U.S. patent application number 16/379456 was filed with the patent office on 2019-10-10 for methods and compositions for determining the presence or absence of dna aberrations.
This patent application is currently assigned to Inguran, LLC. The applicant listed for this patent is Inguran, LLC. Invention is credited to Daniela do Amaral Grossi, Kenneth Michael Evans, Thomas B. Gilligan.
Application Number | 20190310243 16/379456 |
Document ID | / |
Family ID | 68096363 |
Filed Date | 2019-10-10 |
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United States Patent
Application |
20190310243 |
Kind Code |
A1 |
Gilligan; Thomas B. ; et
al. |
October 10, 2019 |
METHODS AND COMPOSITIONS FOR DETERMINING THE PRESENCE OR ABSENCE OF
DNA ABERRATIONS
Abstract
The invention consists of methods and compositions for detecting
the presence or absence of a DNA aberration by analyzing
fluorescence emission characteristics in sperm cells or sperm
nuclei, which generally consists of entraining sperm cells or sperm
nuclei stained with a DNA selective dye in sheath fluid; exposing
the entrained sperm cells or sperm nuclei to electromagnetic
radiation; determining a forward fluorescence characteristic and a
side fluorescence characteristic of individual events associated
with the exposed sperm cells or sperm nuclei; and gating the
individual events based on the forward fluorescence characteristic
and the side fluorescence characteristic with a criterion.
Inventors: |
Gilligan; Thomas B.;
(College Station, TX) ; Evans; Kenneth Michael;
(College Station, TX) ; do Amaral Grossi; Daniela;
(Saskatoon, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Inguran, LLC |
Navasota |
TX |
US |
|
|
Assignee: |
Inguran, LLC
Navasota
TX
|
Family ID: |
68096363 |
Appl. No.: |
16/379456 |
Filed: |
April 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62655040 |
Apr 9, 2018 |
|
|
|
62673668 |
May 18, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 15/1459 20130101;
G01N 15/14 20130101; G01N 15/1012 20130101; G01N 1/30 20130101;
G01N 2001/302 20130101; C12Q 1/6879 20130101; G01N 2015/149
20130101; G01N 2015/1006 20130101; C12N 5/061 20130101; G01N
21/6486 20130101; G01N 33/5005 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 21/64 20060101 G01N021/64 |
Claims
1. A method of analyzing fluorescence emission characteristics in
sperm cells or sperm nuclei, comprising: entraining sperm cells or
sperm nuclei stained with a DNA selective dye in sheath fluid;
exposing the entrained sperm cells or sperm nuclei to
electromagnetic radiation; determining a forward fluorescence
characteristic and a side fluorescence characteristic of individual
events associated with the exposed sperm cells or sperm nuclei;
gating the individual events based on the forward fluorescence
characteristic and the side fluorescence characteristic with a
criterion; and determining the presence or absence of a DNA
aberration from the gated individual events.
2. The method of claim 1, wherein the DNA selective dye is Hoechst
33342.
3. The method of claim 1, further comprising the step of orienting
the entrained sperm cells or sperm nuclei.
4. The method of claim 1, wherein the step of exposing the sperm
cells or sperm nuclei to electromagnetic radiation comprises
exposing the sperm cells or sperm nuclei to a laser beam with
modified beam profile.
5. The method of claim 1, wherein the criterion encompasses a
subpopulation of oriented sperm cells or sperm nuclei.
6. The method of claim 1, wherein the sperm cells or sperm nuclei
comprise sperm cells or sperm nuclei from a first and a second
mammalian species.
7. The method of claim 1, wherein determining the presence of a DNA
aberration from the gated individual events comprises detecting
more than two peaks or modes, or a peak to valley ratio of 50% or
less, 60% or less, 70% or less or 80% or less, on a histogram of
fluorescence intensities.
8. The method of claim 6, wherein determining the presence of a DNA
aberration from the gated individual events comprises detecting a
difference between a peak to valley ratio on a histogram of
fluorescence intensities of sperm cells or sperm nuclei from the
first mammalian species and a peak to valley ratio on a histogram
of fluorescence intensities of sperm cells or sperm nuclei from the
second mammalian species.
9. A method of analyzing fluorescence emission characteristics in
sperm cells or sperm nuclei, comprising: entraining sperm cells or
sperm nuclei stained with a DNA selective dye in sheath fluid;
exposing the entrained sperm cells or sperm nuclei to
electromagnetic radiation; determining two or more fluorescence
emission characteristics of individual events associated with the
exposed sperm cells or sperm nuclei; gating the individual events
based on the two or more fluorescence emission characteristics with
a first criterion for further processing; gating the further
processed individual events with a second criterion; and
determining the presence or absence of a DNA aberration from the
twice gated individual events.
10. The method of claim 9, wherein the first criterion encompasses
a first subpopulation of oriented sperm cells or sperm nuclei.
11. The method of claim 10, wherein the second criterion
encompasses a second subpopulation of sperm cells or sperm nuclei
comprising 25% to 75% of the first subpopulation.
12. The method of claim 11, wherein the step of determining the
presence or absence of DNA aberrations in the sperm cells or sperm
nuclei from the twice gated individual events further comprises
evaluating a third subpopulation of sperm cells or sperm nuclei
within the first subpopulation, wherein the third subpopulation of
sperm cells or sperm nuclei excludes the second subpopulation of
sperm cells or sperm nuclei.
13. The method of claim 10, wherein the second criterion
encompasses 25% to 75% of sperm cells or sperm nuclei around a
median value of a fluorescence emission characteristic.
14. The method of claim 9, wherein the step of determining the
presence or absence of a DNA aberration in the sperm cells or sperm
nuclei from the twice gated individual events further comprises
determining the quantity of peaks or modes in a twice gated
fluorescence emission characteristic.
15. The method of claim 9, wherein the step of determining the
presence or absence of a DNA aberration in the sperm cells or sperm
nuclei from the twice gated individual events further comprises
analyzing a coefficient of variation of a fluorescence emission
characteristic associated with the first subpopulation of sperm
cells or sperm nuclei or the second subpopulation of sperm cells or
sperm nuclei.
16. The method of claim 9, wherein the step of determining the
presence or absence of a DNA aberration in the sperm cells or sperm
nuclei from the twice gated individual events further comprises
generating a first univariate plot based on a fluorescence emission
characteristic of the first subpopulation or generating a second
univariate plot based on a fluorescence emission characteristic of
the second subpopulation.
17. The method of claim 16, wherein the step of determining the
presence or absence of a DNA aberration in the sperm cells or sperm
nuclei from the twice gated individual events further comprises
analyzing a peak to valley ratio of the first univariate plot or a
peak to valley ratio of the second univariate plot.
