammalian offspring
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.
Inventors: |
Bhattacharya; Bhairab C.
(Princeton, NJ) |
Assignee: |
Bhattacharya; Bhairab C.
(Princeton, NJ)
Bhattacharya; Manju (Princeton, NJ)
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Family
ID: |
27503099 |
Appl.
No.: |
06/373,143 |
Filed: |
April 29, 1982 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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641501 |
Dec 17, 1975 |
4067465 |
|
|
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526378 |
Nov 22, 1974 |
3976197 |
|
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Reissue of: |
734243 |
Oct 20, 1976 |
04092229 |
May 30, 1978 |
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Current U.S.
Class: |
435/2;
435/173.9 |
Current CPC
Class: |
A01K
67/02 (20130101); A61K 35/52 (20130101); G01N
27/44721 (20130101); B03B 13/02 (20130101); B03B
5/28 (20130101) |
Current International
Class: |
A01K
67/02 (20060101); A01K 67/00 (20060101); A61K
35/48 (20060101); A61K 35/52 (20060101); B03B
13/00 (20060101); B03B 13/02 (20060101); B03B
5/28 (20060101); G01N 27/447 (20060101); B01D
057/02 (); C25B 007/00 (); G01N 027/26 () |
Field of
Search: |
;204/18R |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
"Electrophoretic Characteristics of Ram and Rabbit Spermatozoa", A.
D. Bangham, Proceedings of the Royal Society, B., vol. 155, pp.
292-305 (1961). .
"Galvanic Separation of X- and Y-Bearing Human Spermatozoa", S.
Shishito et al, Int. J. Fertil 20:13-16 (1975). .
"Galvanic Separation of X- and Y- Chromosome-Bearing Sperm", H. D.
Hafs et al., Michigan Agricultural Experiment Station Journal
Article No. 5201. .
"Sex Control in Cattle", Harold D. Hafs, Michigan State University,
Lansing, Michigan..
|
Primary Examiner: Williams; Howard S.
Attorney, Agent or Firm: Bacon & Thomas
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of application Ser. No.
641,501, filed Dec. 17, 1975 .Iadd.now U.S. Pat. No. 4,067,465
.Iaddend.in the name of Bhairab C. Bhattacharya which is in turn a
division of application Ser. No. 526,378 filed Nov. 22, 1974 by the
same inventor, now U.S. Pat. No. 3,976,197.
Claims
What is claimed is:
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.
Description
Reference is made to the applications of Bhairab Chandra
Bhattacharya, Ser. No. 443,473 filed Mar. 29, 1965 and now
abandoned; Ser. No. 873,795 filed Nov. 4, 1969 by the same
inventor, now U.S. Pat. No. 3,692,897, incorporated herein by
reference; and application Ser. No. 336,454 filed Feb. 28, 1973 by
the same inventor, now U.S. Pat. No. 3,816,249, also incorporated
herein by reference.
BACKGROUND OF THE INVENTION
The present invention relates to a method and apparatus for
controlling the sex of mammalian offspring by separation of
X-chromosome female producing sperm of a certain density and
electric potential from Y-chromosome male producing sperm of a
differing density and electric potential.
As discussed in the aforementioned applications, the sex of
offspring is controlled by the chromosomes of the particular sperm
cell which fertilizes the egg. As further disclosed therein,
X-chromosome-containing sperm which are responsible for producing
female offspring are somewhat more dense than
Y-chromosome-containing sperm which are responsible for producing
male offspring. Additionally, it has been determined that X and
Y-chromosome-containing sperm having a differing electric potential
on their respective cell surfaces. These differences in density and
electric potential make possible the separation of sperm into
fractions containing substantially all of either the X or the Y
sperm. Separation techniques utilizing these density and electric
potential differences are suitable for use with all mammals
including human beings and other primates, cattle, swing, sheep,
rabbits, cats, dogs, goats, horses, donkeys, buffalo, etc. As
previously disclosed, primarily in application Ser. No. 443,473,
the method of separation by density has been to apply a buoyant
force to the sperm to cause the more buoyant sperm to attain a
different level in the separation medium than the less buoyant
sperm where the buoyant force applied has been either positive or
negative or both. As previously practiced, the separation of X and
Y sperm cells by differing electric potential has been difficult
due to the fact that while in the seminal fluid the positive and
negative zeta potential of the female and male spermatozoa create
an equilibrium within the dielectric constant of the medium thus
making it difficult to draw them apart in a galvanic field. This
equilibrium has been mainly due to the substantially equal
populations of X and Y cells of opposite charge which have been
subjected to this process. While various types of electrophoreses
cells have been utilized in the attempt to separate male and female
spermatozoa with applied potentials varying from 200 microvolts to
10 volts DC, the outcome has been disappointing particularly when
the concentration of separated cells was too low to achieve a fair
conception. The use of higher current while possibly increasing the
purity of the separation has been found to lower the viability of
the sperm. These problems have been substantially overcome in the
present invention where sperm fractions containing substantially
unbalanced populations of X and Y cells resulting from a first
separation by thermal convection counter streaming sedimentation
are placed in a forced convection column maintained between the two
electrodes of an electrophoreses cell thus causing the respective
cells of different charge to be preferentially drawn to their
respective electrodes in a more efficient manner than heretofore
obtained.
It has been noted in the past that the presence of foreign
particles in the medium disturbs both the buoyant or sedimentation
velocity of the sperm and their movement due to galvanic forces as
well as their fertilization capacity after separation. The use of
the universal medium disclosed in U.S. Pat. No. 3,816,249
substantially eliminates this problem while promoting control of
cellular hyperactivity and prolonging sperm life as well. The use
of the universal medium as well as the imposition of a low
temperature immobilizing the sperm prevents the small difference in
density (2 to 5%) and electric cell surface potential between male
and female sperm from being neutralized by the high metabolic
activity of the sperm cells.
