U.S. patent application number 14/581142 was filed with the patent office on 2015-04-23 for methods for sorting sperm and producing sexed embryos.
This patent application is currently assigned to XY, LLC. The applicant listed for this patent is XY, LLC. Invention is credited to Lisa A. Herickhoff, John L. Schenk, George E. Seidel.
Application Number | 20150112125 14/581142 |
Document ID | / |
Family ID | 21695819 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150112125 |
Kind Code |
A1 |
Seidel; George E. ; et
al. |
April 23, 2015 |
METHODS FOR SORTING SPERM AND PRODUCING SEXED EMBRYOS
Abstract
A method of sorting sperm cells and a method of producing at
least one sexed embryo. The method of sorting sperm cells includes
the steps of establishing a sheath fluid environment including a
citrate for stained sperm cells, establishing a stream including
stained sperm cells in said sheath fluid environment, sensing a
property of the stained sperm cells, and discriminating between
stained sperm cells for a desired sex characteristic. The method of
producing at least one sexed embryo includes the steps for sorting
sperm cells in addition to fertilizing at least one egg with the
sexed sperm to form at least one sexed embryo.
Inventors: |
Seidel; George E.; (LaPorte,
CO) ; Herickhoff; Lisa A.; (Fort Collins, CO)
; Schenk; John L.; (Fort Collins, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
XY, LLC |
Navasota |
TX |
US |
|
|
Assignee: |
XY, LLC
Navasota
TX
|
Family ID: |
21695819 |
Appl. No.: |
14/581142 |
Filed: |
December 23, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13764408 |
Feb 11, 2013 |
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14581142 |
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11536492 |
Sep 28, 2006 |
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13764408 |
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10378109 |
Feb 25, 2003 |
7195920 |
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11536492 |
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09511959 |
Feb 23, 2000 |
6524860 |
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10378109 |
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09001394 |
Dec 31, 1997 |
6149867 |
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09511959 |
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Current U.S.
Class: |
600/35 ; 435/2;
435/449 |
Current CPC
Class: |
G01N 1/30 20130101; A01K
67/027 20130101; C12N 15/873 20130101; Y10T 436/108331 20150115;
A61D 19/04 20130101; G01N 2015/149 20130101; G01N 33/52 20130101;
C12N 5/0612 20130101; A01N 1/0226 20130101; A61D 19/02 20130101;
Y10T 436/101666 20150115; A01N 1/02 20130101; C12Q 1/04
20130101 |
Class at
Publication: |
600/35 ; 435/2;
435/449 |
International
Class: |
C12N 5/071 20060101
C12N005/071; A61D 19/02 20060101 A61D019/02 |
Claims
1. A method of sorting sperm cells comprising: establishing a
sheath fluid environment for stained sperm cells, the sheath fluid
environment including a citrate; establishing a stream comprising
said stained sperm cells in said sheath fluid environment; sensing
a property of the stained sperm cells; and discriminating between
stained sperm cells for a desired sex characteristic.
2. The method of claim 1, wherein the step of discriminating
between stained sperm cells for a desired sex characteristic
comprises the step of differentiating X chromosome bearing sperm
cells from Y chromosome bearing sperm cells.
3. The method of claim 1, wherein changes in the amount of citrate
are minimized between the sheath fluid environment and a pre-sort
and/or a post-sort fluid environments.
4. The method of claim 1, further comprising: forming drops from
the stream containing the sperm cells; charging drops having sperm
cells with the desired sex characteristic; and collecting drops
having sperm cells with the desired sex characteristic.
5. The method of claim 4, wherein the step of collecting drops
having sperm cells with the desired characteristic further
comprises: collecting drops having sperm cells with the desired
characteristic in a wide collection tube.
6. The method of claim 4, wherein the step of collecting drops
having sperm cells with the desired characteristic further
comprises: collecting drops having sperm cells with the desired
characteristic in a stream matched collection tube.
7. A method of producing a sexed sperm specimen according to the
process of claim 1.
8. The method of claim 7, further comprising preparing an
artificial insemination dosage having less than one half of the
typical number of sperm provided in a typical artificial
insemination dosage.
9. A method of sorting sperm cells comprising: chemically
coordinating a sheath fluid environment for stained sperm cells
with a pre-sort fluid environment or post-sort fluid environment;
supplying stained sperm cells in a stream comprising said
chemically coordinated; sensing a property of said stained sperm
cells; and discriminating between stained sperm cells having a
desired sex characteristic.
10. The method of claim 9, wherein the step of chemically
coordinating a sheath fluid environment with a pre-sort environment
or a post-sort fluid environment further comprises incorporating a
citrate into the sheath fluid.
11. The method of claim 10, wherein changes in the amount of
citrate are minimized between the sheath fluid environment and a
pre-sort and/or a post-sort fluid environments.
12. The method of claim 10, wherein the step of chemically
coordinating a sheath fluid environment with a pre-sort environment
or a post-sort fluid environment further comprises incorporating
comprises a chemical in the citric acid cycle into the sheath
fluid.
13. The method of claim 9, further comprising: forming drops from
the stream containing the sperm cells; charging drops having sperm
cells with the desired sex characteristic; and collecting drops
having sperm cells with the desired sex characteristic.
14. The method of claim 13, wherein the step of collecting drops
having sperm cells with the desired characteristic further
comprises: collecting drops having sperm cells with the desired
characteristic in a wide collection tube.
15. The method of claim 13, wherein the step of collecting drops
having sperm cells with the desired characteristic further
comprises: collecting drops having sperm cells with the desired
characteristic in a stream matched collection tube.
16. A method of producing a sexed sperm specimen according to the
process of claim 1.
17. The method of claim 16, further comprising preparing an
artificial insemination dosage having less than one half of the
typical number of sperm provided in a typical artificial
insemination dosage.
18. A method of producing at least one sexed embryo comprising:
establishing a sheath fluid environment for stained sperm cells,
the sheath fluid environment including a citrate; establishing a
stream comprising said stained sperm cells in said sheath fluid
environment; sensing a property of the stained sperm cells;
discriminating between stained sperm cells having a desired sex
characteristic; and fertilizing at least one egg with the sexed
sperm to form at least one sexed embryo.
19. The method of claim 18, wherein the at least one egg is
fertilized in vitro with the sex selected sperm.
20. The method of claim 18, wherein the step of fertilized in vivo
further comprises the step of delivering sperm cells into both
uterine horns of a uterus.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/764,408, filed Feb. 11, 2013, which is
continuation of U.S. patent application Ser. No. 11/536,492, filed
on Sep. 28, 2006, which is a continuation of U.S. patent
application Ser. No. 10/378,109, filed Feb. 25, 2003, now U.S. Pat.
No. 7,195,920 which is a divisional of U.S. patent application Ser.
No. 09/511,959, filed on Feb. 23, 2000, now U.S. Pat. No.
6,524,860, which is a divisional of U.S. patent application Ser.
No. 09/001,394, filed on Dec. 31, 1997, now U.S. Pat. No.
6,149,867, each of which are incorporated herein by reference.
I. BACKGROUND OF THE INVENTION
[0002] This invention relates generally to the field of sex
selection in mammalian offspring. It is especially relevant to the
aspect of low dose artificial insemination for creating the desired
sex of offspring. Particularly, the invention relates to systems
for sorting sperm via flow cytometry for sex-specific and low dose
efforts at artificial insemination or the like.
[0003] For ages it has been desired to select the sex of specific
offspring. Beyond obvious psychological aspects, the actual sex
selection of mammalian offspring has significant economic
consequences when one considers its application to food producing
animals such as cattle as well as celebrated trophy animals such as
horses and the like. This great desire has resulted in a
significant variety of efforts to achieve sex-selected offspring.
Probably the effort which has appeared most likely to achieve the
desired results has been efforts at sorting and selecting between X
and Y sperm prior to insemination.