18. The method of claim 9, further comprising the step of
separating tails or midpieces from the sperm nuclei by
centrifugation.
19. A method of analyzing a fluorescence emission characteristic in
sperm cells or sperm nuclei comprising: staining the sperm cells or
sperm nuclei with a DNA selective dye; entraining the stained sperm
cells or sperm nuclei in sheath fluid; exposing the entrained sperm
cells or sperm nuclei to electromagnetic radiation; determining a
fluorescence emission characteristic of the exposed sperm cells or
sperm nuclei; generating a first multivariate plot based on the
fluorescence emission characteristic; providing a first gate on the
first multivariate plot; generating a second multivariate plot
based on the first gate; providing a first sort region on the
second multivariate plot encompassing a first subpopulation of
sperm cells or sperm nuclei and a second sort region on the second
multivariate plot encompassing a second subpopulation of sperm
cells or sperm nuclei; separating the first subpopulation of sperm
cells or sperm nuclei and the second subpopulation of sperm cells
or sperm nuclei; generating a first univariate plot based on a
fluorescence emission characteristic of the separated first
subpopulation of sperm cells or sperm nuclei; generating a second
univariate plot based on a fluorescence emission characteristic of
the separated second subpopulation of sperm cells or sperm
nuclei.
20. The method of claim 19, further comprising the step of removing
tails or midpieces from the sperm nuclei by centrifugation.
21. The method of claim 20, wherein the first subpopulation of
sperm cells or sperm nuclei comprises 25% to 75% of the sperm cell
or sperm nuclei population.
22. The method of claim 21, wherein the second subpopulation of
sperm cells or sperm nuclei excludes the first subpopulation of
sperm cells or sperm nuclei.
23. The method of claim 22, wherein the first sort region
encompasses the center of the second multivariate plot.
24. The method of claim 21, further comprising the step of
determining the quantity of peaks or modes of the first univariate
plot or the second univariate plot, analyzing a coefficient of
variation of the first univariate plot or the second univariate
plot, or analyzing a peak to valley ratio of the first univariate
plot or the second univariate plot.
25. A composition comprising unsorted sperm nuclei, a DNA-selective
dye and an aggregation reducing compound, wherein the composition
has been sonicated.
26. The composition of claim 25, wherein the aggregation reducing
compound comprises egg yolk, iodixanol, lecithin, bovine serum
albumin, gelatin, collagen or hydrolyzed collagen, arabinogalactan,
or a chemically defined polyethylene or polypropylene glycol.
27. The composition of claim 25, wherein the sperm nuclei comprise
sperm nuclei from a first non-human mammalian species and sperm
nuclei from a second non-human mammalian species.
28. A method of analyzing a fluorescence emission characteristic in
sperm nuclei comprising: combining a DNA-selective dye and an
aggregation reducing compound with a sperm cell sample to create a
sperm cell mixture; sonicating the sperm cell mixture to create
stained sperm nuclei; determining a forward fluorescence
characteristic and a side fluorescence characteristic of individual
events associated with the exposed stained sperm nuclei;
determining the presence or absence of a DNA aberration from the
individual events.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of United States
Provisional Patent Application No. 62/655,040 filed Apr. 9, 2018
and U.S. Provisional Patent Application No. 62/673,668 filed May
18, 2018. The entire disclosures of which are incorporated herein
by reference.
BACKGROUND
[0002] In mammals, the male (sire) haploid cells (sperm) are
presented by ejaculation in very large numbers comprising millions
or even billions of cells, each of which carry a somewhat random
distribution of autosomes (normally one of a pair of equal sized
autosomes) as well as sex chromosomes (X and Y) that normally
distribute 1/2 of the total haplotype of the male into each sperm.
In normal healthy males, the sperm population is typically two
distinct populations, where variation in total DNA content is based
only on the sex chromosome and sorting of such populations of sperm
by flow cytometry is a well-established and non-invasive method to
preselect sex (create percent of bias of male or female) in
mammals.
[0003] The use of genetic analysis of individual breeding animals
by DNA genotyping or DNA sequencing methods has become a low-cost
method to analyze the genetic microstructure and mathematical
distribution of haplotype inheritance and is used in large scale to
determine the breeding value of an animal. Large stable DNA
macrostructural aberrations such as translocations are not analyzed
by the above-mentioned microstructural DNA analysis methods, but
since they can also lead to poor reproductive performance, such as
reduction in litter size (in multiparous species) or pregnancy
rates (in uniparous species) by mechanism of early embryonic death,
low-cost methods to screen for them are needed.
[0004] A common problem, for example in swine breeding, is created
when translocations or other stable DNA aberrations are carried
forward, particularly in breeding males, by non-lethal inheritance.
When the negative phenotype is litter size, the reliance on that
phenotypical observation to cull out breeding boars and sows is
problematic because progeny carriers are created, and if these
animals are then used to generate new breeding males and females
they may also be carriers and the problem can remain or even be
amplified. For this reason, it has become a common practice to use
the long-standing method of karyotype analysis to screen breeding
animals, in special boars, prior to their use. Karyotype analysis
has the advantage that it can commonly identify large stable
changes in DNA macrostructure by reliable and well defined methods,
and the advent of computer assisted image analysis has made
karyotype analysis routine. Nonetheless, karyotype analysis
typically requires handling of fresh specimens (mainly blood), the
cultivation of clonal cell lines in defined but artificial media
and the analysis of diploid somatic cells as a proxy to direct
analysis of germ line cells. The process typically takes a minimum
of several days, if not even weeks to complete. It is not highly
statistically significant, as the number of cells analyzed is
small. There are also limitations in the threshold of percent of
DNA change that can be determined. Furthermore, karyotype analysis
does not measure any abnormal effects in spermatogenesis that might
lead to materially important changes in DNA distribution within
sperm populations (gamete aneuploidy).
[0005] Additionally, in species such as bovines where artificial
insemination is used extensively but conception rate information is
difficult and time consuming to recover, karyotype analysis is not
reliable and low-cost enough to be used industrially. The current
karyotyping procedures takes approximately 3 weeks. A sample must
be shipped by overnight courier and in some cases shipments are
delayed, and the sample is lost and unable to be analyzed requiring
resampling. Karyotyping is costly and prices are expected to
increase with a lack of sufficient facilities to perform
karyotyping, with some facilities discontinuing service.