SUMMARY OF THE INVENTION
.Iadd.In one embodiment, the invention comprises a method of
separating sperm cells of differing cell surface electrical
potentials from semen by 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. .Iaddend.
In .[.the present invention.]. .Iadd.a preferred embodiment,
.Iaddend.positive and negative buoyant forces in combination with
galvanic forces are used to achieve a more efficient separation of
male and female sperm from a mixture of semen and particle-free
medium. In the first part of .[.the present.]. .Iadd.this
.Iaddend.method, the medium is held in a vertical sedimentation
column under the influence of a thermal convection counter stream
at low temperatures thus inducing separation of the differing sperm
cells according to density. Subsequent to this separation step each
of the lighter and heavier fractions, containing unbalanced sperm
populations predominating in Y and X chromosome sperm,
respectively, are separately processed in the forced convection
galvanization apparatus by separately injecting each fraction into
a central convection column located between the two arms of an
electrophoreses cell containing, respectively, positive and
negative electrodes. Galvanic potential is applied to the sperm
cells while they are circulated in the column in a manner similar
to the circulation achieved previously in the thermal convection
counter stream. The forced convection circulation in combination
with controlled conditions of temperature, ionic strength and pH of
the medium, and voltage make possible separations of X and Y sperm
cells to a purity of as much as 92% or more.
The first part of the present invention wherein X and Y sperm cells
are separated according to differing density is based on the theory
that in a closed vessel the molecules contained therein can be
influenced to move in two counter streaming courses when a
temperature differential is created between two adjacent areas. In
a vertical column of fluid when the peripheral temperature is
maintained lower than the axial temperature, a counter stream will
be formed moving a peripheral portion of the liquid downwards and
the axial portion upwards due to the difference in temperature. The
rate of flow of these streams in either direction is dependent on
the difference in densities created between the sections. Thus any
change in the temperature differential will influence the densities
and the rate of flow of the two streams.
If two classes of particles of different density and volumne are
introduced into a constant counter current stream in a vertical
column, the particles will initially be influenced by the
velocities of the counter streams; then positive and negative
buoyant forces acting on the particles will follow a definite
physical law, carrying them apart.
According to the present invention a thermal convection counter
stream is produced within the sedimentation column causing one
portion of the medium therein to move at velocity V in an upwards
direction and another portion of the medium to move with velocity V
in a downwards direction. X-sperm and Y-sperm suspended within the
medium will be caused to move at velocities V.sub.x and V.sub.y,
respectively, which velocities will be affected by the velocity of
the medium V, the direction of the movement of the medium upwards
or downwards, and gravity which will apply a different force on
X-sperm particles than on Y-sperm particles according to the
differing densities of the two types. As a result of these factors
a Y-sperm particle which is less dense than an X-sperm particle
tends to rise faster in that portion of the medium moving in an
upwards direction and to settle slower in that portion of the
medium moving in a downwards direction. Conversely, an X-sperm
particle which is more dense tends to rise more slowly than a
Y-sperm particle when the medium is moving in an upwards direction
and tends to settle more quickly than the Y-sperm particle when the
medium is moving in a downwards direction. Over a period of time
these circumstances cause the less dense Y-sperm particles to
accumulate near the top of the sedimentation column and the more
dense X-sperm particles to accumulate near the bottom of the
sedimentation column.
The convectional currents serve to aid and accelerate the process
of separating the two classes of particles. Therefore, the minute
density differential effect is used in conjunction with the
convectional counter stream to obtain a purer and optimum
separation of the two classes in a shorter time.
From the observational data, it has been noticed that the average
velocity of the particles in pure sedimentation field at 5.degree.
C. is 33 m.per second, whereas, the average velocity in convection
counter stream is 120 m.per second. When the height of the column
was selected at 18.2 cm, the average time taken for pure
sedimentation amounts to 15.4 hours in contrast to 24.3 minutes
when convection current was used.
In the second portion of the present invention, fractions of medium
having unbalanced populations predominating in either X or Y
chromosome containing sperm resulting from thermal convection
counter streaming sedimentation are further separated and
concentrated by convection galvanization taking advantage of the
differing electric cell surface potentials of the X and Y
chromosome containing sperm cells.
It has been generally agreed that both X and Y containing sperm
possess a net negative charge at neutral pH (pH 7.0) and would thus
both migrate towards the anode during electrophoreses. It has been
found, however, that male or Y chromosome containing sperm have a
more negative charge on the head than on the tail and are thus
drawn toward the anode head first while female or X chromosome
containing sperm are drawn toward the anode tail first due to a
higher negative charge on the tail than on the head. Thus, active
or motile sperm are found to be swimming in different directions
during such electrophoreses with the male or Y chromosomes
containing sperm swimming in the direction of the anode, thus
adding electrophoretic velocity to swimming velocity while the
female or X chromosome containing sperm swim toward the cathode
with their electrophoretic progress being opposed by their swimming
progress. On the other hand, immobilized sperm such as those at a
low temperature (e.g. 3.degree.-5.degree. C.) contribute
substantially little swimming velocity to their movement which is
then determined mainly by their electrophoretic velocity found to
be substantially equal in neutral buffer solutions, as mentioned
above. It has been found, that X and Y chromosome bearing sperm can
be made to differ in their net surface charges according to the
type of buffer involved and the pH thereof. The separation of X and
Y chromosome-containing sperm is thus dependent on the adjustment
of the net surface charge of the respective X and Y cells which
vary according to pH, ionic strength and concentration of divalent
ions in the buffer, as well as temperature, current and voltage
utilized in the electrophoretic cell. Although greater separation
velocity in the electrophoretic cell may be expected if the sperm
cells are motile, since they swim in opposite directions in
practice it has been found that the characteristics of motile cells
may alter quickly due to their activity. It has thus been found
necessary for maximum efficiency of the separation as practiced in
the present invention to immobilize the sperm cells immediately
subsequent to ejaculation so as to prevent the cells from absorbing
materials from the surrounding fluid or producing metabolic
by-products, either one of which would substantially alter the
pheno-typical differences which allow separation by net difference
in electric potential as well as the separation described above
according to differing density. It will thus be obvious that the
use of the particle-free universal medium disclosed in U.S. Pat.