[0004] One of the challenges the sperm sorting effort faces is the
large numbers of sorted sperm required. In natural insemination
sperm are produced in some species by the billions; in artificial
insemination less, but still significantly large numbers of sperm
are used. For instance, artificial insemination techniques commonly
use ten million to five hundred million sperm (depending on
species). Thus a significant number of sperm are necessary even in
an artificial insemination environment.
[0005] Many methods have been attempted to achieve the separation
of X- and Y-chromosome bearing sperm. These methods have ranged
from magnetic techniques such as appears disclosed in U.S. Pat. No.
4,276,139 to columnar techniques as appears disclosed in U.S. Pat.
No. 5,514,537 to gravimetric techniques as discussed in U.S. Pat.
No. 3,894,529, reissue Pat. No. 32,350, U.S. Pat. Nos. 4,092,229,
4,067,965, and 4,155,831. Electrical properties have also been
attempted as shown in U.S. Pat. No. 4,083,957 as well as a
combination of electrical and gravimetric properties as discussed
in U.S. Pat. Nos. 4,225,405, 4,698,142, and 4,749,458. Motility
efforts have also been attempted as shown in U.S. Pat. Nos.
4,009,260 and 4,339,434. Chemical techniques such as those shown in
U.S. Pat. Nos. 4,511,661 and 4,999,283 (involving monoclonal
antibodies) and U.S. Pat. Nos. 5,021,244, 5,346,990, 5,439,362, and
5,660,997 (involving membrane proteins), and U.S. Pat. Nos.
3,687,803, 4,191,749, 4,448,767, and 4,680,258 (involving
antibodies) as well as the addition of serum components as shown in
U.S. Pat. No. 4,085,205. While each of these techniques has been
presented as if to be highly efficient, in fact at present none of
those techniques yield the desired level of sex preselection.
[0006] At present, the only quantitative technique used to achieve
the separation of X- and Y-chromosome bearing sperm has been that
involving individual discrimination and separation of the sperm
through the techniques of flow cytometry. This technique appeared
possible as a result of advances and discoveries involving the
differential dye absorption of X- and Y-chromosome bearing sperm.
This was discussed early in U.S. Pat. No. 4,362,246 and
significantly expanded upon through the techniques disclosed by
Lawrence Johnson in U.S. Pat. No. 5,135,759. The Johnson technique
of utilizing flow cytometry to separate X- and Y-chromosome bearing
sperm has been so significant an advancement that it has for the
first time made the commercial separation of such sperm feasible.
While still experimental, separation has been significantly
enhanced through the utilization of high speed flow cytometers such
as the MoFlo.RTM. flow cytometer produced by Cytomation, Inc. and
discussed in a variety of other patents including U.S. Pat. Nos.
5,150,313, 5,602,039, 5,602,349, and 5,643,796 as well as
international PCT patent publication WO 96/12171. While the
utilization of Cytomation's MoFlo.RTM. cytometers has permitted
great increases in speed and while these speed increases are
particularly relevant given the high number of sperm often used,
certain problems have still remained. In spite of the almost
ten-fold advances in speed possible by the MoFlo.RTM. flow
cytometer, shorter and shorter sorting times have been desired for
several reasons. First, it has been discovered that as a practical
matter, the sperm are time-critical cells. They lose their
effectiveness the longer they remain unused. Second, the
collection, sorting, and insemination timings has made speed an
item of high commercial importance. Thus, the time critical nature
of the sperm cells and the process has made speed an essential
element in achieving high efficacy and success rates.
[0007] Other problems also exist ranging from the practical to the
theoretical. On the practical side, it has been desired to achieve
sex-sorted sperm samples using inexpensive disposable components
and substances. Also on the expense side, it has been desired to be
able to achieve sorting (as well as collection and insemination) in
as efficient a labor event as possible. Thus, for commercial
production and success in this field, improvements which might only
represent an increase in efficiency may still be significant.
Related to the practical aspect of expense, is the practical aspect
of the delicateness and sensitivity of the entire process. In this
regard, it has been desired to simplify the process and make it as
procedurally robust as possible so that operator error or skill can
play an ever decreasing role.
[0008] In addition to the delicateness of the process, it has
always been known that the sperm themselves are extremely delicate
cells. While this factor at first glance seems like it might be
considered easily understood, in fact, the full extent of the
cells' sensitivities have not yet been fully explored. In the
context of flow cytometry in general, most sorted cells or
particles have often been spherical or otherwise physically able to
withstand a variety of abuses. This is not the case for sperm
cells. In fact, as the present invention discloses, the processing
through normal flow cytometer techniques may, in fact, be
unacceptable for cytometric sorting of sperm cells in certain
applications. The sensitivities range from dilution problems and
the flow cytometer's inherent need to isolate and distinguish each
cell individually as well as the pressure and other stresses which
typical flow cytometry has, prior to the present invention, imposed
upon the cells or other substances that it was sorting. This may
also represent a unique factor for sperm cells because it appears
that even though the sperm cell may appear to pass through the flow
cytometer and be sorted with no visually discernable side-effects,
in fact, the cells themselves may have been stressed to the point
that they perform less than optimally in the insemination process.
Thus, an interplay of factors seems involved and has raised unusual
problems from the perspective of sperm cell sorting and ultimate
use for artificial insemination.
[0009] Another problem which has remained--in spite of the great
advances achieved through the Johnson patent and related
technology--is the fact that prior to the present invention it has
been extremely difficult to achieve lower dosage insemination with
sexed sperm. While historically, some achievement of low dose
insemination has occurred, it has appeared to be more on a
theoretical or laboratory environment rather than from environments
which are likely to be experienced in or applicable to a commercial
application. In this regard, the desire has not been merely to
achieve low dose insemination but rather to achieve low dose
insemination with pregnancy success rates which are comparable to
existing unsexed, high dosage artificial insemination efforts.
Thus, the advances achieved by the present inventors in both sexed
and low dose artificial insemination represent significant advances
which may, for the first time, make commercial applications
feasible.
[0010] Another problem which has been faced by those in the
industry--again, in spite of the great advances by the Johnson
patent and related technology--is the fact that the problem itself,
namely, artificial insemination with a high success rate is one of
a statistical nature in which a multitude of factors seem to
interplay. Thus, the solutions proposed may to some degree involve
a combination of factors which, when thoroughly statistically
studied, will be shown to be necessary either in isolation or in
combination with other factors. Such a determination is further
compounded by the fact that the results themselves vary by species
and may be difficult to ascertain due to the fact that testing and
statistical sampling on a large enough data base is not likely to
be worth the effort at the initial stages. For these reasons the
invention can also involve a combination of factors which may,
individually or in combination, represent the appropriate solutions
for a given application. This disclosure is thus to be considered
broad enough so that the various combinations and permeations of
the techniques disclosed may be achieved. Undiscovered synergies
may exist with other factors. Such factors may range from factors
within the sorting or flow cytometer steps to those in the
collection as well as insemination steps. At present, studies have
been primarily achieved on bovine species, however, it is not
believed that these techniques will be limited to such species or,
for that matter to only sperm cells. It appears that the techniques
used may have application beyond just sperm cells into areas which
involve either sensitive items to be sorted or merely minimization
of the impacts of the stresses of flow cytometry upon the item
sorted.