[0006] Translocations happen at a rate of 1% in swine--therefore a
very high number of tests are normal at a high cost. The effect of
a translocated boar's semen is a loss of about 4-5 piglets per
litter. One single boar can produce thousands of pigs in a
lifetime, so if those litter sizes are smaller there is lost
productivity. Because of the problem, which is that approximately
1/3 of the sperm produced by a translocated boar will be non-viable
due to reduced amount of DNA, decreased fecundity occurs.
SUMMARY OF THE INVENTION
[0007] One embodiment of the invention encompasses a method of
analyzing fluorescence emission characteristics in sperm cells or
sperm nuclei (i.e., sperm heads), comprising entraining sperm cells
or sperm nuclei stained with a DNA selective dye in sheath fluid;
exposing the entrained sperm cells or sperm nuclei to
electromagnetic radiation; determining a forward fluorescence
characteristic and a side fluorescence characteristic of individual
events associated with the exposed sperm cells or sperm nuclei;
gating the individual events based on the forward fluorescence
characteristic and the side fluorescence characteristic with a
criterion; and determining the presence or absence of a DNA
aberration from the gated individual events. In a further
embodiment, the DNA selective dye is Hoechst 33342. Another
embodiment further comprises the step of orienting the entrained
sperm cells or sperm nuclei. In another embodiment, the step of
exposing the sperm cells or sperm nuclei to electromagnetic
radiation comprises exposing the sperm cells or sperm nuclei to a
laser beam with modified beam profile. In yet another embodiment,
the criterion encompasses a subpopulation of oriented sperm cells
or sperm nuclei. In these embodiments, the sperm may comprise sperm
cells or sperm nuclei from a first and a second mammalian species.
In a further embodiment, determining the presence of a DNA
aberration from the gated individual events comprises detecting
more than two peaks or modes, or a peak to valley ratio of 80% or
less, 70% or less, 60% or less, or 50% or less, on a histogram of
fluorescence intensities, or measureable differences in
co-efficient of variation in two or more modes. In a yet further
embodiment, determining the presence of a DNA aberration from the
gated individual events comprises detecting a difference between a
peak to valley ratio on a histogram of fluorescence intensities of
sperm cells or sperm nuclei from the first mammalian species and a
peak to valley ratio on a histogram of fluorescence intensities of
sperm cells or sperm nuclei from the second mammalian species or
measureable differences in co-efficient of variation between two or
more modes created by the first and second mammalian species.
Another embodiment of the invention comprises subjecting the
entrained sperm cells or sperm nuclei to orienting hydrodynamic
forces such as those imparted by an orienting nozzle.
[0008] The invention also encompasses a method of analyzing
fluorescence emission characteristics in sperm cells or sperm
nuclei, comprising entraining sperm cells or sperm nuclei stained
with a DNA selective dye in sheath fluid; exposing the entrained
sperm cells or sperm nuclei to electromagnetic radiation;
determining two or more fluorescence emission characteristics of
individual events associated with the exposed sperm cells or sperm
nuclei; gating the individual events based on the two or more
fluorescence emission characteristics with a first criterion for
further processing; gating the further processed individual events
with a second criterion; and determining the presence or absence of
a DNA aberration from the twice gated individual events. In a
further embodiment, the first criterion encompasses a first
subpopulation of oriented sperm cells or sperm nuclei. In a yet
further embodiment, the second criterion encompasses a second
subpopulation of sperm cells or sperm nuclei comprising 25% to 75%
of the first subpopulation. In an even further embodiment, the step
of determining the presence or absence of DNA aberrations in the
sperm cells or sperm nuclei from the twice gated individual events
further comprises evaluating a third subpopulation of sperm cells
or sperm nuclei within the first subpopulation, wherein the third
subpopulation of sperm cells or sperm nuclei excludes the second
subpopulation of sperm cells or sperm nuclei. In an additional
embodiment, the second criterion encompasses 25% to 75% of sperm
around a median value of a fluorescence emission characteristic. In
a further embodiment, the step of determining the presence or
absence of a DNA aberration in the sperm cells or sperm nuclei from
the twice gated individual events further comprises determining the
quantity of peaks or modes in a twice gated fluorescence emission
characteristic. In an even further embodiment, the step of
determining the presence or absence of a DNA aberration in the
sperm cells or sperm nuclei from the twice gated individual events
further comprises analyzing a coefficient of variation of a
fluorescence emission characteristic associated with the first
subpopulation of sperm cells or sperm nuclei or the second
subpopulation of sperm cells or sperm nuclei. In an additional
embodiment, the step of determining the presence or absence of a
DNA aberration in the sperm cells or sperm nuclei from the twice
gated individual events further comprises generating a first
univariate plot based on a fluorescence emission characteristic of
the first subpopulation or generating and a second univariate plot
based on a fluorescence emission characteristic of the second
subpopulation. In a further embodiment, the step of determining the
presence or absence of a DNA aberration in the sperm cells or sperm
nuclei from the twice gated individual events further comprises
analyzing a peak to valley ratio of the first univariate plot or a
peak to valley ratio of the second univariate plot or measureable
differences in co-efficient of variation between modes in
multivariate plots. In an even further embodiment, the method
further comprises separating tails or midpieces from the sperm
nuclei by centrifugation. In a further embodiment, the DNA
selective dye is Hoechst 33342. Another embodiment further
comprises the step of orienting the entrained sperm cells or sperm
nuclei. In another embodiment, the step of exposing the sperm cells
or sperm nuclei to electromagnetic radiation comprises exposing the
sperm cells or sperm nuclei to a laser beam with modified beam
profile. In these embodiments, the sperm cells or sperm nuclei may
comprise sperm cells or sperm nuclei from a first and a second
mammalian species. In a further embodiment, determining the
presence of a DNA aberration comprises detecting more than two
peaks or modes, or a peak to valley ratio of 80% or less, 70% or
less, 60% or less, or 50% or less, on a histogram of fluorescence
intensities or measureable differences in co-efficient of variation
between modes in multivariate plots. In a yet further embodiment,
determining the presence of a DNA aberration comprises detecting a
difference between a peak to valley ratio on a histogram of
fluorescence intensities of sperm cells or sperm nuclei from the
first mammalian species and a peak to valley ratio or co-efficient
of variation in modes on a histogram of fluorescence intensities of
sperm cells or sperm nuclei from the second mammalian species.
Another embodiment of the invention comprises subjecting the
entrained sperm cells or sperm nuclei to orienting hydrodynamic
forces such as those imparted by an orienting nozzle.
