U.S. patent number 3,894,529 [Application Number 04/814,906] was granted by the patent office on 1975-07-15 for method and means for controlling the sex of mammalian offspring and product therefor.
This patent grant is currently assigned to Bio-Controls, Inc.. Invention is credited to Wallace Shrimpton.
United States Patent |
3,894,529 |
Shrimpton |
July 15, 1975 |
Method and means for controlling the sex of mammalian offspring and
product therefor
Abstract
A method of controlling the sex of mammalian offspring by
separating spermatozoa into fractions having the desired sex
characteristics and artificially inseminating the female to produce
offspring of the desired sex. The sperm is separated by applying a
buoyant force or forces to a mixture of sperm in nutrient media so
that separation occurs according to density of the sperm. The
nutrient media is controlled as to density characteristics and can
have a uniform density gradient from top to bottom so that buoyant
forces within such media are selectively applied to sperm of
differing density to effect separation of the sperm and to hold
sperm fractions of different density in suspended relation within
the nutrient media. Substantially pure sperm fractions (having the
desired male or female sex characteristics) are isolated at the top
or at the bottom of a separation column for use in artificially
inseminating the female. Under certain circumstances, separation of
the sperm into fractions is enhanced by the application of gas
pressure (positive or negative) above the mixture of sperm and
nutrient media in the column.
Inventors: |
Shrimpton; Wallace (San
Francisco, CA) |
Assignee: |
Bio-Controls, Inc. (San
Francisco, CA)
|
Family
ID: |
25216316 |
Appl.
No.: |
04/814,906 |
Filed: |
April 10, 1969 |
Current U.S.
Class: |
600/35;
435/2 |
Current CPC
Class: |
A61K
35/52 (20130101) |
Current International
Class: |
B03B
7/00 (20060101); A61b 019/00 () |
Field of
Search: |
;128/1 ;195/1.8 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Bhattacharya - Zeitschrift fur Wissenschaftliche Zoologie -
Arademische Verlagsgesselschaft Geest & Portig K.G., Leipzig,
pp. 203-250, 1962..
|
Primary Examiner: Truluck; Dalton L.
Claims
I claim:
1. In a method of controlling the sex of mammalian offspring, the
steps of mixing fresh sperm with a nutrient medium; cooling the
mixture of sperm and medium to a low temperature to immobilize the
sperm; introducing the cooled mixture of sperm and medium to a
separation medium in the form of a separate body of nutrient
medium, at least part of said separation medium being substantially
equivalent in density to the density of said mixture and having a
uniform density gradient extending from a lightest density at the
top to a heaviest density at the bottom; applying buoyant forces to
the sperm introduced to said separation medium tending to separate
the sperm at levels of suspension within the separation medium
according to individual sperm density; separating a portion of the
separation medium of known density containing a suspended sperm
fraction of equivalent density and desired predetermined sex
characteristics; and artificially inseminating a female with the
separated sperm fraction whereby conception occurs to produce
offspring of the desired sex.
2. A method as in claim 1 wherein the fresh sperm is introduced to
the separation media at an intermediate point as respects the
density gradient so that less dense sperm rise to upper portions of
the separation medium while more dense sperm sediment into the
lower portions of the separation medium.
3. A method as in claim 1 wherein a bottom portion of the
separation medium is separated to thereby isolate a bottom sperm
fraction comprising substantially pure X-sperm.
4. A method as in claim 3 wherein the separated portion of the
separation medium contains less than 10 percent of the total sperm
population.
5. A method as in claim 1 wherein a top portion of the separation
medium is separated to thereby isolate a top sperm fraction
comprising substantially pure Y-sperm.
6. A method as in claim 5 wherein the separated portion of the
separation contains less than 10 percent of the total sperm
population.
7. A method as in claim 1 wherein an intermediate portion of the
separation medium is separated to thereby obtain a sperm fraction
containing intermixed X- and Y- sperm.
8. A method as in claim 1 wherein said separation media contains at
least one member of the group consisting of whole mammalian milk,
cream, nonfat milk, egg yolk, dextrose, coconut cream, tomato
juice, glucose, fructose, sugar alcohols, lecithin, amino acids,
living body fluids and tissue extracts.
9. A method as in claim 8 wherein all portions of said separation
media have a density between about 1.010 and 1.150 grams per cc
when measured at 0.degree.C.
10. A method as in claim 1 wherein said separation media has an
osmolility within the range from about 276 to 280 milliosmos.
11. A method as in claim 1 wherein said separation media has a
viscosity between about 2.00 to about 9.0 centipoise when measured
at 0.degree.C.
12. A method as in claim 1 wherein said sperm is a member of the
group consisting of primates, cattles, pigs, sheep, rabbit,
buffalo, goat and horse sperm.
13. A method as in claim 12 wherein said sperm is cattle sperm.
14. A method as in claim 12 wherein said sperm is human sperm.
15. In a method of controlling the sex of mammalian offspring the
steps of mixing fresh sperm with a nutrient medium; cooling the
mixture of sperm and medium to a low temperature to immobilize the
sperm; introducing the cooled mixture of sperm and medium to a
separation medium in the form of a separate body of nutrient
medium, at least part of said separation medium being substantially
equivalent in density to the density of said mixture and wherein
the separation medium is subject to a negative gas pressure to
thereby decrease the apparent density of the sperm relative to the
separation medium; applying buoyant forces to the sperm introduced
to said separation medium tending to separate the sperm at levels
of suspension within the separation medium according to individual
sperm density; separating a portion of the separation medium of
known density containing a suspended sperm fraction of equivalent
density and desired predetermined sex characteristics; and
artificially inseminating a female with the separated sperm
fraction whereby conception occurs to produce offspring of the
desired sex.
16. A method as in claim 15 wherein said negative gas pressure is
achieved by application of a vacuum ranging up to 20 inches of
mercury.
17. In a method of controlling the sex of mammalian offspring, the
steps of mixing fresh sperm with a nutrient medium; cooling the
mixture of sperm and medium to a low temperature to immobilize the
sperm; introducing the cooled mixture of sperm and medium to a
separation medium in the form of a separate body of nutrient
medium, at least part of said separation medium being substantially
equivalent in density to the density of said mixture and wherein
the separation medium is subjected to a positive gas pressure to
thereby increase the apparent density of the sperm relative to the
separation media; applying buoyant forces to the sperm introduced
to said separation medium tending to separate the sperm at levels
of suspension within the separation medium according to individual
sperm density; separating a portion of the separation medium of
known density containing a suspended sperm fraction of equivalent
density and desired predetermined sex characteristics; and
artificially inseminating a female with the separated sperm
fraction whereby conception occurs to produce offspring of the
desired sex.
18. In a method of separating the sperm population in an ejaculate
of a mammalian male into two portions, one portion containing
substantially pure X-sperm or Y-sperm, the steps of preparing a
separation media from nutrient liquids, said separation media
having predetermined density characteristics; cooling the
separation media to a temperature between about -5.degree. C. to
about +2.degree. C.; independently collecting fresh sperm and
mixing the same with a nutrient medium of the same general type as
said separation media; gradually lowering the temperature of the
resultant mixture of sperm and nutrient medium to a temperature
between about -5.degree.C. and about +2.degree.C.; introducing the
mixture of sperm and nutrient medium to said separation media
whereby the latter subjects the sperm to buoyant forces tending to
cause separation of the sperm according to individual sperm density
and wherein said separation media comprises a mixture of at least
two of said nutrient liquids, said mixture of nutrient liquids
being characterized by a uniform density gradient extending from a
lightest density of at least 1.010 grams per cc to a heaviest
density no greater than about 1.150 grams per cc, measured at
0.degree.C; continuing the application of said buoyant forces to
the sperm in said separation media until such time as the sperm
achieves a state of relative suspension in said separation media;
separating a portion of said separation media containing a
suspended sperm fraction of equivalent density and desired
predetermined X- or Y- sperm characteristics; and then inseminating
a female with the separated sperm fraction to achieve conception
and offspring of the desired sex.