[0011] Interestingly, while the present invention takes an approach
to minimize the impacts and stresses upon the sperm cells, others
appear to have actually taken steps away from this direction by
increasing pressures and demands for speed and other such
performance. Essentially, the drive for low dose insemination and
high speed processing may, in an individual or perhaps interrelated
fashion have posed problems which limited one another. Thus, while
there has been a long felt but unsatisfied need for high speed, low
dose sexed insemination, and while the implementing arts and
elements have long been available, prior to the present invention
the advances or perhaps combinations of advances had apparently
been overlooked by those skilled in the art. Perhaps to some degree
they failed to appreciate that the problem involved an interplay of
factors as well as peculiar necessities for the types of cells
(sperm cells or perhaps species-specific sperm cells) involved in
this field. Interestingly, as the listing of efforts earlier in
this discussion shows, substantial attempts had been made but they
apparently failed to understand the problem inherent in such an
area as low dose, sexed insemination and had perhaps assumed that
because the natural service event involves perhaps billions of
sperm, there may have been physical limitations to the achievement
of artificial insemination with numbers which are as many as four
orders of magnitude less in number. Thus, it may not be surprising
that there was to some extent an actual teaching away from the
technical direction in which the present inventors went. Perhaps
the results may even be considered unexpected to a degree because
they have shown that sexed, low dose artificial insemination can be
achieved with success rates comparable to those of unsexed, high
dose artificial insemination. It might even be surprising to some
that the techniques and advances of the present invention in fact
combine to achieve the great results shown. While each technique
could, in isolation, be viewed by some as unremarkable, in fact,
the subtle changes appear to afford significant advances in the end
result whether considered alone or in combination with other subtle
changes.
[0012] Thus, until the present invention the achievement of success
rates for low dose, sexed artificial insemination has not been
possible with levels of performance necessary or simplified
procedures likely to be necessary to achieve commercial
implementation. The present invention discloses techniques which
permit the achievement of improved performances and thus facilitate
the end result desired, namely, low dose, sexed artificial
insemination on a commercial basis.
II. SUMMARY OF THE INVENTION
[0013] Accordingly, the present invention provides improved sheath
and collector systems for sorting of sperm cells to determine their
sex through a flow cytometer. The sheath fluid as typically used in
a flow cytometer is replaced with a fluid which minimizes the
stress on the sperm cells as they are sorted. Furthermore, the
collection system is improved to minimize both the physical and
chemical stress to which the sperm cells are subjected. Various
techniques and substances are represented but as those skilled in
the art will readily understand, various combinations and
permutations can be used in the manner which may be optimized for
performance based in the species, goals and other parameters
involved in a specific processing application.
[0014] An object of the invention is thus to achieve better sorting
for substances such as sperm cells. A goal is to minimize the
impact the sorting function itself has on the cells or other
sensitive items which may be sorted. A particular goal is to
minimize the impact the sheath fluid imposes upon the cells and to
potentially provide a sheath fluid which affirmatively acts to
assist the cells in handling the various stresses involved. A
parallel goal is to provide substances and techniques which are
especially suited for sperm cells in general, for bovine sperm
cells, for equine sperm cells, and for the separation of such sperm
cells into X- and Y-chromosome bearing components. Similarly a goal
is to minimize the impacts that the collection phase (e.g., after
sorting) has upon the cells and to minimize the physical impact as
well as chemical impacts on such sex sorted sperm cells. Thus a
goal is to achieve as unaffected a sorted result as possible.
[0015] Another object of the invention is to achieve low dose,
sorted insemination on levels and with success rates which are
comparable to those of the typical unsexed, high dose artificial
insemination. Thus the prior goals of minimizing the stress or
potential damage upon the sperm cells is important. Sorting in a
manner which affords both high speed and low stress sorting, and
which is especially adapted for sperm cell sorting in a low dose
context is an important goal as well. The goals of providing sheath
and other fluids which do not negatively affect the fertility of
the sperm and which are compatible with artificial insemination are
also important.
[0016] Naturally further objects of the invention are disclosed
throughout other areas of the specification and claims.
III. BRIEF DESCRIPTION OF DRAWINGS
[0017] FIG. 1 is a schematic diagram of a sorter system according
to the present invention.
[0018] FIG. 2 is a diagram of the entrained cells in the free fall
area of a typical flow cytometer.
[0019] FIG. 3 is a conceptual diagram showing differences as they
roughly appear as a result of the present invention.
[0020] FIG. 4 is a diagram of the sorted cell stream as they are
collected in the landing zone area.
IV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0021] As will be seen, the basic concepts of the present invention
can be combined and embodied in a variety of ways. The invention
involves both improved flow cytometer systems as well a systems for
the creation of sex-specific sperm samples which may be used in
artificial insemination and the animals produced by such
techniques. Furthermore, the techniques are disclosed in a general
fashion so that they may be applied to specific systems and
applications once the general principals are understood. While
device enhancements are disclosed it should be understood that
these enhancements not only accomplish certain methods but also can
be varied and combined in a number of ways. Importantly, as to all
of the foregoing, each of these facets should be understood to be
encompassed by this disclosure.
[0022] As mentioned, the basic goal is that of separating the
X-bearing sperm from the Y-bearing sperm. This is done in a manner
which isolates the two types of sperm so that each can be
separately packaged and dealt with. The isolation is preferably
done through the use of flow cytometry. Flow cytometry in general
is a technique which is well understood. For instance, the basic
aspects of it are shown and discussed in a variety of patents to
Cytomation, Inc. such as the U.S. Patents and other publications
listed earlier. Each of these patents and the references cited
therein, are incorporated by reference, thus those skilled in the
art can easily understand the basic principles involved.
[0023] Essentially, flow cytometry involves sorting items, such as
cells, which are provided to the flow cytometer instrument through
some type of cell source. A conceptual instrument is shown in FIG.
1. The flow cytometer instrument includes a cell source (1) which
acts to establish or supply cells or some other type of item to be
analyzed by the flow cytometer. The cells are deposited within a
nozzle (2) in a manner such that the cells are surrounded by a
sheath fluid (3). The sheath fluid (3) is usually supplied by some
sheath fluid source (4) so that as the cell source (1) supplies its
cells, the sheath fluid (3) is concurrently fed through the nozzle
(2). In this manner it can be easily understood how the sheath
fluid (3) forms a sheath fluid environment for the cells. Since the
various fluids are provided to the flow cytometer at some pressure,
they flow out of nozzle (2) and exit at the nozzle orifice (5). By
providing some type of oscillator (6) which may be very precisely
controlled through an oscillator control (19), pressure waves may
be established within the nozzle (2) and transmitted to the fluids
exiting the nozzle (2) at nozzle orifice (5). Since the oscillator
(6) thus acts upon the sheath fluid (3), the stream (7) exiting the
nozzle orifice (5) eventually and regularly forms drops (8).
Because the cells are surrounded by a sheath fluid environment, the
drops (8) may contain within them individually isolated (generally)
cells or other items.
[0024] Since the drops (8) generally contain isolated cells, the
flow cytometer can distinguish and separate droplets based upon
whether or not the appropriate cell or cells is/are contained
within the drop. This is accomplished through a cell sensing system
(9). The cell sensing system involves at least some type of sensor
(10) which responds to the cells contained within each drop (8) as
discussed at length in the seminal work (no pun intended) by Larry
Johnson, namely, U.S. Pat. No. 5,135,759. As the Johnson patent
explains for sperm cells, the cell sensing system (9) may cause an
action depending upon the relative presence or relative absence of
a particular dye which may be excited by some stimulant such as the
laser exciter (11). While each type of sperm cell is stained by the
dye, the differing length of the X-chromosome and the Y-chromosome
causes different levels of staining Thus, by sensing the degree of
dye present in the sperm cells it is possible to discriminate
between X-bearing sperm and Y-bearing sperm by their differing
emission levels.
[0025] In order to achieve the ultimate separation and isolation of
the appropriate cells, the signals received by sensor (10) are fed
to some type of sorter discrimination system (12) which very
rapidly makes the decision and can differentially charge each drop
(8) based upon whether it has decided that the desired cell does or
does not exist within that drop (8). In this manner the sorter
discrimination system (12) acts to permit the electrostatic
deflection plates (13) to deflect drops (8) based on whether or not
they contain the appropriate cell or other item. As a result, the
flow cytometer acts to sort the cells by causing them to land in
one or more collectors (14). Thus by sensing some property of the
cells or other items the flow cytometer can discriminate between
cells based on a particular characteristic and place them in the
appropriate collector (14). In the system presently used to sort
sperm, the X-bearing sperm droplets are charged positively and thus
deflect in one direction, the Y-bearing sperm droplets are charged
negatively and thus deflect the other way, and the wasted stream
(that is unsortable cells) is uncharged and thus is collected in an
undeflected stream into a suction tube or the like.