[0009] The invention is also embodied by a method of analyzing a
fluorescence emission characteristic in sperm cells or sperm
nuclei, comprising staining the sperm cells or sperm nuclei with a
DNA selective dye; entraining the stained sperm cells or sperm
nuclei in sheath fluid; exposing the entrained sperm cells or sperm
nuclei to electromagnetic radiation; determining a fluorescence
emission characteristic of the exposed sperm cells or sperm nuclei;
generating a first multivariate plot based on the fluorescence
emission characteristic; providing a first gate on the first
multivariate plot; generating a second multivariate plot based on
the first gate; providing a first sort region on the second
multivariate plot encompassing a first subpopulation of sperm and a
second sort region on the second multivariate plot encompassing a
second subpopulation of sperm; separating the first subpopulation
of sperm cells or sperm nuclei and the second subpopulation of
sperm cells or sperm nuclei; generating a first univariate plot
based on a fluorescence emission characteristic of the separated
first subpopulation of sperm cells or sperm nuclei; generating a
second univariate plot based on a fluorescence emission
characteristic of the separated second subpopulation of sperm cells
or sperm nuclei; and determining the presence or absence of a DNA
aberration based on the first univariate plot or the second
univariate plot. In another embodiment, the method further
comprises the step of separating tails or midpieces from the sperm
nuclei by centrifugation. In yet another embodiment, the first
subpopulation of sperm cells or sperm nuclei comprises 25% to 75%
of the sperm population. In an even further embodiment, the second
subpopulation of sperm cells or sperm nuclei excludes the first
subpopulation of sperm cells or sperm nuclei. In an additional
embodiment, the first sort region encompasses the center of the
second multivariate plot. In yet another embodiment, the method
comprises the step of determining the quantity of peaks or modes of
the first univariate plot or the second univariate plot, analyzing
a coefficient of variation of the first univariate plot or the
second univariate plot, or analyzing a peak to valley ratio of the
first univariate plot or the second univariate plot. In a further
embodiment, the DNA selective dye is Hoechst 33342. Another
embodiment further comprises the step of orienting the entrained
sperm cells or sperm nuclei. In another embodiment, the step of
exposing the sperm cells or sperm nuclei to electromagnetic
radiation comprises exposing the sperm cells or sperm nuclei to a
laser beam with modified beam profile. In these embodiments, the
sperm cells or sperm nuclei may comprise sperm cells or sperm
nuclei from a first and a second mammalian species--in such
embodiments, the first subpopulation of sperm cells or sperm nuclei
comprises 25% to 75% of the sperm cells or sperm nuclei from the
first mammalian species. In a further embodiment, determining the
presence of a DNA aberration comprises detecting more than two
peaks or modes, or a peak to valley ratio of 80% or less, 70% or
less, 60% or less, or 50% or less, on a histogram of fluorescence
intensities or measureable differences in co-efficient of variation
between modes in multivariate plots. Another embodiment of the
invention comprises subjecting the entrained sperm cells or sperm
nuclei to orienting hydrodynamic forces such as those imparted by
an orienting nozzle.
[0010] Another embodiment of the invention comprises a method of
analyzing a fluorescence emission characteristic in sperm cells or
sperm nuclei, comprising staining the sperm cells or sperm nuclei
with a DNA selective dye; entraining the stained sperm cells or
sperm nuclei in sheath fluid; exposing the entrained sperm cells or
sperm nuclei to electromagnetic radiation; determining a
fluorescence emission characteristic of the exposed sperm cells or
sperm nuclei; generating a first multivariate plot based on the
fluorescence emission characteristic; providing a first gate on the
first multivariate plot; generating a second multivariate plot
based on the first gate; providing a first sort region on the
second multivariate plot encompassing a first subpopulation of
sperm cells or sperm nuclei and a second sort region on the second
multivariate plot encompassing a second subpopulation of sperm
cells or sperm nuclei, wherein the first sort region encompasses
the center of the second multivariate plot. In a further
embodiment, the first subpopulation of sperm cells or sperm nuclei
comprises 25% to 75% of the sperm cell or sperm nuclei population.
In a yet further embodiment, the second subpopulation of sperm
cells or sperm nuclei excludes the first subpopulation of sperm
cells or sperm nuclei. Another embodiment of the invention
comprises subjecting the entrained sperm cells or sperm nuclei to
orienting hydrodynamic forces such as those imparted by an
orienting nozzle.
[0011] In a yet further embodiment, determining the presence of a
DNA aberration comprises detecting a difference between a peak to
valley ratio on a histogram of fluorescence intensities of sperm
cells or sperm nuclei from the first mammalian species and a peak
to valley ratio or co-efficient of variation in modes on a
histogram of fluorescence intensities of sperm cells or sperm
nuclei from the second mammalian species.
[0012] Another embodiment of the invention comprises a composition
comprising sonicated sperm cells or sperm nuclei, a DNA selective
dye, an aggregation reducing compound and a buffer. In a further
embodiment, the aggregation reducing compound comprises egg yolk,
iodixanol, lecithin, bovine serum albumin, gelatin, collagen or
hydrolyzed collagen, arabinogalactan, or a chemically defined
polyethylene or polypropylene glycol. In a yet further embodiment,
the sonicated sperm cells or sperm nuclei comprise sperm cells or
sperm nuclei from a first non-human mammalian species and sperm
cells or sperm nuclei from a second non-human mammalian species. In
a further embodiment, the DNA selective dye is Hoechst 33342.
[0013] In a further embodiment of the invention, any of the methods
for determining the presence or absence of a DNA aberration
disclosed herein can further comprise the step of culling the male
from whom the sperm cells or sperm nuclei are obtained or derived
based on the determined presence of a DNA aberration in the male.
In another embodiment of the invention, any of the methods for
determining the presence or absence of a DNA aberration disclosed
herein can further comprise the step removing the male, from whom
the sperm cells or sperm nuclei are obtained or derived, from a
breeding program in a genetic nucleus or a herd based on the
determined presence of a DNA aberration in the male. In another
embodiment, the method further comprises karyotyping the male from
whom the sperm cells or sperm nuclei are obtained or derived based
on the determined presence of a DNA aberration in the male. In an
additional embodiment, the method further comprises karyotyping the
dam of the male from whom the sperm cells or sperm nuclei are
obtained or derived based on the determined presence of a DNA
aberration in the male. In an additional embodiment, the method
further comprises culling the dam of the male from whom the sperm
cells or sperm nuclei are obtained or derived based on the
determined presence of a DNA aberration in the male or an abnormal
karyotype in the dam. In a further embodiment, the method further
comprises determining the presence or absence of a DNA aberration
(using any of the methods disclosed herein) in, or karyotyping, the
progeny of the dam of the male from whom the sperm cells or sperm
nuclei are obtained or derived based on the determined presence of
a DNA aberration in the male. In a further embodiment, the method
further comprises culling progeny of the dam of the male from whom
the sperm cells or sperm nuclei are obtained based on the
determined presence of a DNA aberration or abnormal karyotype in
the progeny or the determined presence of a DNA aberration in the
male.