19. A method claim as in claim 18 wherein the nutrient liquids
employed in preparing said separation media comprise at least one
member of the group consisting of whole mammalian milk, cream,
nonfat milk, egg yolk, dextrose, coconut cream, tomato juice,
glucose, fructose, sugar alcohols, lecithin, amino acids, living
body fluids, tissue extracts, and mixtures thereof.
20. A method as in claim 18 wherein said fresh sperm is intermixed
with a nutrient medium of the character described having a density
within the range of about 1.025 to 1.038 grams per cc, measured at
0.degree.C.
21. A method as in claim 18 wherein said nutrient medium and said
separation media, when measured at 0.degree.C., each have a pH
between about 6.0 and 8.0, a viscosity between about 2.00 and 9.00
centipoise, and an osmolality between about 276 and 280
milliosmos.
22. A method as in claim 18 wherein said separation media is
prepared from mixtures of whole mammalian milk and its components,
and said fresh sperm is likewise intermixed with a nutrient medium
prepared from a mixture of whole mammalian milk and its components,
said separation media having a density within the range from 1.010
to 1.150 grams per cc and said nutrient medium having a uniform
density of the order of 1.028 grams per cc when measured at
0.degree.C.
23. In a method of separating from the sperm population of an
ejaculate of a mammalian male a sperm fraction containing sex
chromosomes of only one type, the steps of preparing a separation
media, said separation media comprising a density controlled
mixture of nutrient liquids at least two of which are selected from
the group consisting of whole mammalian milk, cream, nonfat milk,
egg yolk, dextrose, coconut cream, tomato juice, glucose, fructose,
lecithin, amino acids, living body fluids, tissue extracts and
mixtures thereof, said separation media being held as a
substantially vertical column and having a uniform density gradient
ranging from a lightest density of the order of 1.010 grams per cc
at the top to a heaviest density of the order of 1.150 grams per cc
at the bottom, cooling said separation media to a temperature of
the order of 0.8.degree.C., collecting fresh sperm and mixing the
same with a nutrient media prepared from nutrient liquids
corresponding to those selected for preparation of said separation
media, the density of said nutrient medium being of the order of
1.028 grams per cc, gradually cooling the intermixed sperm and
nutrient medium to a temperature of the order of 0.8.degree.C.,
introducing the intermixed sperm and nutrient medium to the
separation media at an intermediate point as respects the density
gradient and corresponding in density to that of the nutrient
medium at the point of introduction, applying buoyant forces to the
sperm introduced to said separation media to effect upward movement
of less dense sperm and downward movement of more dense sperm as
relates to the density of the separation media at the point of
introduction, whereby less dense sperm rise to upper portions of
the separation media while more dense sperm sediment into lower
portions of the separation medium, continuing the application of
said buoyant forces to the sperm to effect a suspended state of
separation of the sperm according to individual sperm densities,
separating a portion of the separation media containing a sperm
fraction of equivalent density and desired predetermined density
and sex characteristics, and inseminating a female with the
separated sperm fraction to achieve conception and production of
offspring of desired sex.
24. A method as in claim 23 wherein said sperm fraction is
separated from the bottom of said column of separation media to
obtain a substantially pure sperm fraction containing X-sperm.
25. A method as in claim 23 wherein said sperm fraction is
separated from the top of said column of separation media to obtain
a substantially pure sperm fraction containing Y-sperm.
26. A method as in claim 23 wherein said buoyant forces are applied
for a period of from 1/2 to 24 hours.
27. A method as in claim 26 wherein said buoyant forces are applied
for a period of the order of 21/2 hours.
28. A method as in claim 23 wherein a vacuum of the order of 15
inches of Hg is applied adjacent the top of said substantially
vertical column of separation medium, to thereby decrease the
apparent density of sperm within said separation medium and
particularly adjacent the top of said column.
Description
BACKGROUND OF THE INVENTION
It has been determined that the sex of offspring is controlled by
the chromosomes of the particular spermatozoon or sperm cell which
fertilizes the egg. More specifically, some of the spermatozoa
(hereinafter called "sperm") are genotypically known to contain X
chromosomes, which carry female producing genes, while the others
contain Y chromosomes, which carry male producing genes. In
microscopes, the X chromosomes appear larger in size than the Y
chromosomes. When a sperm containing X chromosomes (hereinafter
called X-sperm) combines with the egg (which contains X
chromosomes), female offspring results. When a sperm containing the
Y chromosomes (hereinafter called Y-sperm) combines with the egg,
male offspring results. The sperm population in an ejaculate of a
mammalian male contains both X-sperm and Y-sperm. Heretofore,
separation of these sperm in X and Y components has not been
satisfactorily achieved. It is evident, however, that a
satisfactory procedure for separating the two kinds of sperm, to
isolate substantially pure X and Y-sperm fractions, would permit a
choice or selection of the ultimate sex of the offspring.
SUMMARY OF THE INVENTION AND OBJECTS
This invention relates generally to a method and means for
controlling the sex of mammalian offspring, and to compositions
useful in providing offspring of one sex. More particularly, the
invention relates to a method and means for separating spermatozoa
containing X chromosomes from those containing Y chromosomes, to
obtain substantially pure fractions of spermatozoa containing
either X chromosomes or Y chromosomes.
The present invention is predicated on my discovery that the two
sperm genotypes of mammals (X and Y) may be separated according to
density characteristics by application of buoyant forces within a
liquid separation medium to cause more buoyant sperm to attain a
different level in the separation medium than less buoyant sperm.
The term "buoyant force" is used herein to include both positive
buoyant forces which cause the sperm to rise or float in the medium
and negative buoyant forces which cause the sperm to fall or
sediment in the medium. In a preferred practice of the invention,
use is made of a separation medium (or media) arranged as a
substantially vertical column and having a uniform density gradient
from a lightest density at the top to a heaviest density at the
bottom. Alternatively, the separation medium is in the form of
layers or zones of compatible liquids, each of slightly different
density, to similarly provide a separation media which varies from
a lightest density at the top to a heaviest density at the bottom
of a column. Separation is achieved by introducing a sperm
population to the separation medium at a point intermediate the
ends of the column, in media equivalent in density to that of the
point of introduction, so that the sperm are separated according to
density by the simultaneous application of positive and negative
buoyancy.
It is possible to predicate the rise or fall of an inert particle
in a liquid medium upon Stokes' law, which relates the velocity of
an inert particle to the forces of buoyancy within the liquid,
viscosity of the liquid, gravitational force and the difference in
density between the medium and the particle. As a deduction from
Stokes' law: ##EQU1## v = particle velocity (cm sec.sup..sup.-1) k
= numerical constant in Stokes' law multiplied by those particle
factors related to hydrodynamic efficiency in the medium d.sub.1 =
density of particle d.sub.2 = density of media g = acceleration of
gravity (981 sec.sup..sup.-2) w = viscosity at the medium
temperature, in centipoise.