[0026] Referring to FIG. 2, the process can be even further
understood. As shown in that figure, the nozzle (2) emits a stream
(7) which because of the oscillator (6) (not shown in FIG. 2) forms
drops (8). Since the cell source (1) (not shown in FIG. 2) may
supply sperm cells (15) which have been stained according to the
Johnson technique, the light stimulation by laser exciter (11) is
differentially determined by sensor (10) so that the existence or
nonexistence of a charge on each drop (8) as it separates from
stream (7) can be controlled by the flow cytometer. This control
results in positively charged, negatively charged, and uncharged
drops (8) based upon their content. As shown in FIG. 2, certain
drops are shown as deflected drops (16). These deflected drops (16)
are those containing sperm cells (15) of the one or the other sex.
They are then deposited in the appropriate collector (14) for later
use.
[0027] One of the aspects of flow cytometry which is particularly
important to its application for sperm sorting is the high speed
operation of a flow cytometer. Advances have been particularly made
by the flow cytometers available through Cytomation, Inc. under the
MoFlo.RTM. trademark. These flow cytometers have increased sorting
speeds extraordinarily and have thus made flow cytometry a
technique which is likely to make feasible the commercial
application of sperm sorting (among other commercial applications).
They act to achieve high speed sorting, that is at a speed which is
notably higher than those otherwise utilized. Specifically,
Cytomation's MoFlo.RTM. flow cytometers act with oscillator
frequencies of greater than about five kilohertz and more
specifically can be operated in the 10 to 30 or even the 50
kilohertz ranges. Thus, droplets are formed at very high
frequencies and the cells contained within the sheath fluid
environment can be emitted very rapidly from the nozzle (2). As a
result, each of the components such as the nozzle (2) oscillator
(6), and the like which make up and are part of a flow cytometer
system result in a high speed cell sorter.
[0028] In the application of a high speed cell sorter to the
sorting of sperm cells, sorting at rates of greater than about 500
sorts per second is achieved. In fact, rates of sorting in the
thousand and twelve hundred ranges have already been achieved
through a high speed cell sorter. Importantly, it should be
understood that the term "high speed" is a relative term such that
as other advances in flow cytometry and specific applications are
achieved, the aspect which is considered "high" may be varied or
may remain absolute. In either definition, the general principle is
that the sorting may occur at rates at which the parameters and
physical characteristics of the flow cytometer are significant to
the cells themselves when sorting particular cells such as sperm
cells.
[0029] One aspect of high speed sorting which appears to come into
play when sorting sperm cells is that of the pressures and other
stresses to which the sperm cells are subjected within the flow
cytometer. For instance, when operating at high speeds (and an
alternative definition of "high speed"), flow cytometers can be
operated at a pressure of 50 pounds per square inch and even 60 and
higher pounds per square inch. These pressures may be considered
high because they may result in effects upon the cells being
sorted. The key as disclosed in the present invention for this
facet is the fact that the stress thresholds of the particular
cells are the determining factor. Additionally as further knowledge
is gained it may be shown that the stress thresholds are a function
of combined effects such as the particular species or the
particular prior or subsequent handling of the cells. The key in
this regard is that the stress imposed upon the cells can, in fact,
alter their viability and their ability to achieve the desired
result. In the pressure case, it may be that merely subjecting the
sperm cells to a higher pressure as a result of the operation of
the flow cytometer at that pressure may result in decreased
performance of the cells. The present invention in one regard acts
to minimize these stresses and thus results in greater efficacies
as well as lower dosages as discussed later.
[0030] In considering the stress aspect of the cells, the present
invention acts in a fashion which minimizes the stresses. These
stresses can be minimized at any point in the overall cycle or
process of collecting, sorting or even inseminating the animal.
Importantly, the stress imposed by the handling of the cells within
the flow cytometer appears significant for this application. In one
embodiment of the invention, the sheath fluid is specifically
selected so that it can serve in a coordinated fashion with both
(or either) the pre-sort cell fluid environment or the post-sort
cell fluid environment. While naturally it is possible to adjust
either the pre- or post-sort fluids, in one embodiment the
invention adjusts the sheath fluid (3) so that it imposes
significantly less stress upon the cells than was previously
accomplished. In one regard the invention is remarkable in that it
removes the total focus from that of operation of the flow
cytometer to a focus on handling and removing stress from the cells
themselves. For instance, while it has been known to utilize fluids
having a proper pH factor or osmolality, the present invention
recognizes that there may be certain chemical compositions to which
the cells may be hyper-responsive. These hyper-responsive chemical
compositions may naturally vary based upon the cells or even the
prior handling of the cells. Importantly at present it appears that
for sperm cells certain metabolic chemical compositions such as
citrate seem to prevent unusually high stresses upon the cells.
Thus, the hyper-responsive chemical compositions can be defined as
those to which the cells are particularly responsive in the context
of their functionality and the then-existing handling techniques.
As to sperm cells it appears that metabolic compositions,
specifically citrate constancy for bovine sperm cells and hepes
buffer constancy for equine sperm cells may be very important. Thus
the present invention acts to minimize the changes through the type
of operation or the selection of substances which may act as a
means for minimizing the changes which the cells experience.
[0031] For the sheath fluid, a substance is selected according to
one embodiment of the invention so that it may be chemically
coordinated to prevent minimal changes. Thus, by selecting the
appropriate sheath fluid not only in context of flow cytometry
parameters, but rather also in context of the cell parameters
themselves, the changes experienced by the cells and the overall
result of the sorting can be enhanced. This is shown conceptually
in FIG. 3. FIG. 3 shows some type of chemical factor (such as
citrate or other factors) as it may exist throughout the various
phases of the process. For instance, the four phases shown might
represent the following: phase I may represent the existence of the
cells within the cell source (1), phase II might show the existence
of the cells as they are sorted in the sheath fluid environment,
phase III might show the cells as they are collected after sorting
and phase IV might show the reconstituted cells in a storage medium
after sorting. These four phases as shown for the prior art may
experience vastly different chemical factor environments. As shown
conceptually, however, in the present invention the cells may
experience very little change, most notably the dip or drop
experienced between phases I and II may be virtually absent. This
is as a result of the selection of the appropriate sheath fluid as
mentioned above. Thus, as a result of being subjected to an
appropriate sheath fluid, the cells in the present invention may
experience a much lower level of stress.
[0032] One of the potential generalities that may exist with
respect to this phenomenon is the fact that certain chemical
compositions may represent more hyper-responsive chemical
compositions than others. While naturally this may vary based upon
the species of sperm, the handling, or even the type of cell
involved, it appears that the viability of the cells for their
intended purpose (here, artificial insemination) varies greatly,
naturally or because of sorting or both, and so the cells exhibit a
hyper-responsive character with respect to that chemical
composition. By selecting certain metabolic chemical compositions,
most notably citrates or chemicals which are within the citric acid
cycle, great advances appear possible. Thus for the bovine sperm
application, the sheath fluid (3) is selected and coordinated so
that it presents about a 2.9 percent sodium citrate composition.
Specifically, the 2.9 percent sodium citrate solution may be
created as follows: [0033] 1. Place 29.0 grams of sodium citrate
dihydrate (Na.sub.3C.sub.6H.sub.5O.sub.7.2H.sub.20) in a 1,000 ml
volumetric flask [0034] a. Dissolve sodium citrate in 3/4 of water
batch, then add water to volume. [0035] 2. Add deionized or
Nanopure water to make 1,000 ml final volume. [0036] 3. Transfer to
bottles and autoclave at 15 lbs pressure (245.degree. F.) for at
least 30 minutes [0037] a. Autoclave solution using conditions to
minimize evaporation (loose cover) [0038] b. Be careful that water
does not boil away. [0039] 4. Cool slowly at room temperature.