[0014] It should be understood that any of the embodiments
disclosed herein for determining the presence or absence of a DNA
aberration can be employed to detect the presence or absence of the
aberration in either sperm cells or sperm nuclei (i.e., sperm with
their tails and midpieces removed, by for example,
sonication)--that is, either sperm or sperm nuclei can be analyzed
with the methods of the invention.
[0015] The invention also encompasses an improved method for making
sperm nuclei comprising combining a DNA-selective dye and an
aggregation reducing compound with a sperm cell sample to create a
sperm cell mixture; and sonicating the sperm cell mixture to create
stained sperm nuclei.
[0016] Another embodiment of the invention comprises a composition
comprising unsorted sperm nuclei, an aggregation-reducing compound
and a DNA selective dye, wherein the composition has been
sonicated. In a further embodiment, the DNA selective dye is
Hoechst 33342. In another embodiment, the sperm nuclei are derived
from sperm cells from one male. In another embodiment, the
composition has a temperature of 45.degree. C. or greater. In a
particular embodiment, the aggregation-reducing compound is egg
yolk.
[0017] An additional embodiment of the invention comprises a method
of processing sperm cells comprising providing an unsorted sperm
cell sample; combining a DNA selective dye and an
aggregation-reducing compound with the unsorted sperm cell sample
to create a sperm cell mixture; and sonicating the sperm cell
mixture to create stained sperm nuclei. In another embodiment, the
DNA selective dye is Hoechst 33342. In a yet further embodiment,
the unsorted sperm cell sample is obtained from one male. In
another embodiment, the sperm cell mixture has a temperature of
45.degree. C. or greater during the step of sonication. In a yet
further embodiment, the aggregation-reducing compound is egg yolk.
In an additional embodiment, the method is completed in 20 minutes
or less.
[0018] The improved sperm nuclei of the invention can be used in
any of the aforementioned methods of determining the presence or
absence of a DNA aberration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows examples of multivariate and univariate plots
as well as gates used for logical gating of abnormal boar sperm
nuclei (i.e., comprising a DNA aberration).
[0020] FIG. 2 illustrates part of a flow cytometer used to analyze
and then sort a sperm composition to form one or more
subpopulations.
[0021] FIG. 3 illustrates a microfluidic chip used to analyze and
then sort a sperm composition.
[0022] FIG. 4 illustrates a univariate plot in the form of a
histogram generated by an analyzer or sorter.
[0023] FIGS. 5-11 are flow cytometer images showing exemplary
histograms for the five different categories of stable chromosomal
translocation found in boars, produced using the parse sorting
method of the invention.
[0024] FIGS. 12-18 are flow cytometer images showing histograms for
boar sperm nuclei analyzed using the parse sorting method of the
invention.
[0025] FIGS. 19-22 are flow cytometer images showing histograms for
boar sperm nuclei analyzed using the logical gating method of the
invention.
[0026] FIGS. 23-33 are flow cytometer images showing histograms for
boar sperm nuclei analyzed using the parse sorting method of the
invention.
[0027] FIG. 34 shows flow cytometer images showing histograms for
bull sperm nuclei analyzed using the parse sorting method of the
invention.
[0028] FIG. 35 shows a screenshot from a flow cytometer showing
multivariate plots and a univariate plot of forward fluorescence
intensities of a stained sperm cell sample indicating the presence
of a DNA aberration (Category IV translocation) in a boar.
[0029] FIG. 36 shows a screenshot from a flow cytometer showing
multivariate plots and a univariate plot of forward fluorescence
intensities of a stained sperm cell sample indicating the presence
of a DNA aberration (Category III translocation) in a boar.
[0030] FIG. 37 shows a screenshot from a flow cytometer showing
multivariate plots and a univariate plot of forward fluorescence
intensities of a stained sperm cell sample indicating the presence
of a DNA aberration (Category IV translocation) in a boar.
[0031] FIG. 38 shows a flow cytometer image showing a histogram for
a subpopulation from the center sort region indicating the presence
of a DNA aberration in a bull.
[0032] FIG. 39 shows a flow cytometer image showing a histogram for
a subpopulation from the flanking sort region indicating the
presence of a DNA aberration in a bull.
DETAILED DESCRIPTION OF THE INVENTION
[0033] One embodiment of the invention encompasses a method that
uses a low-cost and readily available biomaterial in sperm,
facilitates the rapid analysis of said sperm (or derived sperm
nuclei) in a matter of minutes (in contrast to days or weeks,) and
depending on the level of precision and accuracy of the equipment
and operator, can generate statistically significant proof and
quantification of changes in total DNA that may be as low as 0.1%.
Parallelization of the invention in sample handling and total
per-sample analysis time of less than one hour can facilitate the
analysis of about 10 samples or more per day and relatedly as many
as 2500 samples per year at costs per sample that may be as low as
20% of karyotype analysis costs. The invention is also capable of
yielding a more sensitive measurement of the size of DNA
aberrations than is typical with karyotyping. Using the invention,
identity mapping to show identical genotypes (related carrier
animals) is facilitated, and the study of the way such aberrations
arise and inherit is also facilitated. It should be understood that
the invention in any of its embodiments can be used to determine
the presence or absence of a DNA aberration using either sperm or
sperm nuclei.
[0034] Although the invention may substitute or replace higher cost
and time consuming karyotyping, it is also complementary, as the
measured feature is distribution of total DNA in different
subpopulations without specification of which chromosomes are
affected. In some embodiments, the invention can be performed using
state-of-the-art equipment used in the sex-selection of mammals in
industrial scale and can therefore be introduced to existing users
of sex-selection technology with limited new capital
investment.
[0035] Since in some embodiments, the invention can use frozen
sperm and in further embodiments be micro-scaled to use as few as
50 million sperm, it can be performed using one or more
cryopreserved semen straws, even from animals that are not living.
In this way, even though in some embodiments the invention may be
concentrated at locations that specialize in sex-selection in
general, the shipment and contract analysis of samples will be
possible.
[0036] Since the invention uses flow cytometry, future advances in
miniaturization, automation and sensitivity of sperm analysis by
flow cytometry will facilitate future reduction in cost of using
the invention and parallelization of analysis, if needed. As used
herein, the term "flow cytometry" also encompasses microfluidics,
and the term "flow cytometers" encompasses both flow cytometers and
microfluidic devices generally. Certain embodiments of the
invention may further comprise the use of kits, where ready-to-use
reagents and consumable materials can allow the dissemination of
the method to large numbers of different locations with same
quality of analysis.