Under Stokes' law, the velocity of rise or fall of the particle
varies with the hydrodynamic efficiency which is related to the
size and shape of the particle, the difference in density between
the medium and the particle and the characteristics of the medium
as a Newtonian solution at the temperature of separation. When the
velocity of rise or fall in a particle class is closely related to
Stokes' law, particles in a liquid of uniform density tend to rise
or fall in conformity with the factors outlined above. Where the
separation liquid varies in density, or has a density gradient, one
can expect the velocity of rise or fall to reduce to zero at the
point where the density of the particle equals the density of the
liquid.
I have found that under carefully controlled conditions a sperm
population introduced to a separation column at an intermediate
point can be separated, at least in part, by the rise and fall of
the sperm related to the individual sperm densities. Moreover,
where the separation media has a uniform density gradient, the
sperm will achieve a suspended state of separation related to
slight density variations in the individual sperm so that desired
sperm fractions or layers can be easily separated from the whole.
When the range of liquid densities within the density gradient
completely encompasses the range or span of individual sperm
densities, the sperm fractions or populations at the top and bottom
of the column will, when inseminated, produce offspring of
different sex. Finally, the observed phenotypical differences in
the sperm obtained from such top and bottom fractions (up to 10
percent of the total sperm population) have been found to be
related to sex genotypes, that is, the top fraction has been found
to contain substantially all Y-sperm whereas the bottom fraction
has been found to contain substantially all X-sperm.
From the foregoing, it will be apparent that the present invention
has utility wherever it is desired to control the sex of mammalian
offspring. It is of extreme practical and commercial importance 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 offpsring from superior animals in the herd
while obtaining male calves from the remainder of the herd, thereby
increasing both his commercial return from the sale of calves and
the genetic quality of his herd while also increasing milk
production from improved cows.
As respects human procreation, it is well known that the rapid
increase in human population poses a serious threat to eventual
over-population. An existing strong incentive to human procreation
is the desire to have offspring of a particular sex, which, when
frustated, leads to further procreation. The present invention
enables parents to select or control the sex of offspring to
quickly satisfy the desire to have a child of a particular sex,
thereby providing the opportunity to reduce the total number of
children desired.
The present invention therefore has important application and
potential in solving the food problems in the hungry world by
making possible:
1. An increase in the production of animal food protein through
selective breeding programs to obtain livestock which predominate
in a particular desired sex.
2. A reduction in the human birth rate.
In general, a principal object of the present invention is to
provide a truly successful method for controlling the sex of
mammalian offspring.
A further object of the present invention is to provide a method
for separating the X-sperm and Y-sperm in the ejaculate of a
mammalian male, to obtain substantially pure fractions containing
either X-sperm or Y-sperm, useful in artificial insemination of the
female to obtain the desired sex.
A further object of the invention is to provide novel compositions
containing either substantially pure X-sperm or substantially pure
Y-sperm, capable of producing mammalian offspring of the desired
sex.
A further object of the invention is to provide novel means for
carrying out the sperm separation method of the present
invention.
A further object of the invention is to provide a novel method and
means of the above character which makes possible the separation of
substantially pure fractions of X-sperm and Y-sperm, capable of
producing normal fertilization.
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.
FIGS. 2, 3 and 4 are schematic representations of a particular step
in the processing, at various points in time.
FIG. 5 is a graphical representation corresponding in point of time
to the schematic representation of FIG. 4.
FIG. 6 is a schematic representation of one system of apparatus
useful in carrying out the method of the present invention.
FIG. 7 is a fragmentary view in section and elevation of another
embodiment of apparatus useful in carrying out the method of the
present invention.
FIG. 8 is an enlarged detail view in section and elevation of a
portion of the apparatus shown in FIG. 7.
FIG. 9 is a view in horizontal section along the lines 9--9 of FIG.
8.
FIG. 10 is a schematic representation of a further embodiment of
apparatus useful in carrying out the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
FIG. 1 is a flow sheet illustrating the method of the present
invention. In step 11, fresh sperm is collected from the male,
containing approximately equal amounts of X-sperm and Y-sperm. This
sperm is mixed with a nutrient medium in step 12, following which
the mixture of sperm and medium is gradually cooled in step 13. The
cooled mixture of sperm and medium is next subjected to processing
in steps 16 and 17 to effect separation of the sperm according to
density. This latter processing is carried out in the presence of a
separation media which has been previously prepared to provide
predetermined desired density characteristics, as in step 14. In a
preferred practice of the invention, the processing in step 14
provides the separation media with a uniform density gradient
sufficient to span the range of individual sperm densities.
Following preparation of the separation media, it is cooled to a
temperature to immobilize the sperm, in step 15. The intermixed
sperm and nutrient medium are also cooled in step 13 to insure
substantial immobilization of the sperm, which is then introduced
to the separation media in step 16. In the case of a separation
media having a uniform density gradient, the sperm is introduced at
an intermediate point of the density gradient. Thereafter the sperm
is subjected to the buoyant forces within the separation media, in
step 17, to cause separation of the sperm according to the
individual sperm density. In a density gradient system, the desired
sperm fractions achieve a suspended state of separation in the
separation media adjacent the top and bottom of the column, thereby
facilitating separation of a sperm fraction of desired sex
characteristics in step 18. The separated sperm fraction (X-sperm
and Y-sperm) can then be employed to inseminate the female, as
represented by step 19, to obtain the desired offspring.
The present invention is suitable for use with all mammals. Of
particular interest are cattle, swine (i.e., hogs and pigs), human
beings and other primates, sheep rabbits, cats, dogs, goats,
horses, donkeys and buffalo. It will be understood that the sperm
fraction separated in step 18 is inseminated in step 19 into the
female of the species from which the sperm is taken. Of course,
horse may be crossed with ass and zebra as may wolf with dog.
The present invention is predicated on determination that there is
a difference between the average or mean density of the X-sperm and
Y-sperm of mammalian species, the X-sperm being generally more
dense than the Y-sperm. For example, in bulls this difference in
average density between sperm containing the X- and Y-chromosomes
is believed to be about 0.5 percent at 0.degree. C. In human males
and in rabbits, the difference appears to be greater than in
bulls.
In general, the medium selected for use in steps 16 and 17 must
have a density sufficiently close to that of the sperm so that the
slight difference in density between the X-sperm and the Y-sperm
will result in the separation of at least part of the sperm into
separate fractions containing predominantly X-sperm or Y-sperm.
Related to density, viscosity must also be appropriate for
controlling the separation. In addition, the medium must not impair
the viability or the fertility rate of the sperm, that is, the
medium must not harm or destroy the sperm. To the contrary, the
medium must provide nutrients to keep the sperm alive. The medium
must also have a suitable pH (i.e., within the range from 6.0 to
8.0) to permit it to act as a buffer and to avoid toxic effects or
impairment to the fertility of the sperm. The final consideration
is that the medium should have the characteristics of a normal body
fluid, and, in particular, its osmotic pressure should be within
the range from about 277 to 280 milliosmos to avoid any possibility
of harmful compression or expansion of the sperm. Throughout the
specification and claims, measurement of pH density and viscosity
are at 0.degree. C.