[0040] 5. Store sealed in a 5.degree. C. cold room.
[0041] Further, for a sheath fluid, the sodium citrate solution may
be filtered. [0042] 6. Filter with a 0.22 micron filter using
aseptic techniques.
[0043] Interestingly, for equine sperm cells such a composition
does not perform as well. Rather, it has been discovered that for
equine sperm cells, a hepes buffered medium such as a hepes bovine
gamete medium--particularly HBGM3 as previously created by J. J.
Parrish for a bovine application--works well. This medium is
discussed in the article "Capacitation of Bovine Sperm by Heparin",
38 Biology of Reproduction 1171 (1988) hereby incorporated by
reference. Not only is this surprising because it is not the same
type of substance as is utilized for bovine sperm, but the actual
buffer, originally was developed for a bovine application. Thus in
the equine application the sheath fluid is selected which contains
the hepes buffer. This solution may have a pH at room temperature
of about 7.54 (pH at 39.degree. C.=7.4) with the following
composition:
TABLE-US-00001 Chemical Dry weight (g/500 ml) CaCl.sub.2 0.145
KCl.sub.2 0.115 MgCl.sub.2.cndot.6H.sub.20 0.004
NaH.sub.2PO.sub.4.cndot.H.sub.2O 0.018 NaCl 2.525 NaPyruvate 0.011
Lactic Acid (60%) 1.84 ml HEPES 4.765 NaHCO.sub.3 0.420 BSA
(fraction V) 3.0
[0044] One other aspect which may interplay in the present
invention is the fact that the cells involved may experience
unusual sensitivities. In one regard this may be due to the fact
that sperm cells are in a class of cells which are non-repairing
cells. That is, they do not have the ability to repair themselves
and hence, they may need to be treated much more sensitively than
is typical for flow cytometers or other handling equipment. Thus,
it may be appropriate that the enhancement is particularly
applicable when the flow cytometer acts to establish a source of
sperm cells. Another potentially related aspect which may be unique
to a class of cells such as sperm cells is the fact that their DNA
is non-repairing, non-replicating, and non-transcribing. Either of
these factors may come into play and so they may be relevant either
individually or together. Thus, it may be that the teachings of the
present invention apply to all gamete cells or even to viruses and
the like which are non-repairing, non-translating, non-transcribing
cells.
[0045] A separate aspect of the flow cytometer processing which may
also be important is the fact of properly treating the cells both
chemically and physically after they are sorted. As shown in FIG.
4, as the cells within drops (8) land in collector (14), it may be
important that the container which makes up the collector be
properly sized so that it acts as some means of avoiding an impact
between the cells and the container itself. While it has been known
to place an initial collector fluid (17) in the bottom of the
container to collect the cells so that they do not hit the bottom
of the container, it appears that a simple widening of the
container to address variations in stream presentation as well as
the inevitable splashing due to the impact of the cells into the
container can be used to enhance the result. In one regard this can
act as a cushioning element so that cells which may be mechanically
delicate, that is, they may break or be damaged by an impact can be
treated appropriately. Thus when the cytometer source establishes
cells which are physically delicate cells as the cells to be
sorted, it may be important to provide some type of cushioning
element such as a wide collection tube for which the opening width
(18) serves to position the walls of the container in a manner
which avoids contact with the cells. Thus the tube does not present
side walls so close that there is any significant probability of
contact between those cells being sorted and the walls of the tube.
In this manner, in addition to the collector fluid (17), it may be
desirable to include a wide collection tube as well. Perhaps merely
providing a wide opening to the container which serves as part of
the collector (14) may be sufficient. For applications utilizing
high speed sorting of sperm cells, it has been found that providing
a container having an inner diameter opening of at least 15
millimeters is believed to be sufficient. Specifically when
utilizing a 14 ml Falcon test tube in such an application, minimal
physical damage to the cells as a result of the collector (14) has
been discovered.
[0046] It should be noted that even the 14 ml Falcon test tube may
not be optimum. Specifically, it is believed that designing a
collection container which matches the geometry of the stream (that
is, a "stream-matched container") may be most optimal. This
stream-matched container may have any or all of the following
characteristics: a relatively wide orifice, an elliptically shaped
orifice, a lesser height to width ratio than currently involved, an
angled or otherwise coordinated presentation such as may present
side walls which are parallel to the falling streams, and the like.
It may also be desirable to provide a mounting element such as a
movable element or medium like ball bearings or the like to permit
variable orientation of the tube to match the falling stream
desired to be collected. In addition, the physical characteristics
for the class of containers such as the existing tube (described as
a "Falcon-type" test tube) may include not only the width of the
tube but also the material (such polystyrene to which the cells do
not stick) out of which it is made and the like. (These material
options are well known for the 14 ml Falcon tube.) Thus the
container and it collection fluid may also serve as a cushioning
element to minimize physical damage to the cells. It also can
serve, by its size, to facilitate collection of adequate numbers of
sperm without a significant dilution effect.
[0047] Another aspect of the collector fluid (17) can be the fact
that it, too, may serve to minimize chemical stresses upon the
cells. In one regard, since it may be important to provide a
nutrient to the cells both before and after sorting, the collector
fluid (17) may be selected so as to provide a coordinated level of
nutrient so that the levels are balanced both before and after
sorting. For bovine sperm in which a nutrient of egg yolk citrate
is utilized at a two percent egg yolk level, it has been discovered
that utilizing a six percent egg yolk citrate level (that is six
percent egg yolk content in a citrate solution) provides good
results. This is as result of the volumes existing before and after
the sorting event. The collector fluid (17) may start (before
sorting) with about 2 ml of volume. The sorting event may add about
double this volume (ending at three times the initial starting
volume) with very little egg yolk citrate in solution (due to
clogging and other flow cytometer considerations). Thus, the end
result in terms of the level of the amount of egg yolk citrate
present may be equivalent to the starting result, namely, two
percent egg yolk content in a citrate solution due to the volumes
involved. Thus the collector fluid (17) may be selected so as to
create an ending collector fluid environment which is balanced with
the initial nutrient or other fluid environment. In this manner, it
may serve to minimize the time and changed level of composition to
which the cells are subjected. Naturally, these fluid environments
may be presented within the flow cytometer or may exist at some
other prior time, the important point being merely minimizing the
stress to which the cells are subjected at any time in their life
cycle. Furthermore, since the initial chemical substance content
can be varied (for instance the percent egg yolk content in the
citrate may be varied up or down), likewise the starting collection
fluid environment or various volumes may also be varied so that the
ending result is the same. Thus, prior to commencing the sorting
process, the collector fluid exists with a six percent egg yolk
content in the citrate solution and after completion of the sorting
event the collector fluid with the sex-specific sperm may result in
a two percent egg yolk content in the citrate solution similar to
the initial nutrient content.
[0048] Note that in later use these sperm cells may be treated to a
20% egg yolk content in the citrate fluid for other reasons,
however these changes are not deemed to provide stress to the cells
as they are merely a known part of the total insemination process.
While naturally the levels may be varied as those skilled in the
art readily understand, a 20% egg yolk citrate buffer may be
constituted as follows:
I. Final Composition:
[0049] 80% sodium citrate solution (72 mM)
[0050] 20% (vol/vol) egg-yolk
II. Preparation for 1 Liter:
[0051] A. Sodium citrate solution [0052] 1. Place 29.0 grams of
sodium citrate dihydrate (Na.sub.3C.sub.6H.sub.5O.sub.7.2H.sub.2O)
in a 1,000 ml volumetric flask [0053] 2. Add deionized or Nanopure
water to make 1,000 ml final volume. [0054] 3. Transfer to bottles
and autoclave at 15 lbs pressure (245.degree. F.) for at least 30
minutes. [0055] a. Autoclave solution using conditions to minimize
evaporation (loose cover) [0056] b. Be careful that water does not
boil away. [0057] 4. Cool slowly at room temperature. [0058] 5.