[0037] In certain embodiments, the invention does not require the
use of computational analysis of histogramatic data produced in
flow cytometry (as exemplified in flow cytometry standard ["FCS"]
files), but since such data can easily be produced during the
analysis, the precision with which data can be analyzed can be
improved by using a large number of already existing software
packages for analyzing flow cytometry data. In other embodiments,
computational analysis of the data, including histogramatic data,
produced by the invention may be utilized.
[0038] It is contemplated that the invention can be applied to many
hundreds of thousands or millions of breeding sires (e.g., boars
and bulls) and can readily become a standard and essential method
in the screening of breeding value and reproductive performance of
livestock or other animals, including humans. Additionally,
although methods for artificial chromosomes (synthetic autosomes)
are still emerging, cattle and swine geneticists are developing and
utilizing safe and effective methods of artificial transgenesis. In
certain embodiments, the analysis of stability and integrity of
heritability of large DNA transgenes that increase the total DNA
content may be analyzed or studied by the invention, and it is
contemplated that the use of artificial transgenes may be
facilitated by sorting methods similar to the sex-selection methods
used today.
[0039] Generally, in one aspect of the invention, a fresh or frozen
ejaculate of sperm is the raw material to be processed. A DNA
selective dye or stain (including, but not limited to, Hoechst
33342) is combined in a liquid media with appropriate pH, modifying
buffers and aggregation reducing chemicals. The sperm mixture is
then optionally sonicated to simultaneously provide heat that
accelerates the rate of stain penetration and saturation and to
disrupt sperm cell structure to remove tails and midpieces, thereby
creating stained sperm nuclei.
[0040] One embodiment of the invention comprises entraining the
stained sperm or sperm nuclei in sheath fluid and exposing the
entrained sperm or sperm nuclei to electromagnetic radiation. In
connection with individual events associated with the exposed sperm
cells or sperm nuclei, the invention further comprises determining
a forward fluorescence characteristic, which in certain embodiments
correlates to the quantity of DNA in a sperm cell or a sperm nuclei
and a side fluorescence characteristic, which in certain
embodiments correlates to the orientation of a sperm cell or sperm
nuclei. Next, the invention contemplates gating the individual
events based on the forward fluorescence characteristic and side
fluorescence characteristic with one or more criterion, e.g.,
oriented sperm or sperm nuclei, or DNA content. In certain
embodiments of the invention, one can determine the presence or
absence of a DNA aberration from the gated individual events.
"Chromosomal aberration" or "DNA aberration" as used herein
encompasses a chromosomal translocation, a transgene, or any other
state in which the DNA content of sperm from an individual varies
from cell to cell other than variation attributable to sex
chromosomes. For example, a univariate plot can be generated based
on forward fluorescence intensity of the gated individual events.
Typically, in normal mammalian sperm or sperm nuclei, the
univariate plot would show two distinct peaks (each peak
corresponding to X-bearing or Y-bearing sperm). In an individual
with a chromosomal or DNA aberration, however, the univariate plot
may show more than two peaks. Additionally, in certain embodiments
of the invention, a peak to valley ratio is calculated for the
peaks shown on the univariate plot, with a peak to value ratio of
50% or less indicating the presence of a chromosomal aberration,
for example. In related embodiments, comparison of co-efficient of
variation between two or more peaks or modes using mathematical
analysis (Gaussian or the like) from histogramatic data is
used.
[0041] In a further embodiment of the invention, the sperm cells or
sperm nuclei are analyzed using a "logical gating" method using a
flow cytometer or microfluidic device. This embodiment comprises
gating a subpopulation of oriented sperm or sperm nuclei on a
multivariate plot with a first gate and then generating a second
multivariate plot based on the first gate. The method further
comprises creating a first "center gate" on the second multivariate
plot that selects a percentage of sperm cells or sperm nuclei
distributed near and around the mean of population distribution
(two or more "peaks"), and a second "flanking gate" on the second
multivariate plot that selects for the remaining sperm cells or
sperm nuclei outside of the center gate or a substantial portion
thereof. A univariate plot can be generated based on forward
fluorescence intensity for each of the center gate and the flanking
gate. Normal sperm cell or nuclei samples that comprise two peaks
(normal X and normal Y) result in center and flanked populations
that show the same two peaks on each of the univariate plots with
similar peak to valley ratios, typically greater than 50%. In
contrast, abnormal sperm cell or nuclei samples that contain
smaller or larger autosomes or sets of autosomes will result in
differences between the number of peaks that appear in the center
and flanked populations or peak to valley ratios of 50% or less,
for example.
[0042] FIG. 1 shows examples of multivariate and univariate plots
as well as gates used for logical gating of abnormal boar sperm
nuclei (i.e., comprising a DNA aberration). Plot 1 represents a
multivariate plot showing forward fluorescence along the y-axis and
side fluorescence along the x-axis. A gate 5 has been placed around
events that are oriented well enough to capture optimal resolution.
Each dot represents an event detected by the flow cytometer. Events
outside of gate 5 are low resolution or unresolvable. The events
within gate 5 are then plotted on plot 2, which shows forward
fluorescence along the y axis and an integral of forward
fluorescence along the x-axis. Essentially, plot 2 depicts the
gated events in plot 1 rotated approximately 90.degree. towards the
viewer. In plot 2, a center gate 7 and a flanking gate 9 are
placed. The events within flanking gate 9 are then plotted on plot
3, which shows forward fluorescence intensity along the y axis and
an integral of forward fluorescence intensity along the x-axis, and
plot 4, which is a histogram showing the forward fluorescence
intensities of events gated by the flanking gate 9. The events
within center gate 7 are plotted on plot 5, which shows forward
fluorescence intensity along the y axis and an integration of
forward fluorescence intensity along the x-axis, and plot 6, which
is a histogram showing the forward fluorescence intensities of
events gated by the center gate 7. As can be seen in FIG. 1, the
histogram (plot 4) generated by events in the flanking gate 7 shows
an abnormal number of modes, or peaks (normal being two modes, or
peaks, in mammalian species) and corresponds to a boar that has a
chromosomal translocation.