I have found that a highly satisfactory nutrient medium for
carrying out the separation technique of the present invention is
derived from whole mammalian milk and its components. Control of
density, for example, in preparing a separation column having a
uniform density gradient, can be readily obtained through use of
the components of commercially available types of cow's milk. In
practice, three media are initially prepared having average
densities, respectfully, of about 1.025, 1.035, and 1.044. The
average density of the lightest media is achieved by using a milk
product known as "half-and-half" (meaning that it contains
approximately equal portions of homogenized milk and separated
cream, mixed with ordinary homogenized milk). Thus, an average
density of about 1.025 is obtained by mixing half-and-half with
small amounts of homogenized milk, as necessary. An intermediate
density (e.g., from about 1.034 to about 1.038) is similarly
obtained by mixing homogenized milk with small amounts of dialyzed
distilled nonfat milk. A relatively dense media of average density
1.044 is prepared by mixing a larger proportion of dialyzed
distilled nonfat milk with homogenized milk. Antibiotics to protect
the sperm introduced to the medium can also be employed.
Fresh mammalian milk such as cow's milk and its components have a
suitable viscosity, density and pH (about 6.4 to 6.8) for use in
carrying out my separation process. Cow's milk is also a normal
body fluid, making it appropriate for use generally as a medium.
Since mammalian milk is approximately isotonic with the blood of
the animal from which it is drawn, its osmotic pressure or
osmolality is also more easily adjusted to a value compatible with
that of the sperm (i.e., between about 276 and 300 milliosmos). As
hereinafter described (see specific examples), specific desired
values of density, viscosity, osmolality, and pH can easily be
obtained during the operations to prepare the separation media for
use in the sperm separation steps.
Satisfactory nutrient liquids are also derived from other mammalian
sources (e.g., human milk) or derivatives of mammalian milk (e.g.,
milk powder). Nutrient liquids based on egg yolk, dextrose, coconut
cream (derived from green coconuts), tomato juice, glucose,
fructose, lecithin, amino acids, living body fluids and extracts,
tissue extracts (e.g., liver extract), and mixtures of these, are
also satisfactory. Egg yolk particularly includes glucose,
fructose, and amino acids which provide nutrients and assist in
fertility. When used in conjunction with glycine, desired values
for density, viscosity, osmolality and pH can be obtained. The
glycine (preferably an aqueous solution of 2 to 5 percent glycine)
serves to buffer the pH and to depress the freezing point of the
egg yolk in the medium. In general, the ratio of glycine to egg
yolk depends principally upon the initial density, viscosity, and
osmolality of the egg yolk. The ratio can be varied, of course, as
may be necessary in preparing a uniform density gradient. Since the
glycine solution has a lower viscosity than the egg yolk, more will
be required where a viscosity depressant is also needed. Generally,
based on a 4 percent glycine solution, one part of glycine will be
required for each one to four parts of egg yolk, depending on
density requirements.
As previously noted, the separation method of the present invention
is best carried out in conjunction with a separation media having a
uniform density gradient. While various procedures are known for
the preparation of density gradient columns, one particularly
satisfactory procedure is carried out in connection with apparatus
as generally represented at 20 in Figure. Thus, assuming the use of
three separate media based on cow's milk, each adjusted to an
average desired density in the manner previously described, the
medium of lightest average density (e.g., 1.025) is placed in the
uppermost container 21. The media with an intermediate average
density (e.g., 1.035) is placed in the lower container 22. As
illustrated, the two containers, 21, 22, are suspended on a pulley
arrangement which functions to raise the container 22 at the same
rate that it lowers the container 21. More specifically, the
separate containers are suspended from the ends of a chain 23
reeved about the rotary supports 24 and the drive pulley 25. The
entire unit is powered by the motor 26 through the drive take-off
27 and worm gear arrangement 28. As will be understood, the
illustrated apparatus serves to simultaneously introduce the
liquids from the containers 21 and 22 to a mixing chamber 29, at
controlled rates of flow determined by the hydrostatic or liquid
head for the media in each container as respects the mixing
chamber. Thus, the flow of the less dense nutrient media in
container 21 begins at a maximum and decreases to a minimum, as
represented by the dotted line 30. In like fashion the flow of the
denser nutrient medium in container 22 begins at a minimum and
increases to a maximum, as represented by the dotted line 31. On
the other hand, the total flow at any time through the mixing
chamber 29 remains constant. The mixture discharged from the bottom
of the mixing chamber 29 will therefore have a linear density
gradient ranging from the density of the lightest media in
container 21 to that of the intermediate media in container 22. A
desired intermixing of the media introduced to the mixing chamber
29 is accomplished by conventional agitation means, for example, as
illustrated in FIG. 6.
In the use of the apparatus shown in FIG. 6, a media having the
described linear density gradient passes from the mixing chamber 29
through a surge coil 32 to a vented manifold 33, whence it is
discharged in equal proportionate amounts to the bottom of the
various separation columns, represented at 34. To facilitate use of
the apparatus, a medium of relatively heavier average density
(e.g., 1.044) is placed in a third container 35 from which it can
be introduced to the bottoms of the columns 34 through the separate
vented manifold 36, under control of the valving means 37. As
hereinafter explained, the heavier medium in container 35 is used
to adjust the height of the density gradient columns within the
separation devices 34.
In carrying out the separation method of the invention, the
separation media is introduced to form substantially uniform
density gradient columns within each of the separation devices 34
and thereafter is gradually cooled to a temperature which will
immobilize without harming the sperm. In the illustrated apparatus,
this cooling is accomplished by a circulatory refrigeration
apparatus including the refrigeration unit 38, the cooling tank 39
surrounding the separations devices, and a body of cooling water 40
containing glycerol which is circulated between the refrigeration
unit and a tank by the inlet and outlet conduits 41 and 42,
respectively. With conventional cooling equipment, cooling of the
density gradient columns within the separation devices from room
temperatures to a temperature below about 1.degree. C. is
accomplished in a very short period of time, ranging from a few
minutes up to a few hours, at the most.
Prior to initiating the separation, fresh sperm is collected and
intermixed with a nutrient medium of the same type and
corresponding in density to an average of median density of the
media forming the density gradient column (e.g., about 1.028 in the
system described). The sperm sample is also pre-cooled to a
temperature below 1.degree.C. and thereafter, as illustrated in
FIGS. 2 and 6, is introduced to the separation column at an
intermediate point of the density gradient column. For best
results, the density of the sperm plus extender sample should
correspond closely to the density of the separation media at the
point of introduction. To insure that this occurs, a small amount
of the nutrient medium for the sperm can be initially introduced to
the separation column, preferably containing a small amount of a
coloring ingredient so its relative density with respect to that of
the column can be readily determined by the movement of the colored
segment within the column. Thus if the position of the colored
insert indicates that the density gradient column in a particular
device 34 should be adjusted upward, a small amount of the heavier
medium in the container 35 can be introduced through the manifold
36 and valving means 37 to the bottom of the density gradient
column in question. On the other hand, if a particular density
gradient column is too high within its separation device 34, a
small amount of the separation media originally present can be
discharged through the stopcocks 43 positioned at the bottom of
each of the separation columns 34. The important consideration is
that the density of the separation medium opposite the point of
sperm introduction (represented at 44 in FIGS. 2 and 5) be
approximately equal to that of the nutrient medium containing the
introduced sperm.
With reference to FIG. 6, each of the separation columns 34 is
provided with inlet valving means 45 operable by the valve controls
46. At such time as the sperm is to be introduced to the separation
columns, the sperm within refrigerated containers 47 is moved to
positions adjacent the filling spouts 48. The valve controls 46 are
then operated to introduce the sperm through the filling lines 49
to the midpoints of the density gradient columns contained within
the separation devices 34.