Store sealed in a 5.degree. C. cold room.
[0059] B. Egg preparation [0060] 1. Obtain fresh hen's eggs from a
good commercial source. [0061] 2. Wash the eggs free of dirt (do
not use too much detergent) and rinse. [0062] 3. Immerse eggs in
70% ethanol for 2-5 minutes. [0063] 4. Remove eggs and allow to dry
(or wipe dry) and store on a clean towel.
[0064] C. Preparation of extender [0065] 1. Use sterile, clean
glassware [0066] 2. A-fraction (non-glycerol fraction) [0067] a.
Place 800 ml of 2.9% sodium citrate solution in a 1,000 ml
graduated cylinder. [0068] b. Antibiotic levels for the
non-glycerol containing fraction (A-fraction) of the extender may
be as follows: [0069] i. Tylosin=100 .mu.g/ml [0070] ii.
Gentamicin=500 .mu.g/ml [0071] iii. Linco-spectin=300/600 .mu.g/ml
[0072] c. Add 200 ml of fresh egg-yolk as outlined below (Section
D) [0073] i. Mix very thoroughly. [0074] d. This provides
A-fraction extender based on 2.9% sodium citrate, with 20% egg-yolk
and antibiotics at concentrations known to be non-toxic to bull
sperm. [0075] e. Extender can be stored overnight at 5.degree. C.
[0076] f Decant supernatant (upper 800 ml) the next day. [0077] g.
Warm to 37.degree. C. prior to use the next day.
[0078] D. To add egg-yolk to a buffered solution, the following
procedure works well. [0079] 1. Wash egg and clean the eggs (see B
above) [0080] 2. Open egg and separate yolk from albumin using a
yolk separator. Alternatively, pour yolk back and forth 2-3 times
between the two half shells. Do not rupture the membrane around the
yolk. [0081] 3. Place the yolk onto a sterile piece of 15 cm filter
paper. [0082] 4. Hold the filter paper over the graduated cylinder
containing buffer and squeeze the yolk (rupturing the membrane) and
allow the yolk to run out of the folded filter paper into the
cylinder. Typically about 12-15 ml of the yolk can be obtained from
one egg.
[0083] Another aspect which may interplay in the various factors of
the present invention is that of utilizing low dose amounts of
sperm for artificial insemination or the like. Additional
background on the aspect of sexed, artificial insemination may be
found in "Prospects for Sorting Mammalian Sperm" by Rupert P. Amman
and George E. Seidel, Jr., Colorado Associated University Press
(1982) hereby incorporated by reference. As mentioned, natural
insemination involves numbers of sperm on the order of billions of
sperm. Typical artificial insemination is presently conducted with
millions of sperm for bovine species and hundreds of millions of
sperm for equine species. By the term "low dose" it is meant that
the dosage of sperm utilized in the insemination event are less
than one-half or preferably even less than about 10% of the typical
number of sperm provided in a typical artificial insemination
event. Thus, the term "low dose" is to be viewed in the context of
the typical artificial insemination dosage or also as an absolute
number. For bovine sperm where currently 1 to 10 million sperm are
provided, a low dose process may be considered an absolute number
of about 500,000 sperm or perhaps as low as 300,000 sperm or lower.
In fact, through utilization of the techniques of the present
invention, artificial insemination with good percentages of success
has been shown with levels of insemination of sperm at 100,000 and
250,000 sperm (41% and 50%, respectively pregnancy rates). As shown
in the article "Uterine Horn Insemination of Heifers With Very Low
Numbers of Non-frozen and Sexed Spermatozoa" as published in 48
Theriogenology 1255 (1997) hereby incorporated by reference. Since
sperm cells appear to display a sensitivity to dilution, these
results may display particular interdependence on the utilization
of low dose sperm samples with regards to various techniques of the
present invention. The absolute numbers may be species dependent,
for equine species, merely less than about ten, five, or even one
million sperm may be considered a low dose process.
[0084] Another aspect which may be important is the fact that the
sperm sexed through the present invention techniques is utilized in
an artificial insemination system. Thus, when the collector (14) is
used to provide sperm for artificial insemination the techniques of
the present invention may be particularly relevant. Further, it is
possible that the combination of both artificial insemination use
and the use in a low dose environment may together create synergies
which makes the various techniques of the present invention
particularly appropriate. Naturally, the sexed sperm can be
utilized not just in an artificial insemination mode, but in other
techniques such as in vitro fertilization and the like.
[0085] The process of collecting, sorting, and eventually
inseminating an animal through the use of flow cytometry involves a
variety of steps. In the context of bovine insemination, first the
semen is collected from the bull through the use of an artificial
vagina. This occurs at rates of approximately 1.5 billion sperm per
ml. This neat semen may be checked through the use of a
spectrophotometer to assess concentration and may be
microscopically evaluated to assure that it meets appropriate
motility and viability standards. Antibiotics may then added. As a
result the initial sample may have approximately 60 to 70 percent
of the progressively motile sperm per ejaculate. For processing, a
dilution through of some type TALP (tyrode albumin lactate
pyruvate) may be used to get the numbers of sperm at a manageable
level (for flow analysis) of approximately 100 million per ml. The
TALP not only nurtures the sperm cells, but it may make them
hyper-activated for the staining step. Prior to staining, in some
species such as the equine species, centrifugation may be
accomplished. Staining may be accomplished according to a
multi-stained or single-stained protocol, the latter, the subject
of the Johnson Patent and related technology. The staining may be
accomplished while also adjusting the extender to create the
appropriate nutrient environment. In bovine applications this may
involve adding approximately 20% egg yolk content in a citrate
solution immediately after staining. Further, in staining the sperm
cells, it has been discovered that by using higher amounts of stain
than might to some extent be expected better results may be
achieved. This high concentration staining may involve using
amounts of stain in the tens of micro-molar content such as
discussed in the examples below where 38 micro-molar content of
Hoechst 33342 stain was used.
[0086] After adding the stain, an incubation period may be used
such as incubating at one hour at 34.degree. C. to hasten the dye
uptake with concentrations at about 100 million sperm cells per ml.
Filtration may then be accomplished to remove clumps of sperm cells
and then dilution or extending may or may not be accomplished to
the desired sort concentration of approximately 100 million sperm
cells per ml may be accomplished. Sorting according to the various
techniques discussed earlier may then be accomplished from which
sperm cells may be recovered in the collection phase. As mentioned
earlier, the collection may result in samples with approximately 2%
egg yolk citrate concentrate content (for bovine species). This
sample may then be concentrated to about 3-5 million sperm cells
per ml through the use of centrifugation after which the sheath
fluid and preserving fluid may be removed. A final extension may
then be accomplished with either 20% egg yolk citrate or a Cornell
Universal Extender or the like. The Cornell Universal Extender may
have the following composition for 1000 ml: [0087] 14.5 g sodium
citrate dihydrate [0088] 2.1 g NaHCO.sub.3 [0089] 0.4 g KCl [0090]
3.0 g glucose [0091] 9.37 g glycine [0092] 0.87 g citric acid
[0093] For 20% egg-yolk using 800 ml of above preparation and may
include about 200 ml of egg-yolk composition.
[0094] After this last extending, 3 to 5 million sperm per ml (for
bovine species) may result. This sample may then be cooled to slow
the sperm's metabolism and to permit use over longer periods of
time. In the equine species the sample may then be used in
oviductal or other insemination processes as those skilled in the
art well understand. In bovine sperm, the sample may be diluted yet
one more time to the desired dosage level. It has been discovered
that dilution may create an effect upon the sperm cell's viability
and so it may be appropriate to avoid too large a level of dilution
by providing a smaller sample. At present, low dosages of
approximately 300,000 sperm per 0.184 ml may be achieved.