[0043] In another embodiment of the invention, sperm cells or sperm
nuclei are actually "parse sorted" using a flow cytometer or
microfluidic device, where "parse sorting" encompasses sorting a
first "center sort region," selecting for a percentage of sperm
cells or sperm nuclei distributed near and around the mean of
population distribution (two or more "peaks"), and a second
"flanking sort region," which typically selects for the remaining
sperm cells or sperm nuclei outside of the center sort region. In
some embodiments, more than two subpopulations may be sorted, for
example, a center sort region and two separate flanking sort
regions. The separate, parse sorted sperm cell or sperm nuclei
subpopulations are then analyzed and compared. Normal sperm cell or
nuclei samples that comprise two peaks (normal X and normal Y)
result in center and flanking populations that show the same two
peaks. Abnormal samples that contain smaller or larger autosomes or
sets of autosomes will result in differences in the number and
relative DNA content of peaks that appear in the center and
flanking populations.
[0044] In a further embodiment of parse sorting, the separate
populations that are sorted may include further dividing the
flanking sort region to separate the high-total-DNA content sperm
cells or sperm nuclei on one side of the flank from the
low-total-DNA content sperm cells or sperm nuclei on the other side
of the flank, while also separating a population from the center
where average-total-DNA content sperm cells or sperm nuclei are
selected.
[0045] In a further embodiment of the invention, sperm cells or
sperm nuclei of a second species may be added to the sperm cell or
nuclei sample to provide an internal standard to establish a
reference of known particle count, known total fluorescence
intensity, known percent DNA differential between sex chromosomes,
known peak to value ratio, or reference standard deviation of
Gaussian distribution. In certain embodiments, a method for
creating the internal standard comprises the addition of sperm or
sperm nuclei of a known quantity from a secondary mammalian species
to a staining mixture comprising a DNA selective dye with
appropriate conditions of sonication, which can then be added as an
internal standard to the sperm cell or nuclei sample of the first
mammalian species that is to be analyzed for chromosomal
aberrations. For example, FIG. 9 shows histograms for parsed center
and parsed flank subpopulations of sperm nuclei from a boar that
includes an internal standard comprising bovine sperm nuclei. The
histogram for the parsed center subpopulation in FIG. 9 clearly
indicates the presence of a DNA aberration in the boar's sperm
nuclei since the peak to valley ratios for that subpopulation is
significantly lower than the peak to valley ratio shown on the
histogram of the internal standard.
[0046] In one embodiment, mathematical analysis of the Gaussian
distributions of normal and abnormal peaks within single samples
and across groups of samples can be used to make a differential
determination of normal vs abnormal (i.e., the presence or absence
of a DNA aberration) more sensitive and precise.
[0047] Generally, the invention is rapid. However, certain
embodiments of the invention may take longer than other
embodiments. For example, in some embodiments, sperm nuclei
populations may be partially or fully purified away from tails and
midpieces prior to staining. Additionally, in certain embodiments,
staining may be performed at temperatures lower than in some
embodiments of the invention. In some embodiments, the invention
can use slower and more benign sample handling methods if
maintaining the viability of sperm is required, such as foregoing
sonication.
[0048] Some embodiments of the invention may specify a target
temperature at which sonication is completed/terminated. The target
temperature can be 5.degree. C., 45.degree. C., or 70.degree. C.,
or can be in the range between 5-70.degree. C., 30-70.degree. C.,
or 55-65.degree. C. In some embodiments of the invention the target
temperature may be reached in a time between 1 minute and 5
minutes, with staining completed in that time.
[0049] In certain embodiments, the invention uses flow cytometry
sorters or microfluidic devices that are identical to or materially
similar to flow sorters used to sort live sperm for total DNA
content in industrial sex selection. In certain embodiments of the
invention, the flow cytometer or microfluidic device utilizes an
event rate between 50 to 500 events per second, 5,000 to 30,000
events per second, or less than 30,000 events per second and sort
rates of about 10-35% of the event rate. In certain embodiments,
where sperm are not sorted before analysis, the analysis rates may
be as low as 2-10% of the event rate.
[0050] For those embodiments encompassing parse sorting, the
invention comprises sorting into two or more subpopulations,
centered around the mean and flanked around the center, where the
percentage of parsed center cells or nuclei (sort region
percentage) is between 25-60% of total sperm cells or sperm nuclei,
and the percentage of parsed flanked cells or nuclei is the
complement/balance of cells or nuclei remaining and in some cases
further dividing the flanking sort region to separate the
high-total-DNA content sperm or sperm nuclei on one side of the
flank from the low-total-DNA content sperm or sperm nuclei on the
other side of the flank. Alternate embodiments that sort into more
than two subpopulations or require the combinatorial blending of
parse samples are contemplated for use in special situations.
[0051] Certain embodiments of the invention encompass extended
sorting times to provide larger quantities of parse sorted samples
that may then be analyzed by microstructural DNA methods such as
genotyping or sequencing are anticipated.
[0052] In certain embodiments, the invention encompasses the
separate analysis of the two or more subpopulations of parse sorted
sperm or sperm nuclei (generally corresponding to center and
flanking subpopulations). Any of the above methods can be applied
to these separated subpopulations. For example, one may entrain the
sperm cells or sperm nuclei, expose them to electromagnetic
radiation, determine a forward fluorescence characteristic and a
side fluorescence characteristic, gate the individual events based
on the forward fluorescence characteristic and side fluorescence
characteristic with one or more criterion and generate a univariate
plot based on forward fluorescence intensity to determine the
number of peaks or the peak to valley ratio. Alternatively, a parse
sorted sperm cell or nuclei subpopulation can be reprocessed using
the logical gating method or parse sorting method. Alternatively,
the parse sorted sperm cells or sperm nuclei may be analyzed by
micro-analysis of DNA such as gene chip analysis or next-generation
sequencing (NGS).
[0053] In certain embodiments, it is contemplated that univariate
plots (e.g., graphical histograms) are visually inspected for
qualitative comparison of native and parse sorted samples. In other
embodiments of the invention, it is contemplated that flow
cytometry data from TRACE files or related FCS files is
mathematically analyzed for quantitative comparison of native and
parse sorted samples. Although the value of mathematical analysis
of flow cytometry data from TRACE files or related FCS files may
facilitate superior comparative quantification of and identity of
related samples, embodiments of the invention that forego
mathematical data analysis (e.g., using only the human eye) may be
more sensitive than existing karyotype methods.
Sperm Collection
[0054] It is contemplated that intact viable bovine, porcine,
equine, ovine, cervine, murine or other mammalian sperm, may be
collected for use with the invention. Various methods of collection
of viable sperm cells are known and include, for example, the
gloved-hand method, use of an artificial vagina, and
electro-ejaculation. As an example, a bovine sperm sample,
typically containing about 0.5 to about 10 billion sperm per
milliliter, may be collected directly from the source mammal, or
from more than one source mammal of the same species, into a vessel
containing an extender to form a sperm cell composition. An
extender may optionally comprise one or more antioxidants, which
may be present as constituents of the extender prior to contacting
with the sperm, or which may be added to the sperm cell
composition, each antioxidant in the concentration range of 0.01
mg/ml to 5 mg/ml.