Within the density gradient columns, represented at 50 in FIGS. 2
and 4, positive and negative buoyant forces are applied to the
sperm to cause the less dense sperm to rise into upper portions of
the column and the more dense sperm to sediment into lower portions
of the column. Thus as particularly illustrated in FIG. 2, the less
dense sperm move at a relatively rapid rate into the upper portions
of the column due to substantial relative density differences
existing at the point of introduction. As the lighter sperm rise in
the column they encounter separation media of gradually decreasing
density within the density gradient (FIG. 3), with the result that
the individual sperm eventually achieve a state of suspension
within the separation medium related to its own density. In similar
fashion, the more dense sperm rapidly sediment into the bottom
portions of the column, gradually losing sedimentation velocity
until such time as they likewise achieve a state of suspension in
the lower portions of the separation column.
While it has been determined that the X-sperm are generally heavier
than the Y-sperm, this is not uniformly so. Instead, I have found
that there is a normal Gaussian distribution of the two populations
so that the difference in density between the X and Y sperm, as
previously indicated, represents a difference between the mean or
average densities of the two populations. Thus, referring to FIG.
5, which is a plot of the distribution of the different populations
with reference to varying density within the columns, this
difference in mean density is represented by the dimension A. On
the other hand, as represented in both Figures 4 and 5, the
separation by density of the X and Y populations according to
relative density makes possible the isolation of substantially pure
X and Y sperm populations at the top and bottom of the separation
columns. These isolated substantially pure fractions are
represented by the sperm populations shown in zones 51 and 52. As a
practical matter, I have found that the sperm capable of being
isolated in either of these zones generally comprise less than 10
percent of the total sperm population. On the other hand, since the
ejaculate of a mammalian male normally contains many millions of
sperm (e.g., a portion of the ejaculate will average 400 million
sperm for a typical male bull), the number of sperm present in one
or more of the isolated fractions (i.e., 20 to 60 million sperm) is
sufficiently large under normal circumstances to permit
insemination and conception of offspring of the desired sex.
Moreover, the intermixed sperm contained within the intermediate
portion of the column, represented at 53 in FIG. 4, is not lost,
but remains available for use by animal breeders and the like to
effect normal artificial insemination wherever sex predetermination
or control is not desired.
It will be understood that the separate indications in FIGS. 2 to 4
represent different points in time. Thus, FIG. 2 illustrates the
point of introducing the sperm to the columns. FIG. 3 represents a
point in time where the sperm are still in motion, due to the
continuing effect of the buoyant forces within the separation media
(positive and negative) upon the individual sperm. FIG. 4
represents a final equilibrium condition wherein the individual
sperm have achieved a state of suspension within the density
gradient of the separation medium, according to individual
densities. With any particular separation media satisfactory for
use in my process, I have found that the time required to reach the
equilibrium state may vary only slightly from one separation to the
next. On the other hand, slight differences in temperature or in
the viscosity of the separation media may cause the time necessary
to completely carry out the separation process to vary
considerably. Generally, no more than 24 hours are needed in any
event, to carry the separation process to the equilibrium state. In
fact, about 1/2 to 4 hours is usually sufficient, with about 21/2
hours being indicated as optimum.
It will be evident that if insufficient time is used for
separation, the equilibrium state may not be achieved and the
separation of pure sperm fractions may not result. On the other
hand, if more than about 24 hours are taken in the separation step,
the medium itself may tend to separate. That is, the inorganic ions
and other heavy particles in the colloid medium may separate after
long periods of time. Moreover, the particles of the medium may
tend to "salt out" or precipitate from the fluid if substantially
more than 24 hours are used.
For best results, assuming the use of a density gradient column,
the median density of the separation medium (i.e., at the midpoint)
should be close to the median density of the sperm of the mammal
from which the sperm is taken. With respect to humans and bulls,
the density range is about 1.01 to about 1.19 grams per cubic
centimeter, with the median density falling in the range from about
1.028 to about 1.036. In practice, I have found that a density
gradient column for use in separating bull and human sperm should
range from about 1.010 to about 1.150 grams per cubic centimeter,
with a median density of the order of 1.028. If the median density
of the density gradient column approaches the upper limits of the
density range, the lighter X-sperm will tend to commingle with the
Y-sperm at the upper end of the column so that separation of a pure
Y-sperm fraction becomes difficult if not impossible. In like
fashion, if the median density of the density gradient column falls
too close to the lower limits of the density range, the heavier
Y-sperm will tend to commingle with the X-sperm so that separation
of pure X-sperm fraction is impaired. Consequently, in order to
insure a separation of substantially pure fractions of X-sperm and
Y-sperm, the median density of the density gradient column should
closely approximate the average density of the sperm sample. In
addition, the range of densities in the density gradient column
should be sufficient to insure that a full range of rise and fall
of the sperm is permitted, particularly as respects the lightest
Y-sperm and the heaviest X-sperm at the extreme density ranges of
the sperm sample.
The viscosity of the separation medium should also fall within a
predetermined viscosity range for satisfactory separation. In
general, the viscosity of the separation medium is related to the
density. For humans, rabbits and cattle, viscosity should be
between 2 to 9 centipoise measured at 0.degree.C.
Table 1 sets forth the range of densities and viscosities for
various species of mammals, and also indicates a medium density and
viscosity for satisfactory separation in a density gradient column.
All measurements taken in Table 1 are at 0.degree.C. with the
density in grams per cc and viscosity in centipoise.
Table 1 ______________________________________ Density Low High
Median Viscosity at 0.degree.C.
______________________________________ Human 1.015 1.09 1.017-1.030
2 - 9 Centipoise Rabbit 1.015 1.09 1.022-1.030 2 - 9 " Bull 1.020
1.10 1.027-1.032 2 - 9 " ______________________________________
While the osmotic pressure of the medium may vary between a lower
limit of about 276 and an upper limit of about 300 milliosmos, as a
practical matter I have found that best results are obtained when
the osmotic pressure of the medium is about 280 milliosmos.
Depending on the medium employed, a temperature between about
-5.degree.C. and about 2.degree.C. is required. Below this
temperature range, the physical properties of the medium are
changed to such an extent that the desired rise and fall of the
sperm to achieve a desired state of suspended separation will not
result. At higher temperatures, the sperm tend to swim or move on
their own through the medium so that the buoyant forces within the
separation medium fail to separate the sperm as substantially inert
particles. Accordingly, a preferred temperature for use of a
density gradient column prepared from whole mammalian milk is
0.8.degree.C.
During the process care must be taken to avoid excessive vibration.
Violent shaking tends to tear the tails from the sperm. Moreover,
during the separation sequence, steps 16 and 17, even slight
vibrations will effect the rise and fall of the individual sperm so
that they do not act as inert particles. In the steps of separating
the sperm fractions, step 19, particular care is required to avoid
vibration and resultant intermixing and contamination of adjacent
fractions.
Another factor to be considered is the avoidance of visible light.
Light affects the fertility of the sperm. Fertility can also be
affected by extremely high or low pH of the medium (outside the
range 6.0 to 6.8 for most species), age of the sperm, and number of
motile sperm.
The use of a density gradient column results in a suspended state
of separation of the sperm according to sperm density. Once the
sperm have achieved the equilibrium state, represented in FIG. 4,
the desired substantially pure sperm fractions should be
immediately separated from the top or bottom of the column and
preserved for artificial insemination. With the apparatus of FIG.