Furthermore, it may be desirable to maintain a level of seminal
plasma at approximately a five percent level, although the results
of this requirement are, at present, mixed. The sperm cell specimen
may then be placed in a straw for use in artificial insemination
and may be transported to the cows or heifers to be
inseminated.
[0095] In order to achieve conveniently timed artificial
insemination, heifer or cow estrus may be synchronized using known
techniques such as the utilization of prostaglandin F2.sub..alpha.,
according to techniques well known in the art. This latter
substance may be particularly valuable in that it has been reported
to potentially achieve enhanced fertility in heifers as discussed
in the article "Prostoglandin F2.sub..alpha., --A Fertility Drug in
Dairy Cattle?", 18 Theriogenology 245 (1982) hereby incorporated by
reference. While recent results have not maintained this premise,
it may be that the present invention demonstrates its particular
viability in situations of sexed, low dose insemination. For bovine
species, artificial insemination may then be accomplished through
the use of embryo transfer equipment with placement of the sperm
cells deep within the uterine horns. This may be accomplished not
at the peak moment as typically used in artificial insemination,
but rather at a somewhat later moment such as 12 hours after that
time since there is some possibility that fertility for sexed
artificial insemination may occur slightly later. The utilization
of embryo transfer equipment may be used because there may be high
sensitivity of the uterine wall for such low dose, sexed
inseminations.
[0096] Interestingly, rather than inseminating within the uterine
body where such insemination are usually placed, by insemination
deep within the uterine horn, better results may be achieved.
Perhaps it is also surprising that the samples thus far studied
have shown no difference between ipsi- and contra-lateral
inseminations when accomplished deep within the uterine horn. By
deep, it should be understood that the insertion is placed well
into the uterine horn using the embryo transfer equipment. The fact
that results do not appear significantly different using ipsi- and
contra-lateral inseminations has led the present inventors to
propose the use of insemination in both so that the process of
identifying the appropriate uterine horn may no longer be
needed.
[0097] As a result of the insemination, it is of course desired
that an animal of the desired sex be produced. This animal may be
produced according to the systems discussed earlier through the use
of the sexed sperm specimen. It should also be understood that the
techniques of the present invention may find application in other
techniques such as laproscopic insemination, oviductal
insemination, or the like.
[0098] As examples, the following experiments have been conducted.
While not all use every aspect of the inventions described here,
they do show the performance enhancements possible through
differing aspects of the invention. Further, a summary of some
experiments is contained in the article "Uterine Horn Insemination
of Heifers With Very Low Numbers of Non-frozen and Sexed
Spermatozoa" as referenced earlier. This article summarizes some of
the data showing the efficacy of the present invention. As to the
experiments, one has been conducted with sexed, unfrozen sperm
cells with high success as follows:
Example 1
[0099] Angus heifers, 13-14 mo of age and in moderate body
condition, were synchronized with 25 mg of prostaglandin F-2 alpha
at 12-day intervals and inseminated 6-26 h after observed standing
estrus. Freshly collected semen from three 14-26 mo old bulls was
incubated in 38 .mu.M Hoechst 33342 at 75.times.10.sup.6 sperm/ml
in a TALP medium for 1 h at 34.degree. C. Sperm were sorted by sex
chromosomes on the basis of epiflourescence from laser excitation
at 351 and 364 nm at 150 mW using a MoFlo.RTM. flow cytometer/cell
sorter operating at 50 psi and using 2.9% Na citrate as sheath
fluid. X chromosome-bearing sperm (.about.90% purity as verified by
resorting sonicated sperm aliquots) were collected at .about.500
live sperm/sec into 2-ml Eppendorf tubes containing 100 .mu.l
Cornell Universal Extender (CUE) with 20% egg yolk. Collected sperm
were centrifuged at 600.times.g for 10 min and resuspended to
1.63.times.10.sup.6 live sperm/ml in CUE. For a liquid semen
unsexed control; Hoechst 33342-stained sperm were diluted with
sheath fluid to 9.times.10.sup.5 sperm/ml and centrifuged and
resuspended to 1.63.times.10.sup.6 progressively motile sperm/ml in
CUE. Sexed semen and liquid control semen were cooled to 5.degree.
C. over 75 min and loaded into 0.25-ml straws (184 .mu.l/straw).
Straws were transported at 3 to 5.degree. C. in a
temperature-controlled beverage cooler 240 km for insemination 5 to
9 h after sorting. Sexed semen and liquid control semen were
inseminated using side-opening blue sheaths (IMV), one half of each
straw into each uterine horn (3.times.10.sup.5 live sperm/heifer).
As a standard control, semen from the same bulls had been frozen in
0.5-cc straws by standard procedures (mean 15.6.times.10.sup.6
motile sperm/dose post-thaw), thawed at 35.degree. C. for 30 sec,
and inseminated into the uterine body. Treatments were balanced
over the 3 bulls and 2 inseminators in a ratio of 3:2:2
inseminations for the sexed semen and two controls. Pregnancy was
determined ultrasonically 31-34 days after insemination and
confirmed 64-67 days later when fetuses also were sexed (blindly).
Data are presented in the table.
TABLE-US-00002 No. Heifers No. Pregnant No. Pregnant No female
Treatment bred d31-34 d64-67 fetuses Sexed semen 45 20 (44%) 19
(42%) 18 (95%).sup.a Liquid control 28 15 (54%) 15 (54%) 8
(53%).sup.b Frozen control 29 16 (55%) 15 (52%) 12 (80%).sup.c
.sup.a,bSex ratios of values with different superscripts differ (P
< 0.02).
[0100] Although the pregnancy rate with sexed semen was only 80% of
controls, this difference was not statistically significant
(>0.1). One pregnancy was lost by 64-67 d in each of the sexed
and frozen control groups; 18 of 19 fetuses (95%) were female in
the sexed group, and 20 of 30 (67%) were female in the control
groups. The liquid semen control yielded a virtually identical
pregnancy rate to the frozen semen control containing over 50 times
more motile sperm (over 120 times more total sperm), demonstrating
the efficacy of low-dose insemination into the uterine horns. We
have altered the sex ratio in cattle significantly using flow
cytometer technology and artificial insemination.
Similarly, an experiment was conducted with unsexed, unfrozen sperm
cells and may be reported as follows:
Example 2
[0101] The objective was to determine pregnancy rates when heifers
are inseminated with extremely low numbers of frozen sperm under
ideal field conditions. Semen from three Holstein bulls of above
average fertility was extended in homogenized milk, 7% glycerol
(CSS) extender plus 5% homologous seminal plasma to
2.times.10.sup.5, 5.times.10.sup.5 or 10.times.10.sup.6 (control)
total sperm per 0.25 ml French straw and frozen in moving liquid
nitrogen vapor. Semen was thawed in 37.degree. C. water for 20 sec.
Holstein heifers 13-15 mo of age weighing 350-450 kg were injected
with 25 mg prostaglandin F-2-alpha (Lutalyse.RTM.) twice at a
12-day interval and inseminated with an embryo transfer straw gun
and side-opening sheath, half of the semen deep into each uterine
horn 12 or 24 h after detection of estrus. The experiment was done
in five replicates over 5 months, and balanced over two
insemination technicians. Ambient temperature at breeding was
frequently -10 to -20.degree. C., so care was taken to keep
insemination equipment warm. Pregnancy was determined by detection
of a viable fetus using ultrasound 40-44 days post-estrus and
confirmed 55-62 days post-estrus; 4 of 202 conceptuses were lost
between these times. Day 55-62 pregnancy rates were 55/103 (53%),
71/101, (70%), and 72/102 (71%) for 2.times.10.sup.5,
5.times.10.sup.5 and 10.times.10.sup.6 total sperm/inseminate
(P<0.1). Pregnancy rates were different (P<0.05) among bulls
(59, 62, and 74%), but not between technicians (64 and 65%) or
inseminations times post-estrus (65% for 12 h and 64% for 24 h,
N=153 at each time). With the methods described, pregnancy rates in
heifers were similar with 5.times.10.sup.5 and 10.times.10.sup.6
total sperm per inseminate.