No. 3,816,249 is significant in both aspects of the present
invention in order to control the metabolism and hyperactivity of
the sperm cell so as to allow phenotypical differences in density
and electric cell potential to be utilized.
It has furthermore been found that the use of an unbalanced
population in the convection galvanization step, that is the use of
a fraction of medium containing substantially more of one type of
sperm cell than the other type is of substantial significance to
the degree of success of galvanic separation and concentration. The
electrostatic state between the cells having a positive zeta
potential (X chromosome or female sperm cells), the cells with
negative zeta potential (Y chromosome or male sperm cells), and the
medium dielectric constant is advantageously unbalanced in order to
permit efficient separation in a galvanic field. The thermal
convection counter streaming sedimentation process described above
augments the galvanic separation step and supplies the unbalanced
population condition necessary for most efficient separation by
galvanic means.
It will be clear from the above that the combination of thermal
convection counter streaming sedimentation and convection
galvanization provides substantially more efficient separation of X
and Y chromosome containing sperm than either the counter streaming
sedimentation process or the galvanization process utilized
separately. It should be noted at this point that while thermal
convection counter streaming sedimentation is the preferred method
for creating an unbalanced sperm population for use in the forced
convection galvanization step of the present invention, various
other methods of preliminary X and Y cell separation may be
utilized in combination with the forced convection galvanization
disclosed herein with varying degrees of success proportional to
the degree of separation achieved by the first method utilized.
Thermal convection counter streaming sedimentation has been found
to be the most efficient of these methods of primary separation and
is preferred in the present invention for combination with forced
convection galvanization separation.
It should also be noted that the convection circulation which is
produced during convection galvanization may be either forced
convection circulation or thermal convection circulation. In the
following specification it should be clear that thermal convection
may be used instead of forced convection.
In one embodiment of the present method using forced convection
galvanization, fractions of universal medium containing desired X
and Y chromosome sperm ratios are injected into the central forced
convection column and due to the narrower diameter of the top of
the column utilized, a backflow in the opposite direction creates
the forced convection circulation. Circulation of the medium has
been found to aid in the galvanic separation of the unbalanced
sperm population.
Alternatively to the above, a convection may be achieved in the
galvanic cell convection column by producing a thermal gradient,
substantially as described for the thermal convection produced in
the sedimentation column. This would provide the desired convection
circulation in the galvanic cell.
It is furthermore possible, as will be described, to combine the
thermal convection counterstream sedimentation column and the
forced convection galvanization cell in one apparatus and carry out
both processes in the same column. In this embodiment, the thermal
convection sedimentation would be performed until the desired
separation by density was achieved, and then galvanic forces could
be applied to the medium in the column, without any transfer to
another apparatus. The thermal convection could be continued during
galvanization as desired.
While any ionic buffer solution known in the prior art can be
utilized in the forced convection galvanization method of the
present invention, the universal medium as disclosed in U.S. Pat.
No. 3,816,249 is preferably utilized as the sole medium in the
forced convection galvanization cell. The universal medium, as
previously disclosed, may have a pH of from about 6 to about 8, and
an osmolality generally between 250 to 350 mos/kg. Preferably,
universal medium composition will be utilized having a pH in the
range of from about 6.8 to about 7.0 and an osmolality of
approximately 300 mos/kg. It has been found that use of a slightly
acid medium during forced convection galvanization is most
preferable in that it accentuates the net cell surface potential
differences between X and Y chromosome containing sperm.
The apparatus of the present invention utilized in one embodiment
for forced convection galvanization comprises an electrophoreses
cell having a central forced convection column and two arms of the
cell communicating therewith containing the positive and negative
electrodes, respectively. It is to be noted that while the prior
art has experimented with electrophoretic or galvanic separation of
X and Y sperm cells, the use of convection galvanization utilizing
convection circulation of the sperm cells during galvanic
separation, has not been used insofar as is known, prior to the
present invention.
The present invention in addition to apparatus utilized for forced
convection galvanization as described above includes apparatus for
producing thermal convection counter streaming sedimentation,
comprising means adjacent to a sedimentation column for producing
the required temperature differential between two portions of the
medium contained therein. Additionally, means are provided to
determine the extent of accumulation of the two sperm populations
at different levels within the sedimentation column both during and
after thermal convection counter streaming. This may be achieved by
several different means. Small fractions of the medium may be
drained to determine the location and concentration of X-sperm and
Y-sperm cells or a plurality of small hydrometers may be introduced
into the sedimentation column to make the determination by
measurement of density. Alternatively, the determination may be
made by measurement of conductivity at various points within the
column. In the preferred embodiment, the means for determining the
location and concentration of the sedimented layers comprises a
laser and laser detecting means in combination with means for
scanning the laser beam throughout the length of the sedimentation
column. Variations in the opacity of the medium to a particular
wave length are thus determined, without the necessity of
physically disturbing the contents of the sedimentation column in
any way. This also facilitates the recording of variations in
particle distribution and in the location and concentration of
separated layers of X and Y-sperm cells.