6, it is a relatively simple matter to withdraw the X-sperm
fractions from the bottom of the columns 34 through the stopcocks
43. However, to avoid intermixing of adjacent fractions, it is
desirable to drain drop-by-drop with about 5 to 10 seconds per
drop. It will be understood that the Y-sperm fraction at the top of
the column is done by draining the entire column, with the Y-sperm
fraction being the last to be recovered. Alternatively, the Y-sperm
fraction may be initially removed from the top of the column with a
special apparatus for this purpose, for example, a special burette
fitted with a Pasteur pipette.
Referring to FIG. 7, apparatus is shown which is particularly
useful in initially removing the uppermost or Y-sperm fraction from
the top of the column. As particularly shown in FIGS. 8 and 9, the
separation apparatus comprises a cylindrical burette or column 60,
suitably provided with apertures 62 along its length to facilitate
the separation of closely spaced liquid fractions. Each of the
apertures 62 is in communication with a separating or fractionating
valve mechanism 64 which controls the discharge of a sperm fraction
into the receiving vials 66 mounted on the column by means of the
bottom supports 67 and clips 68. Thus, as best seen in FIG. 8, each
aperture 62 is closed by a spring biased valve member 70 operable
by a pull rod 72. The pull rod can be actuated through the pulls 74
to move outwardly, thereby discharging a sperm fraction through the
discharge spout 76 into the vial 66. Since individual valving
mechanisms 64 and vials 66 are spaced along the length of the
column with respect to each aperture 62, the illustrated mechanism
makes possible the separation of minute fractions along the entire
length of the column. Alternatively, of course, a larger specimen
may be withdrawn from a substantially larger portion of the column
by operation of a single valving mechanism 64 at the bottom of a
desired column length. The vials 66 can thus be made sufficiently
large to accommodate the volume of the several fractions, as may be
necessary.
As illustrated in FIG. 7, the individual valving mechanisms 64 are
constructed to be operated in automatic or semi-automatic fashion
by means of the cam actuated rod and actuator mechanisms 76, 78.
The latter are adapted to engage between the actuators 74 of the
pull rods and the body of the valve mechanisms 64, to bias the
valve members 70 outwardly. Thus as particularly illustrated in
FIG. 7, each of the valve actuators 76 and 78 are provided with
spring biased cam followers 79 which cooperate with the rotating
cams 80 and 82 at the top and bottom of the column, respectively.
The cam actuators can be rotated at a predetermined rate to effect
a desired sequential operation of the valving mechanism 64 by
suitable drive means (e.g., a variable speed electric motor) at the
bottom of the column and similar means at the top (not shown). It
will be understood that the number and positioning of the valve
actuators 78 with respect to the camming mechanisms can be
predetermined to facilitate fractional separation of sperm samples
from the interior of the column 60, as may be appropriate to a
particular separation technique.
FIG. 7 illustrates the mounting of the separating column 60 within
a refrigeration enclosure. In view of the use of sperm collecting
vials 66, cooling of the columns in this embodiment is accomplished
through use of a dry gaseous atmosphere, for example, dry cold air
at a temperature below about 1.degree.C. Refrigerated gas from the
cooling operation may be supplied by any appropriate means, as
represented by the refrigeration chamber 86 and impeller 88, which
supply refrigerated gas to the cooling chamber 90 through the
conduit 92. Cooling gas discharged from the outlet 94 may be
recovered for recycling or discharged to the atmosphere, depending
upon the particular cooling system employed. In other respects the
separating column 60 is employed in the separation process in
similar fashion to the processing described with respect to columns
34, the valving mechanisms 96 and 98 being the counterparts of the
manifolds 33 and 36 in the apparatus of FIG. 6. As illustrated, the
valving mechanisms 96, 98 and the associated conduits 100, 102 are
in direct communication with the bottom opening of the columns 60
(i.e., through chamber 104).
In a typical use of the apparatus illustrated in FIGS. 7 to 9, the
columns 60 are prepared in the previous manner by introducing a
separation media of predetermined desired density characteristics.
Each column is then cooled by suitable application of the
refrigeration unit and circulating system 86, 88 to obtain a
desired temperature of the separation media. The sperm samples to
be separated are likewise prepared by intermixing with a nutrient
medium in a manner previously described. Assuming the use of
density gradient columns, the vertical position of the separation
media or columns can be adjusted by the technique previously
described. A sperm sample, after being first gradually cooled to
immobilize the sperm, is then injected through the conduit 49 and
valving mechanism 45 at the point of average or median density to
initiate the sperm separation through action of the buoyant forces
within the density gradient column. After equilibrium conditions
have been reached, the camming mechanisms for the valve operators
78 are energized to initiate withdrawal of sperm fractions. In a
preferred technique, the sperm fractions are withdrawn from the top
of the column in descending fashion, to obtain substantially pure
fractions of the lightest sperm (Y-sperm). The central portion of
the column 60 is then discharged in a single operation through a
timed operation of a particular valving mechanism (e.g., 106 in
FIG. 7) obtained by a timed deactivation of the cam motor 84. When
the central portion of the column has been drained, the cam motor
84 is energized to draw off the lowermost fractions, again
descending from the top, to obtain the heaviest sperm fractions
(X-sperm). In other words, the actuation of the cams in the upper
portion of the column serve to draw off the sperm fraction in the
zone 52 (see FIG. 4), whereas the actuation of the valve mechanisms
adjacent the bottom of the column serve to separate the sperm
fraction in zone 51.
Under certain conditions, the separation processing is facilitated
by use of a gas under positive pressure (i.e., 0.1 to 10 psi) in
the head space above the separation media in the column 60. Such
gaseous pressure which may be exerted by a cold dry air or an inert
gas such as N.sub.2 or CO.sub.2, supplements the hydrostatic head
serving to discharge the sperm fractions into the vials 66. Such
use of gas under pressure is particularly useful, for example, in
facilitating discharge of the intermediate sperm fractions through
the valving mechanism 106.
In preparing the separation media in the form of a density gradient
column for use in operations just described, various techniques can
be employed in addition to those shown and described with reference
to the apparatus in FIG. 5. For example, a milk media can be
appropriately prepared to a desired density gradient by subjecting
commercially available homogenized milk to the substantial
centrifugal forces possible with modern ultracentrifuges. Such
devices, which rotate at speeds in excess of 1,000 revolutions per
minute, generate forces enormously greater than gravity. The result
of such centrifugation is to distribute the molecules in solution
in the centrifuge cell in such way that the density is higher the
greater the distance from the center of the rotor. I have found
that a milk media with a desired density gradient can be prepared
in relatively short time with ordinary laboratory centrifuges,
without any change in the osmolality of the milk media which
remains substantially constant throughout the density gradient. The
range of the density gradient can also be varied by using mixtures
of half-and-half and homogenized milk to vary the starting fat
content. Control over the density gradient can additionally be
obtained by variations in the speed and time of centrifugation.
To facilitate practice of the invention in relatively
underdeveloped countries, even simpler procedures are available for
preparation of density gradients. One such procedure is illustrated
in FIG. 10 which illustrates interconnected vessels 110 and 112
which may be simultaneously discharged by valve means 114 and 116.