Prior experiment has also been conducted on sexed, unfrozen sperm
cells and may be reported as follows:
Example 3
[0102] Semen was collected from bulls at Atlantic Breeders
Cooperative, diluted 1:4 with a HEPES-buffered extender+0.1% BSA,
and transported 160 km (.about.2 HR) to Beltsville, Md. where it
was sorted at ambient temperature by flow cytometry into a TEST
yield (20%) extender using methods described previously (Biol
Reprod 41:199). Sorting rates of up to 2.times.10.sup.6 sperm of
each sex per 5-6 h at .about.90% purity were achieved. Sperm were
concentrated by centrifugation (300 g for 4 min) to
2.times.10.sup.6 sperm/ml. Some sperm were sorted into extender
containing homologous seminal plasma (final concentration, 5%).
Sorted sperm were shipped by air to Colorado (.about.2,600 km) and
stored at either ambient temperature or 5.degree. C. (cooled during
shipping over 6 hr in an Equitainer, an insulated device with an
ice-containing compartment). Heifers or dry cows detected in estrus
11 to 36 h earlier were inseminated within 9 to 29 h of the end of
the sperm sorting session. Sperm (1 to 2.times.10.sup.5 in 0.1 ml)
were deposited deep in the uterine horn ipsilateral to the ovary
with the largest follicle as determined by ultrasound at the time
of insemination.
[0103] None of 10 females became pregnant when inseminated with
sperm shipped and stored at ambient temperature. Of 29 females
inseminated with sperm cooled to 5.degree. C. during shipping, 14
were pregnant at 4 weeks of gestation, and 12 (41%) at 8 weeks.
Eleven of the 22 inseminated within 10 h of the end of sorting were
pregnant at 8 weeks, but only 1 of 7 inseminated 17-24 h after
sorting was pregnant. There was no significant effect of adding
seminal plasma. One of the 12 fetuses was not of the predicted sex,
one was unclear, and 10 were of the predicted sex, as determined by
ultrasonography at 60-70 days of gestation.
[0104] Subsequently, 33 additional heifers were inseminated with
0.05 ml (semen extended as described above) into each uterine horn
without using ultrasonography; only 3 were pregnant 4 weeks after
insemination, and only 1 remained pregnant at 8 weeks.
[0105] However, different bulls were used from the previous group,
and all inseminations were done 18-29 h post-sorting. An additional
38 heifers were inseminated similarly (.about.22 h post-sorting)
200 km from our laboratory with sorted sperm from another bull;
none of these was pregnant 8 weeks after insemination.
[0106] To summarize, it is possible to achieve pregnancies in
cattle via artificial insemination of sperm sorted for sex
chromosomes by flow cytometry, and the sex ratio of fetuses
approximates that predicted by reanalysis of sorted sperm for DNA
content (90%). However, pregnancy rates varied greatly in these
preliminary experiments which required shipping sperm long
distances. Fertility decreased drastically by 17 h post-sorting,
but there was some confounding because different bulls were used at
the different times. Further studies are needed to determine
whether variation observed in pregnancy rates was due to bull
differences, insemination techniques, interval between sorting and
insemination, or other factors.
[0107] Finally, an experiment also has been conducted with unsexed,
unfrozen sperm cells and may be reported as follows:
Example 4
[0108] The objective was to determine pregnancy rates when heifers
were inseminated with very low numbers of sperm under ideal
experimental conditions. Semen from three Holstein bulls was
extended in Cornell Universal Extender plus 5% homologous seminal
plasma to 1.times.10.sup.5 or 2.5.times.10.sup.5 sperm per 0.1 ml;
2.5.times.10.sup.6 total sperm per 0.25 ml was used as a control.
Fully extended semen was packaged in modified 0.25 ml plastic
French straws to deliver the 0.1 or 0.25 ml inseminate doses. Semen
was cooled to 5.degree. C. and used 26-57 h after collection.
Holstein heifers 13-15 mo of age weighing 350-450 kg were injected
with 25 mg prostaglandin F-2 alpha (Lutalyse.RTM.) at 12-day
intervals and inseminated with an embryo transfer straw gun and
side-opening sheath into one uterine horn 24 h after detection of
estrus. Insemination was ipsilateral to the side with the largest
follicle determined by ultrasound 12 h after estrus; side of
ovulation was verified by detection of a corpus luteum by
ultrasound 7-9 days post-estrus. Pregnancy was determined by
detection of a fetus by ultrasound 42-45 days post estrus. The
experiment was done in four replicates and balanced over three
insemination technicians. Side of ovulation was determined
correctly in 205 of 225 heifers (91%); surprisingly, pregnancy
rates were nearly identical for ipsilateral and contralateral
inseminates. Pregnancy rates were 38/93 (41%), 45/87 (52%), and
25/45 (56%) for 1.times.10.sup.5, 2.5.times.10.sup.5 and
2.5.times.10.sup.6 sperm/inseminate (P>0.1). There was a
significant difference in pregnancy rate (P<0.05) among
technician, but not among bulls. With the methods described, it may
be possible to reduce sperm numbers per inseminate sufficiently
that sperm sorted by sex with a flow cytometer would have
commercial application.
[0109] As mentioned and as can be seen from the various
experiments, the field is statistically base and thus a variety of
additional experiments may be conducted to show the appropriate
combination and limitation strategies. Thus synergies among various
affects will further be identified, such as instances in which the
dye effects and combined dye effects with laser excitation may be
studied.
[0110] The discussion included in this application is intended to
serve as a basic description. The reader should be aware that the
specific discussion may not explicitly describe all embodiments
possible; many alternatives are implicit. It also may not fully
explain the generic nature of the invention and may not explicitly
show how each feature or element can actually be representative of
a broader function or of a great variety of alternative or
equivalent elements. Again, these are implicitly included in this
disclosure. Where the invention is described in device-oriented
terminology, each element of the device implicitly performs a
function. Apparatus claims may not only be included for the device
described, but also method or process claims may be included to
address the functions the invention and each element performs.
Neither the description nor the terminology is intended to limit
the scope of the claims which may be submitted. It should be
understood that a variety of changes may be made without departing
from the essence of the invention. Such changes are also implicitly
included in the description. They still fall within the scope of
this invention. A broad disclosure encompassing both the explicit
embodiment(s) shown, the great variety of implicit alternative
embodiments, and the broad methods or processes and the like are
encompassed by this disclosure.
[0111] In addition, each of the various elements of the invention
and claims may also be achieved in a variety of manners. This
disclosure should be understood to encompass each such variation,
be it a variation of an embodiment of any apparatus embodiment, a
method or process embodiment, or even merely a variation of any
element of these. Particularly, it should be understood that as the
disclosure relates to elements of the invention, the words for each
element may be expressed by equivalent apparatus terms or method
terms--even if only the function or result is the same. Such
equivalent, broader, or even more generic terms should be
considered to be encompassed in the description of each element or
action. Such terms can be substituted where desired to make
explicit the implicitly broad coverage to which this invention is
entitled. As but one example, it should be understood that all
actions may be expressed as a means for taking that action or as an
element which causes that action. Similarly, each physical element
disclosed should be understood to encompass a disclosure of the
action which that physical element facilitates. As but one example
of this aspect, the disclosure of a "collector" should be
understood to encompass disclosure of the act of
"collecting"--whether explicitly discussed or not--and, conversely,
were there only disclosure of the act of "collecting", such a
disclosure should be understood to encompass disclosure of a
"collector." Such changes and alternative terms are to be
understood to be explicitly included in the description.
[0112] Any references mentioned in the application for this patent
as well as all references listed in any information disclosure
filed with the application are hereby incorporated by reference. In
addition, the table of references as presented below are hereby
incorporated by reference. However, to the extent statements might
be considered inconsistent with the patenting of this/these
invention(s) such statements are expressly not to be considered as
made by the applicant(s).
* * * * *