Accordingly, it is an object of the present invention to provide a
more efficient method for controlling the sex of mammalian
offspring by obtaining a more complete separation of X-sperm and
Y-sperm than has heretofore been possible.
Another object of the present invention is the provision of a
method and apparatus for convection galvanization wherein galvanic
potentials are utilized to separate X and Y chromosome containing
sperm cells according to the differing electric potentials on the
cells surfaces thereof while the sperm cells are being circulated
in a convection column so as to create the unbalanced condition
necessary for more efficient galvanic separation.
It is also an object of the present invention to provide a method
for the separation of X sperm and Y sperm which takes advantage of
the production of an unbalanced sperm population by thermal
convection counter streaming sedimentation and utilizes this
unbalanced population in a successful method of convection galvanic
separation of the sperm cells so as to further separate and
concentrate the unbalanced population resulting from such thermal
convection counter streaming sedimentation.
A further object of the present invention is to provide a method of
separating sperm according to phenotypical differences related to
normal and abnormal genotypes by utilizing differences in their
density and cell surface electrical potential.
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
drawings in which:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram of a thermal convection counter streaming
sedimentation apparatus which may be used in carrying out the first
step of the present invention.
FIG. 2 is a representation of the operation of a sedimentation step
useful in the process of the present invention.
FIG. 3 is a diagram of apparatus which may be utilized in the
forced convection galvanization step of the present invention.
FIG. 4 is a diagram of an embodiment of the present invention
combining the thermal convection counterstream sedimentation
apparatus with the convection galvanization apparatus.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Shown in FIG. 1 is an illustration of the preferred embodiment of
the apparatus used to carry out the first portion of the method of
the present invention, that is, the thermal convection counter
streaming sedimentation process. A sedimentation column 1
containing the universal medium 3 with both X-sperm and Y-sperm
cells suspended therein is surrounded by a water jacket 5 through
which a first water stream 7 of a first temperature is pumped by
water pump 9. The water is drawn through water tank 11 where the
temperature of the water can be controlled by temperature control
means 13. This may comprise a cooling element with precise
thermostatic control. Within the medium 3 is shown a second water
jacket 15 which is coaxial with sedimentation column 1 and which
has inlet 17 and outlet 19. A second stream of water 21 flows
through water jacket 15 and may be pumped by means 22 through water
tank 24 and its temperature controlled by separate means 26. In the
event that the temperature of water stream 7 is the same as water
stream 21 the entire medium within the sedimentation column will be
at a uniform temperature. If a temperature differential is created
between the two water streams, a thermal convection counter stream
will result within the medium contained in the sedimentation
column. It will be understood of course that other fluid heat
exchange mediums may be used in lieu of water.
Inlet means 23 are provided for introducing sperm cells into the
medium contained in the sedimentation column and outlet means 25
are provided for withdrawing fractions of sperm of substantially
one chromosome type after sedimentation has been completed and
collecting them in container 27.
In order to determine the progress of sedimentation as well as the
location and concentration of the different types of sperm cells
within the medium, means are provided for scanning the length of
the sedimentation column and determining the relative opacity at
different points therein. These means are described in detail in
U.S. Pat. No. 3,976,197 which is incorporated herein by
reference.
The apparatus thus described has the capability of efficiently
producing the thermal convection counter stream of the method of
the present invention as well as to efficiently determine the
location and concentration of separated X and Y-sperm cells.
Shown in FIG. 3 is an illustration of a preferred embodiment of
convection galvanization apparatus used to carry out the second
portion of the method of the present invention wherein the
unbalanced sperm population fractions resulting from thermal
convection counter streaming sedimentation are further separated
and concentrated. While forced convection is utilized in the
following description, it should be clear that thermal convection
could be substituted therefor in the practice of the present
invention.
Shown generally as 71 is a forced convection electrophoretic cell
having a central forced convection column 73 with two bent tubes 75
and 77 communicating therewith and containing respectively positive
and negative electrodes 79 and 81. It will be noted that these
anode and cathode containing tubes communicate with the forced
convection column on opposite sides thereof so as to maximize the
movement of oppositely charged sperm cells under galvanic
forces.
Convection tube 73 is comprised of an upper section designated as
83 and a lower section designated as 85 with the upper section
being substantially narrower, 1 centimeter in diameter in the
present embodiment than the lower section which is 1.5 centimeters
in diameter in the present embodiment. The lower end of the
convection tube has inlet means 87 for attachment to peristaltic
pump 89 through tube 91, heat exchange means 129 and tube 92. The
pump is connected through tube 93 to the outlet of semen reservoir
95 which may be an erlenmyer flask. The inlet of the semen
reservoir is connected in turn to tube 97 which communicates with
an outlet 99 at the upper end of convection tube 73. Semen
reservoir 95 is furthermore provided with an inlet 101 for
placement of a semen mixture containing an unbalanced sperm
population of X and Y sperm cells therein. Heat exchange means 129
is provided in close proximity to inlet means 87. The temperature
of the medium flowing through the cell may thus be controlled, so
as to keep the sperm immobile and at a low metabolic rate.