In this procedure, the dense liquid contained in vessel 110 is
introduced into the bottom of the vessel 112 containing the less
dense liquid, where the two liquids are mixed by a simple propeller
agitator 118. The resulting mixture flows out of the vessel 112
through a tube 122 that leads to the bottom of the density gradient
column being formed in the column 120. If the rate at which the
denser liquid flows from the vessel 110 is exactly half the rate at
which the mixture flows out of the vessel 112, a linear density
gradient will be formed in the column 120, with the denser liquid
at the bottom and the less dense liquid at the top. An even simpler
way to produce a density gradient is to pour a lighter liquid
gently over a heavier liquid, initially poured into the bottom of
the column. With time, interdiffusion of the two miscible liquids
will nullify the transition zone between the liquids of different
density to eventually achieve a linear gradient, varying more or
less directly with the height of the column. This technique can be
speeded by the use of very gentle agitation at the transition point
between the liquids of different density.
The processing of the present invention makes possible the
separation of sperm fractions of varying degrees of purity, that
is, fractions containing varying amounts of either X-sperm or
Y-sperm. As represented in FIG. 4, which is typical of conditions
actually achieved with the sperm of the mammalian species herein
mentioned, the separation of substantially pure fractions of
X-sperm or Y-sperm is possible, and has been obtained by the
present method (see specific examples). Thus, as previously
mentioned, the zone 52 in FIG. 4 contains substantially pure
Y-sperm. The fraction contained in zone 51 contains substantially
pure X-sperm. Fractions effective to carry out the purposes of the
present invention should have at least 70 to 80 percent of either
the X-sperm or the Y-sperm (according to the selection) to overcome
the 50--50 ratio existing in nature. For commercial purposes, at
least 70 to 80 percent of the sperm should be of the desired sex,
if the expense involved in carrying out the method is not to be
prohibitive. While a fraction containing at least 90 percent of
sperm of a single type is essential to reduce the change of
obtaining offspring of the opposite sex to a statistically
tolerable rate, a fraction substantially 100 percent pure would, of
course, be the most desirable since there would be no change of
error. The complete separation of a pure fraction, made possible by
the present invention, assures that if there is any conception at
all, the offspring will be of the desired sex. Thus, where
separation of a substantially pure fraction is accomplished, as in
FIG. 4, each of the fractions represented by the sperm in zones 51
and 52 comprises essentially sperm having only the desired
chromosomes in a carrier.
In carrying out the separation procedures herein described, it is
desirable to have a procedure for testing the various fractions to
determine the density of the solution. I have found that one
suitable procedure involves means for measuring density, such as a
plurality of containers containing solutions of known varying
density (e.g., CuSO.sub.4) into which droplets of media may be
introduced to determine density. As an alternative embodiment, a
plurality of small hydrometers might be placed in the separation
column to determine different density levels within the column,
this latter embodiment would avoid the necessity of taking small
fractions and testing each one.
When mammalian milk or body fluid is used as a separation medium,
determination of the density in various zones within the separation
column may be determined without draining by using monochromatic
light or radiant energy of various frequencies that measure the
opacity of the mixture. Thus, with a transparent or translucent
mixture, such as milk, a light source may be placed on one side of
the column and a photoelectric cell on the other side (connected to
an amplifier and a recorder) to measure the opacity of the mixture
to determine where the separated layers of sperm are located.
Location of desired density layers or the desired fractionating
point between the desired layers, may also be determined by
measuring the conductivity at various points in the column. Thus
multiple electrodes are placed in the column to determine the
change in conductivity of the contents of the column at various
points along its length. The conductivity of the separated sperm
fractions is different from that of the supporting medium.
Other means for determining the location of the separated sperm
fractions include a micro-densitometer, which measures density
using polarized light, and microscope techniques which measure the
scattering of polarized light.
In a further embodiment, a gelatin tube may replace the burette
forming the separation column. When the separation process has been
carried to the state of equilibrium represented by FIG. 4, the tube
and its contents are gradually cooled to a temperature well below
freezing, for example -20.degree.C., and the separated sperm
fractions frozen in situ. The frozen tube may then be cut into
appropriate fractions as desired. Storage of the separated sperm
fractions for relatively long periods is then possible.
In those cases where the average or mean density between the
X-spern and Y-sperm of the species being separated is quite small,
for example, in the case of bulls, the degree or range of
effectiveness of the positive and negative buoyant forces within
the density gradient column becomes somewhat more critical. I have
found that this difficulty can be largely overcome through
application of negative gas pressure above the surface of the
separation column, to achieve a Cartesian effect. Specifically, I
have found that by application of a vacuum in the head space above
the separation column (e.g., in the space 130 in FIG. 3), the
apparent density of the sperm being separated within the density
gradient column is decreased, with the apparent density of the
Y-sperm at the top end of the tube decreasing to a greater degree
than the apparent density of X-sperm at the bottom end of the tube.
While the physiological factors underlying this decrease in
apparent density is not clearly understood, it is postulated that
the spermatozoa have an elastic exterior which is therefore
responsive to the negative pressure in the space above the column.
By way of illustration, the individual spermatozoa may be compared
in the initial state to tiny balloons carrying tiny weights so that
the resultant effect of the buoyant forces on the sperm is close to
zero (i.e., neither positive nor negative). In such condition, the
sperm will tend to move up and down in the media in response to any
change in the gas pressure above the media surface. The explanation
for this phenomenon is that the separation media is relatively
incompressible and readily transmits to the compressible sperm the
force of the changing gas pressure, thus causing variations in the
volume of the sperm and consequently its apparent density.
Consequently, in circumstances where the positive buoyancy is less
than desired, so that the Y-sperm do not rise as rapidly as desired
in the upper portion of the density gradient column, imposing a
negative pressure above the column serves to increase the positive
buoyancy and thereby the separation effects of the separation media
upon the Y-sperm. In practice, we have found that separation
efficiencies can be increased as much as 30 to 50 percent by
application of a vacuum of about 10 to 20 inches of mercury in the
head space above the density gradient column. The normal procedure
is to apply the vacuum gradually, for example, reaching a vacuum
equivalent to 15 inches of mercury in a period of about 5 minutes.
Best results are obtained when the vacuum pump is energized shortly
after the introduction of the sperm sample to the separation
column, with the vacuum being drawn continuously until the end of
the separation process. To protect the separated sperm, the vacuum
is reduced to zero over a period of at least two minutes, prior to
separation of the desired sperm fractions.
As a generalized example, illustrating the practice of the
invention, a separation media in the form of a density gradient
column can be prepared from commercially available cow's milk and
its components by initially heating to 90.degree.F., cooling to
room temperature (e.g., 72.degree.F.), filtering all the starting
materials (i.e., homogenized commercial milk, half-and-half,
distilled nonfat and low-fat milk) through sterilized glass wool at
approximately room temperature. After cooling, each component can
be treated with suitable antibiotics (e.g., potassium, penicillin
g, and streptomycin sulfate solution). In the case of nonfat milk,
the value of density can be increased without substantial change or
alteration in the osmotic pressure by dialysis to remove various
organic and inorganic salts and without reduction in protein
content or other impairment of the milk. The milk is redistilled
with vacuum to remove the excess water, thereby achieving the
higher density. I have found, for example, that dialysis in
laboratory dialysis equipment (e.g., Oxford Multiple Dialyser) for
periods of 10 to 15 hours followed by redistillation at
58.degree.C. for about 3 to 10 hours will produce the desired
effect. Using the described procedure, the following are
representative of typical densities and osmotic pressures obtained
with the indicated starting materials, after preparation:
Media Osmolality Density ______________________________________
Half-and-half 280 1.024 Homogenized milk 280 1.034 Low-fat milk 348
1.043 Distilled nonfat milk 214 1.049
______________________________________
Mixtures of the foregoing materials are used to obtain desired
densities and osmolalities. Thus, low-fat milk is mixed with
distilled nonfat milk to obtain a mixture having an osmolality of
approximately 280 and a density substantially above the desired
maximum density of 1.044 grams per centimeter. In like fashion,
half-and-half is mixed with homogenized milk to obtain a mixture
with a compatible osmolality and a desired density of 1.028 grams
per centimeter. These mixtures are now mixed to provide a medium of
desired maximum density of 1.044 grams per centimeter. Following
this procedure media of desired density at 0.degree.C. are readily
obtained for use in forming a density gradient in accordance with
the procedures herein described, for example with reference to FIG.