In further association with electrophoretic cell 71 is a power
supply generally designated at 103 which is comprised of a DC power
source or rectifier 105 which may be plugged into any standard AC
outlet, the DC output of which may be controlled by potentiometer
107 in order to produce any desired voltage as measured by
voltmeter 109. An ampmeter 111 is also provided for the monitoring
of current flowing through the electrophoretic cell by means of
wires 113 and 115 connected to the anode and cathode, respectively,
of said cell. Upon completion of forced convection galvanic
separation of the X and Y chromosome containing sperm cells to the
desired degree, the desired fractions are withdrawn through outlets
117 and 119 which communicate respectively with the tubes
containing anode 79 and cathode 81. The fraction of medium drawn
off through valve 117 from the arm of the cell containing anode 79
will contain substantially completely Y chromosome containing cells
which are male producing and which have been drawn to the positive
anode due to their net negative cell surface potential. The portion
of the medium drawn off through outlet 119 communicating with the
arm of the cell containing cathode 81 contains predominately X
chromosome containing sperm cells which are female producing and
have been drawn to the negative cathode due to their net positive
cell surface potential.
It will be noted that those portions of the electrode containing
arms of the cell shown at 121 and 123 are slightly less than one
centimeter in diameter whereas the portions of the arms
communicating with the electrodes shown at 125 and 127 are of a
wider diameter, in this embodiment 1.5 centimeters. The degree of
constriction at the horizontal portions of these arms, i.e. 121 and
123, as well as the slight constriction in the upper part of the
convection tube at 83 relative to the lower part 85 has been found
to aid in both the production of a convection circulation within
the lower portion 85 of the convection tube and facilitate the
tendency of Y and X chromosome containing sperm cells to be drawn
into electrode tubes 75 and 77.
In an alternate embodiment of the present invention as shown in
FIG. 4, the sedimentation apparatus and galvanization apparatus may
be combined. In FIG. 4, all elements described previously relating
to the thermal convection counter streaming apparatus may be
identical to that shown in FIG. 1. The following additions to the
apparatus are made, however.
The sedimentation column 1 has two bent glass tubes 141 and 143
communicating therewith at different vertical levels. These glass
tubes contain respectively an anode 145 and a cathode 147 and each
has respective outlet means 149 and 151. DC power may be supplied
by means similar to those shown at 103 in FIG. 3. It will be clear
in this embodiment that the sedimentation column 1 performs the
function of convection column 73 in FIG. 3.
The first portion of the method of the present invention as
performed by the thermal convection counter streaming sedimentation
apparatus first described above will now be described with
reference to both FIGS. 1 and 2.
As discussed previously the preferred medium for use in the
sedimentation column in the method of the present invention is the
universal medium described in U.S. Pat. No. 3,816,249. This medium
comprises a mixture of glycine, .alpha.-aminopropionic acid and egg
yolk in amounts effective in aqueous solution to extend the life of
said semen. The preferred composition of this medium comprises an
aqueous solution having a pH in the range of from about 6.0 to 8.0,
and containing, by weight, from about 0.01 percent to about 1.0
percent glycine, from about 0.01 percent to about 1.0 percent
.alpha.-aminopropionic acid, from about 0.1 percent to about 2.0
percent of sodium chloride, potassium chloride or calcium chloride,
from about 30 percent to about 55 percent egg yolk, and from about
30 percent to about 70 percent water and is filtered using
millipore size 0.2.mu. filter. As noted previously, this universal
medium has been found to be ideal for use in the forced convection
galvanization apparatus described above as well as in the
sedimentation step due to the ability to tailor both the pH of the
medium as well as the osmolality to the degree required for most
efficient galvanic separation of the sperm cells. The osmolality of
the universal medium may range from about 250 to about 350 mos/kg.
While this medium is preferred, it is possible to practice either
the first or second portion of the method of the present invention
with other particle-free mediums of appropriate composition and
especially those of appropriate pH and ionic conductivity
concentration for the galvanization portion of the present
method.
Fresh sperm containing equal amounts of X and Y-sperm is collected
from the male and mixed immediately with the universal medium at
22.degree. C. The sperm mixture is then diluted further to 30
million cells per ml and checked microscopically for its quality.
Only mixtures with excellent grading are used in the separation
procedure.
The temperature of the sperm mixture is gradually lowered to
15.degree. centigrade and then introduced into a sedimentation
column, for example, the column 1 of FIG. 1. The outer water stream
7 (FIG. 1) is maintained at a temperature of 3.5.degree. centigrade
throughout the operation, and water stream 21 contained in coaxial
water jacket 15 is maintained at 10.degree. centigrade for one half
hour and then brought down to 3.5.degree. centigrade in another
half hour by simply cutting off circulation within the coaxial
tube. It should be understood that the above temperatures are only
representative. In practice, the process may be carried out at any
temperature which is sufficiently low to prevent the activity of
the sperm cells from interfering with the sedimentation process.
The temperature differential created as described above is also
exemplary and any differential which would create sufficient
convection counter streaming to facilitate sperm cell separation
within a reasonable time would suffice. As previously discussed,
the combination of this low temperature and the use of the particle
free universal medium play an extremely important role in the
invention by immobilizing the sperm so that they effectively become
inert particles. This enables the subsequent positive and negative
buoyant forces applied to use the 2 %-5% difference in density of
the two types of sperm to effect a separation. During the period of
temperature differential between the central and outer portions of
the medium within the sedimentation column a thermal convection
counter stream shown diagrammativelly at 55 of FIG. 2 occurs which
produces the positive buoyant force in this embodiment.
Gravitational sedimentation is the negative buoyant force, and
continues when the temperature differential becomes zero and the
motion of the medium ceases. It takes 1/2 to 8 hours to achieve
satisfactory separation beginning with introduction of the sperm
mixture into the universal medium contained in the sedimentation
column. Throughout this period, distribution of sperm in the
sedimentation column at different times, shown at 57, 59, 61 and 63
of FIG. 2, is determined in a preferred embodiment by use of the
laser scanning system described in detail in U.S. Pat. No.