6.
To prepare sperm for separation, freshly collected sperm are mixed
with a mixture of homogenized milk and half-and-half of known
density (e.g., 1.0295) in proportions to provide a population of
about 200 to 300 million sperm per cc of media, the total sample
providing at least 400 to 600 million sperm for the separation
processing. The intermixed sperm and nutrient media are then placed
in a cold room and gradually cooled to a temperature close to
0.degree.C. (i.e., 0.8.degree.C.).
The sperm sample is introduced to the "midpoint" of the density
gradient column by first determining the location within the column
of the zone of average or median density corresponding to that of
the sperm sample undergoing separation (e.g., 1.028). This point is
easily located by injecting a small amount of color into a separate
portion of the medium of density, say, 1.028, and introducing this
small amount of colored medium through the inlet valve 45 to a
central portion of the column. Thereafter the 1.028 density strata
(as determined by the color) is vertically adjusted within the
column to a point opposite the inlet valve 45 (FIG. 6).
Prior to initiating separation, the density gradient column is
cooled to the desired separation temperature (0.8.degree.C.) and
the sperm sample introduced through the filling spout 48 and tube
49 to the midpoint of the density gradient column. Separation of
the sperm according to density thereafter occurs through the
buoyant effects of the density gradient medium, causing the less
dense sperm to rise and the more dense sperm to fall within the
density gradient column. After a sufficient period of time to
achieve equilibrium (e.g., about 21/2 hours), a sperm fraction is
separated from the top or bottom of the column (according to
selection) to obtain nutrient media containing a sperm fraction
comprising from 4 to 8 percent of the total sperm population in the
sample introduced. In the case of X-sperm, the sperm fraction is
separated from the bottom of the column in any of the manners
previously described. Where Y-sperm are desired, the sperm fraction
is similarly separated from the top of the column. In practice, the
separated fraction is counted for the number of sperm present
therein, and the number plotted against the fraction.
The practice of the invention is exemplified in the following
generalized procedure which was used in a number of separations (in
excess of 50 to obtain the results hereinafter tabulated. In
carrying out the separation specified, using apparatus as in FIG.
6, a series of density gradient columns were simultaneously
prepared in each of the columns 44. The density gradient in each
column ranged from 1.0255 at the top to 1.044 at the bottom
(accomplished by use of a medium of density to 1.0255 in container
21 and 1.036 in container 22). Sufficient time was then allowed to
permit the density gradient columns to cool to the temperature of
the water bath 40 in the refrigeration tank 39 (0.8.degree.C.),
thirty minutes being sufficient for such purpose. The column height
was adjusted to show a density of 1.0295 at the inlet point
opposite the valve mechanism 45.
Prior to initiating separation, fresh bull sperm was collected and
intermixed with a nutrient medium having a density of approximately
1.0295, following which the density of the intermixed sperm and
nutrient medium was adjusted to a final density of 1.0295, adding
higher density or lower density medium as necessary. The sperm and
nutrient medium were then cooled for approximately two hours to
gradually cool the sperm to an immobilization temperature of
1.degree.C. (i.e., 0.8.degree.C.). Sperm samples containing
approximately 480 million sperm (e.g., 1 to 21/2 cc of intermixed
sperm and nutrient medium) were introduced to the individual
separation columns through the side tubes 49 and valve mechanisms
45, using a hypodermic syringe 47 cooled to a sperm immobilizing
temperature of 0.8.degree.C. Sperm was introduced to the column at
a relatively slow rate, not exceeding about 1 cc per minute. To
insure that all the sperm were introduced to the columns, a small
amount of nutrient medium was additionally pushed through the side
tubes 49 thereby clearing the side tubes of sperm. Following the
introduction of the sperm, which was done under infra-red light
conditions, the refrigeration tank and separation columns were
covered to exclude light during the separation processing.
Separation of the sperm occurred through operation of the buoyant
forces within the density gradient columns over a period of 1 to 4
hours (optimum 21/2 hours) and thereafter under subdued light,
sperm fractions were removed from the top of the columns by means
of pipettes equipped with water pumps (hereinbefore described)
following which the more dense sperm fraction was removed from the
bottoms of the column, (as represented at 34), through the
stopcocks 43 at the bottom of the columns. To insure purity of the
separated fractions, the portions removed from the top and bottom
of density gradient columns were maintained at a volume of
approximately 1/2 cc.
Intermixed sperm fractions were also removed from central portions
of the column. All of the sperm samples were then checked for sperm
count, using a standard procedure with a haemacytometer.
The separated fractions of relatively pure sperm (1/2 cc) were
subsequently mixed with a small quantity (0.165 cc) of 15 percent
glycerol in homogenized milk, and the mixture held 15 minutes at
1.degree. to 8.degree.C. After a holding period of approximately 15
minutes, an additional amount of glycerol (0.165 cc) was added and
the mixture held at 1.degree. to 8.degree.C. for an additional 15
minute period, following which a third addition of glycerol
(0.165cc) was added to the mixture. Samples were selected for
insemination from the sperm population at the bottom of the tube
representing less than 41/2 percent of the total population.
Collected samples of sperm were then deep frozen using standard
procedures as normally practiced in the artificial insemination
industry.
As needed, sperm samples prepared as above, were removed from cold
storage and used in artificial insemination of female mammals. In
the specific case of cattle, cows found to be in heat were
inseminated with 1 to 2 ccs containing at least 10 to 20 million
sperm (before freezing), again using procedures standard in the
artificial insemination industry. Sperm purity as determined by
laboratory testing was in excess of 70 percent with the procedure
described.
In the specific procedure being described, 13 of the cows
inseminated became pregnant, and their fetuses killed and examined
after a period of approximately 60 days. The results of these 13
pregnancies are set forth in Table 2 below:
Table 2 ______________________________________ Date Insem. Run. No.
Female No. Govt. Lab. Result ______________________________________
5-15-67 53 H-77 1328 F 6-6-67 70 H-73 1328 F 1-27-68 212 H-62 900 F
6-6-68 314 CH-67 1812 F 7-23-68 361 CH-365 1968 F 7-24-68 358
CH-430 1812 F 7-24-68 362 CH-386 1812 F 7-27-68 365 CH-364 1968 M
.times. 2.sup.1 CH-55 1968 8-1-68 368 CH-431 1968 F .times. 2.sup.2
8-4-68 370 CH-349 1968 F 8-6-68 371 CH-42 1968 M 8-7-68 372 CH-366
1968 F 8-8-68 373 CH-352 1968 M
______________________________________ .sup.1 This run (No. 365)
was inseminated into two cows. Each produced a male calf. .sup.2
This run (No. 368) produced twin female calves.
In analyzing the results of Table 2, run No. 365 was treated as if
one male calf had been produced, and run No. 368 was treated as if
a single female calf had been produced. It is to be noted that 13
females were expected, and that 10 females were actually obtained
as against 3 males. This represents a statistically significant
departure from the normal sex ratio in cows.
* * * * *