3,976,197. As the convection separation is stopped, concentration
of cells by sedimentation continues dragging both lighter and
heavier sperm towards the bottom. By utilizing a chart recorder 51
connected to laser detecting means 33 the distribution of sperm at
different times in the separation period can be recorded and
observed. When the distribution is considered adequate, outlet
means 25 can be opened and the fluid is allowed to drop into
container 27 at a rate of approximately 20 drops per minute. The
first fractions collected from the sedimentation column will
contain the heavier X-chromosome containing sperm and successive
fractions will contain less X-chromosome sperm and more
Y-chromosome sperm until the final fractions collected will contain
substantially all Y-chromosome sperm. At this point it is possible
to centrifuge separately the lighter Y chromosome containing
fraction and the heavier X chromosome fraction so as to concentrate
and purify the products, and both the X and Y fractions may be
utilized at this point for insemination. It is desirable, however,
to further concentrate and purify the X and Y chromosome containing
fractions by forced convection galvanization as described
below.
Subsequent to the removal of the desired fraction of medium from
the sedimentation apparatus containing an unbalanced
X-chromosome/Y-chromosome sperm population, the fractions, which
are preferably on the order of approximately 50 cc. are mixed with
150 cc of fresh medium at 3.degree. to 5.degree. C. This mixture is
added to the erlenmyer flask 95 in FIG. 3 and peristaltic pump 89
is set at a very low rate forcing the fluid from the erlenmyer
flask into the middle of the convection column through inlet means
87. The entire electrophoretic cell including sidearms 75 and 77 as
well as convection column 73 have previously been filled with fresh
medium at the desired temperature which is maintained by activating
heat exchange means 129 and pump 89. As the semen containing
universal medium is forced through inlet means 87 to the middle of
the tube, the narrower portion of the convection column shown at 83
creates a backflow in the opposite direction facilitated by the
wider portion of the tube shown at 85, thus creating a convection
circulation as indicated by the arrows in the lower portion of
convection tube 73. This convection carries oppositely charged
spermatozoa by the opening of side tubes 75 and 77 containing
respectively the anode and cathode of the electrophoretic cell.
Power supply 105 is activated and potentiometer 107 is used to
accurately set the desired voltage and current flow through the
system. While the voltage utilized in the present invention may be
anywhere in the range of from about 1 to about 5 volts with the
current on the order of several hundred microamps, the preferred
range is from about 2 to about 4 volts of galvanic force creating a
current of from about 100 to about 400 microamps depending on the
ionic concentration of the medium utilized. In practice the lower
limit of voltage useful is that necessary to create substantial
migration of the sperm cells towards their respective electrodes
within an efficient amount of time, with the upper limit of voltage
being that which will not permanently affect the motility or
virility of the sperm cells. In one embodiment, a voltage of 2.3
volts was used with a resulting current of 200 microamps.
It should be noted that the circulating fluid in the system
contains a relatively low concentration of sperm (i.e. 15 million
per cc) which low concentration prevents the clumping of the
spermatozoa which are moving in the opposite direction to the
galvanic field.
Upon the application and adjustment of voltage to electrodes 79 and
81, the circulating sperm cells in the convection column are
subjected to galvanic forces. These galvanic forces are accentuated
by the utilization of a medium in the electrophoretic cell having a
pH and ionic concentration of the proper values. While mediums of a
pH of from about 6.0 to about 8.0 may be utilized in the present
invention, it has been found that the net electrical potential
difference on the respective cell surfaces of X chromosome and Y
chromosome containing sperm is accentuated in a medium of pH from
about 6.8 to about 7.0. As noted, the ionic concentration or
osmolality of the medium is also of significance in the present
process and while compositions with osmolalities ranging from about
200 to about 400 mos./kg., or an even greater range may be
utilized, values of from 250 to 350 mos./kg are preferred and the
osmolality found to be useful in the present embodiment was about
300 mos./kg.
During forced convection galvanization, the escaping hydrogen and
oxygen from the respective electrodes is trapped at the top of bent
side tubes 75 and 77 where the gases can easily escape and mix with
the circulating fluid. This is a distinctive advantage over other
cells where nascent hydrogen and oxygen may injure the
spermatozoa.
The slow circulation of medium through the electrophoretic cell as
described above is maintained until the desired degree of
separation is achieved, which under the circumstances of the
present embodiment, may be for about 30 minutes for each fraction
processed. After separation is completed to the desired degree,
circulation is stopped and power to the electrodes disconnected.
The desired male and female medium fractions are drawn off from the
two electrode tubes through outlets 117 and 119 with the amount of
medium drawn off being carefully regulated so as to prevent
substantial amounts of the medium which had still been circulating
in the central convection tube from being drawn off. Subsequently,
the fluid from the central column may be forced back into the
flask. Alternatively to the above, the fluid from the central
portion of the column may first be drawn off into the erlenmyer
flask, it being necessary in this case to ensure that all separated
sperm population has been drawn towards the electrodes past the
bent arms of side tubes 75 and 77 so that when the medium is
drained from the central column, that portion of the medium flowing
out of side tube sections 121 and 123 does not contain substantial
amounts of separated sperm populations which would be mixed upon
flowing back into the central convection column. The fractions in
the lower portions of side arms, that is portions 125 and 127, may
then be drawn off through outlets 117 and 119.
In the event that the combined sedimentation and galvanization
apparatus shown in FIG. 4 is utilized, a procedure similar to that
described above is followed, without the steps of transferring the
sperm cells from one apparatus to the other.
After thermal convection counter streaming sedimentation is
completed, the circulation is slowed and galvanic forces are
applied to electrodes 145 and 147 in FIG. 4. A predominantely Y
chromosome containing population has accumulated near the top of
the sedimentation column, which sperm have a net negative cell
surface electrical potential, and are thus further concentrated by
attraction into side tube 141 towards positive anode 145. A similar
population predominating in X sperm which have a net positive
charge is present near the bottom of the column and is further
concentrated by attraction into side tube 143 towards cathode 147.
The desired fractions may then be drawn off through outlets 149 and
151, respectively.
While the separated fractions may be processed as desired, the
following method is preferably used.
Equal amounts of universal medium (containing about 20% glycerol)
is used to dilute the fractions drawn off from outlets 117 and 119
to the desired volume and sperm cell number which is preferably
approximately 20 million cells per ml. and the mixtures are then
held at 5.degree. to 8.degree. C. for 4 to 6 hours in order to
equilibrate the glycerol with the cells. The material may then be
put into one ml. ampules, sealed, marked as male or female or
mixture in the event of the fraction drawn off from the central
convection tube, frozen and stored in liquid nitrogen.
The purity of male and female fractions obtained by the method
outlined above may be tested either serologically by producing
antibodies or may be checked by the B-body or F-body tests. It
should be pointed out that sperm fractions resulting directly from
any of the above described embodiments or combinations thereof may
be processed as follows. The lighter and heavier fractions from the
sedimentation column or the anode and cathode fractions from the
forced convection galvanization apparatus are centrifuged
repeatedly with fresh medium in order to concentrate and purify the
different sperm types. The sediment in the heavier fractions and
the supernatant in the lighter fractions subsequent to repeated
centrifuging and washing are considered to hold the most pure forms
of the female and male sperm respectively. The subsequent procedure
for serological testing is followed as described in my U.S. Pat.
No. 3,692,897, column 3, lines 11 through 43 and column 5, lines 5
through 55.
Alternatively to the above, the purity of the separated and/or
non-separated semen may be checked with the B-body test. This test
is based on the knowledge that Y spermatozoa of humans and primates
tends to fluoresce with a special brightness when stained with
quinacrine-HCL or quinacrine-mustard with the staining technique
being simple and generally accepted by the prior art. While
attempts have been made in the past to extend this technique in
order to identify male and female spermatozoa in other domestic
animals besides humans and primates, failure has been reported in
many instances. My analysis on the subject encouraged me to develop
a successful technique of staining Y spermatozoa by this method in
other species since human and primate sperm contain a large
quantity of proteolotic enzymes which partially dissolve the sialic
acid-protein complex, glyco- and lypo-protein coating of the sperm
membrane, which has been found to enhance the penetration of the
dye into the chromatin materials. Thus, after the utilization of
different enzymes at different concentrations, temperatures, and
pH's, etc., I have particularly found that papaya protease
(available Sigma Chemicals, U.S.A.) is suitable for the purpose of
performing a similar function artificially on the cell membranes of
the sperm of other domestic animals.
The above process is carried out as follows: Approximately 1
milliliter of semen or medium mixture (containing from about 20
million to 50 million cells) is washed with saline and may be
centrifuged three times at 2,500 grams for 15 minutes. This
centrifuging and washing, if it is in addition to that first
described above, may be optional. After diluting the sediment with
1 milliliter of fresh saline in the present example, 3 drops of
this suspension is mixed with 5 milligrams of protease and allowed
to digest for approximately 10 minutes at room temperature.
Subsequently, one drop of 0.005% quinacrine-mustard is added to 1
drop of the digested mixture, put on a slide, and mounted
immediately for microscopic examination. After allowing 40 minutes
for the dye to enter the inner structure of the spermatozoa, which
is facilitated by the digestion of the outer membrane by protease,
the spermatozoa may be viewed with a Leitz Ortholux microscope
using the KP-490 Eciter Filter at a transmission wave length of 530
namometers with two heat barriers. HP430 and HP 460. Human
spermatozoa treated in the above matter may take a deep stain
without identifying Y chromosome containing sperm; however, bull
and horse spermatozoa with the Y chromosome show a distinct bright
spot with excellent visibility (B-body) with the X bearing
spermatozoa taking a dull and diffused stain. Nonprocessed bull,
human and horse semen and processed bull semen have been checked
for B-bodies as a routine procedure for checking product purity in
the practice of the present invention. The results in 523
experiments are tabulated below in Table I where the results from
B-body tests are compared with that from biological tests.
TABLE I ______________________________________ A Comparative Study
of the Product Purity (Female Fraction) in Relation to B-Body and
Biological Test No. of Cells % % Tests Counted Male Female
______________________________________ B-Body Tests 1.
Non-processed 16 5,242 48.5 51.5 2. Convention Counter- 65 26,000
40.9 59.1 Streaming Sedimentation 3. Convection Counter- 28 11,200
30.3 69.7 Streaming Sedimentation and Galvanization Biological Test
4. Convection Counter- 414 43.5 56.5 Streaming Sedimentation
______________________________________
It will be noted that the combination of convection counter
streaming sedimentation with convection galvanization as shown in
line 3 provides a substantial increase in product purity over
convection counter streaming sedimentation when performed
alone.
It should be noted that the specified enzyme concentrations,
digestion and staining times, etc., as described above, are
variable for individual bulls as well as horses. It is to be noted
once again that the use of the protease enzyme in the digestion of
the cell membrane is not necessary in many cases with human and
primate cells due to the difference in the cell membranes thereof
which enhances penetration of the dye without membrane treatment.
The use of the enzymes is particularly suited to other domestic
animals such as horses and bulls in order to enhance penetration of
the dye through cell membranes.
In several cases in which the above process of thermal convection
counter streaming sedimentation combined with forced convection
galvanization was performed, samples containing as high as 92% X or
Y chromosome containing sperm, with in some cases as many as 25
million viable sperm subsequent to thawing of the samples, have
been achieved.
50 ampules of good-quality, high purity female semen processed as
described in the manner of the present invention have been used for
biological tests, that is, by insemination and conception and the
samples have shown a very high conception rate.
Another method of identifying male sperm of human and primates is
the F-body test which is based on the fact that one arm of the Y
chromosome in summatic cells tends to fluoresce with quinicrine
dye. Unprocessed human ejaculations treated with the F-body
staining procedure have been found to produce almost half of the
population fluoresing. While a biological test is impossible with
humans, an obvious conclusion to be drawn is that the fluoresing
population is synonymous to the fluoresing symmatic Y chromosome
and hence the fluoresing cells are Y chromosome containing or male
producing cells. The reproducibility of the F-body technique has
been verified and is accepted now as a standard technique to
identify male spermatozoa in human semen.
In order to evaluate the comparative efficiencies of F-body and
B-body techniques, a comparative study has been made mixing human
and bull semen and applying both F-body and B-body techniques for a
positive and negative correlation. In particular, it has been found
that the B-body test with bull semen fitted in very well with the
biological test results.
In this comparison, 2 milliliters of human semen and 2 milliliters
of bull semen were mixed and processed in this case by forced
convection galvanic separation alone without the first described
method of thermal convection counter streaming sedimentation.
Subsequent to forced convection galvanic separation and processing
as described above, the frozen male and female fractions containing
bull and human semen were treated with the B-body and F-body tests
respectively, with the following results being recorded.
TABLE II ______________________________________ Comparison of
F-Body and B-Body Test Results B-Body Test F-Body Test Cells Cells
% Male Counter % Male Counted
______________________________________ Bull N.P.* 55.8 446 -- --
Male 57.6 1425 -- -- Female 40.5 646 -- -- Human N.P.* 52.2 221
49.0 200 Male 59.4 223 53.5 200 Female 34.9 245 32.8 280
______________________________________ *Not-processed
It will be noted that the F-Body Test did not differentiate male
and female bull spermatozoa.
It should also be noted that subsequent to the mixing of the human
and bull semen and processing, identification of separate bull and
human spermatozoa was still possible due to the different
characteristics thereof.
The above results would indicate that firstly, the F-body test
results in connection with human spermatozoa find general
correlation with the B-body test results also performed on human
spermatozoa, and secondly that the B-body test results performed on
bull sperm finds general correlation with the B-body test results
performed on human sperm. It may thus be concluded that the B-body
test performed in connection with bull sperm is similar to the
F-body test performed on human sperm. The B-body test (found to be
easier and less complicated) is thus equally good for bull as well
as for human male spermatozoa identification.
In one instance, a biological test was performed utilizing a group
of cows artificially inseminated with female sperm derived by the
first portion of the method of the present invention, that is, the
thermal convection counter streaming sedimentation process as
described first above. After 50 to 60 days of gestation, 9 of the
cows were slaughtered and the observed fetuses were all of the
female sex, thus indicating the substantial utility of thermal
convection counter streaming sedimentation utilized above.
It is feasible by using thermal convection counter streaming
sedimentation as described first above as well as convection
galvanization or a combination of both, to avoid most heavy and
light sperm or sperm or abnormal phenotypical characteristics which
are considered to form only a small fraction of the sperm
population carrying abnormal chromosomes which may cause birth
defects. This would reduce those cases of Klinefelter's and
Turner's syndromes, and autosomal defects caused by nondisjunction
and translocation of chromosomes, this being possible by the
rejection of defective heavier and lighter sperm or sperm of
unusual phenotypical characteristics related to the electrical
potential on the cell surfaces thereof. As noted previously, this
electrical potential difference on the cell surface is directly
related to the chromsome structure in each cell and thus by careful
classification of sperm according to such electrical potential
differences, unusual or abnormal chromosomes may be easily
discovered and rejected.
From the foregoing, it will be apparent that thermal convection
counter streaming sedimentation combined with convection
galvanization in the method of the present invention has utility
whenever it is desired to control the sex of mammalian offspring.
The combination of the above methods as well as either thermal
convection counter streaming sedimentation or forced convection
galvanization utilized separately are of extreme practical and
commercial importance in order to meet the great demand in
increasing herds, cattle and hog herds particularly, by selecting
female offspring. This permits the breeder or farmer to have a
choice of a sex in the animal. By way of illustration, the dairy
farmer and exotic cattle breeder can elect to obtain only female
offspring and thereby advantageously breed only milk producing cows
rather than bulls, or on the other hand, exotic breed bulls rather
than cows, as the case may be. As respects to human procreation,
the present method and apparatus may allow normal parents to select
or control the sex of offspring to quickly satisfy the desire to
have a child of a particular sex, thus providing the opportunity to
reduce the total number of children. The observed high fertility of
sperm cells resulting from the process of the present invention
will help in general to achieve success in artificial insemination.
In the cases of parents carrying defective genes, the present
method provides a sure and definitive manner of providing them with
the opportunity of increasing the chance of having a normal baby by
eliminating defective sperm having phenotypical characteristics of
density and electric cell surface potential out of the norm.
* * * * *