[0055] An aggregation reducing compound, or anti-aggregation
compound, may also be added to the sperm cell composition to
prevent aggregation of sperm. Examples of aggregation reducing
compounds suitable for use in the invention include but are not
limited to egg yolk, iodixanol, lecithin, bovine serum albumin,
gelatin, collagen or hydrolyzed collagen, macromolecules such as
arabinogalactan, and chemically defined polyethylene or
polypropylene glycols.
Staining Sperm Cells for Use in the Invention
[0056] A process of staining sperm for flow cytometric analysis
typically comprises the formation of a staining solution containing
sperm cells and a dye, sometimes referred to as a label. A media or
extender may be contacted with sperm cells to form a sperm
composition, and then the sperm composition contacted with a DNA
selective dye to form a staining solution. Alternatively, a DNA
selective dye may be added to a media or an extender to form a
staining solution, with sperm subsequently added to the staining
solution.
[0057] The sperm to be stained may be a neat semen (i.e., raw
ejaculate), or alternatively, a sperm-containing semen derivative
obtained by centrifugation or the use of other means to separate
semen into fractions.
[0058] The pH of the staining solution may be maintained at any of
a range of pHs; typically this will be in the range of about 5.0 to
about 9.0, or in the range of 5.5 to 7.8. The staining solution may
be maintained at a slightly acid pH, i.e., from about 5.0 to about
7.0. Typically, the pH is from about 6.0 to about 7.0; from about
6.0 to about 6.5; about 6.2, about 6.5; about 6.6; about 6.7; about
6.8; about 6.9; or about 7.0. Alternatively, the staining solution
may be maintained at a slightly basic pH, i.e., from about 7.0 to
about 9.0. Typically, the pH is about 7.0 to about 8.0; about 7.0
to about 7.5; about 7.0; about 7.1; about 7.2; about 7.3; about
7.35; about 7.4; or about 7.5.
[0059] The staining solution may be formed by using one or more UV
or visible light excitable, DNA selective dyes as previously
described in U.S. Pat. No. 5,135,759 and WO 02/41906, the contents
of each of which are hereby incorporated herein by reference.
Exemplary UV light excitable, selective dyes include Hoechst 33342
and Hoechst 33258.
[0060] The concentration of the DNA selective or of any other type
of dye in the staining solution is a function of a range of
variables which include the permeability of the cells to the
selected dye, the temperature of the staining solution, the amount
of time allowed for staining to occur, the concentration of sperm,
and the degree of enrichment desired in the subsequent sorting or
enrichment step. In general, the dye concentration is preferably
sufficient to achieve the desired degree of staining in a
reasonably short period of time. For example, the concentration of
Hoechst 33342 in the staining solution will generally be between
about 0.1 .mu.M and about 1.0M; from about 0.1 .mu.M to about 1000
.mu.M; from about 100 .mu.M to about 500 .mu.M; from about 200
.mu.M to about 500 .mu.M; or from about 300 .mu.M to about 450
.mu.M. Accordingly, under one set of staining conditions, the
concentration of Hoechst 33342 is about 350 .mu.M. Under another
set of staining conditions, the concentration of Hoechst 33342 is
about 400 .mu.M. Under still another set of staining conditions the
concentration is about 450 .mu.M. In some embodiments the
concentration of Hoechst 33342 is between about 250 and about 500
picomoles of Hoechst per 1 million sperm.
[0061] Once formed, the staining solution may be maintained at any
of a range of temperatures; typically, this will be within a range
of about 4.degree. C. to about 50.degree. C., but can also be
frozen (i.e., cryopreserved). For example, the staining solution
may be maintained at a relatively low temperature, i.e., a
temperature of about 4.degree. C. to about 30.degree. C.; in this
embodiment, the temperature is about 20.degree. C. to about
30.degree. C.; from about 25.degree. C. to about 30.degree. C.; or
about 28.degree. C. Alternatively, the staining solution may be
maintained within an intermediate temperature range, i.e., a
temperature of about 30.degree. C. to about 39.degree. C.; in this
embodiment, the temperature is at about 34.degree. C. to about
39.degree. C.; about 35.degree. C.; or about 37.degree. C. In
addition, the staining solution may be maintained within a
relatively high temperature range, i.e., a temperature of about
40.degree. C. to about 50.degree. C.; in this embodiment, the
temperature is from about 41.degree. C. to about 49.degree. C.;
from about 41.degree. C. to about 45.degree. C.; from about
41.degree. C. to about 43.degree. C.; or about 41.degree. C.
Selection of a preferred temperature generally depends upon a range
of variables, including for example, the permeability of the cells
to the dye(s) being used, the concentration of the dye(s) in the
staining solution, the amount of time the cells will be maintained
in the staining solution, and the degree of enrichment desired in
the sorting or enrichment step.
[0062] Uptake of dye by the sperm in the staining solution is
allowed to continue for a period of time sufficient to obtain the
desired degree of DNA staining. That period is typically a period
sufficient for the dye to bind to the DNA of the sperm such that X
and Y chromosome-bearing sperm can be distinguished from one
another based upon the differing and measurable fluorescence
intensity between the two. Generally, this will be no more than
about 24 hours; no more than about 30 hours; no more than about 10
hours; no more than about 2 hours; no more than about 90 minutes;
no more than about 60 minutes; or from about 5 minutes to about 60
minutes. In a particular embodiment, the period is about 30 minutes
or about 55 minutes. In another embodiment, the period is less than
5 minutes, less than 4 minutes, less than 3 minutes, about 2
minutes, or less than 2 minutes.
Creating Stained Sperm Nuclei for Use in the Invention
[0063] One aspect of the invention comprises a staining media for
making stained sperm nuclei. The stained sperm nuclei can in turn
be used in the methods disclosed herein for detecting the presence
or absence of a chromosomal aberration instead of using intact
sperm cells.
[0064] In one embodiment, the staining media comprises a buffer, a
DNA-selective dye and an aggregation-reducing compound. Any
suitable buffer in the art, such as TRIS citrate, sodium citrate,
sodium bicarbonate, HEPES, TRIS, TEST, MOPS, KMT, TALP, and
combinations thereof, can be used.
[0065] Any DNA selective dye known in the art can be used,
including but not limited to Hoechst 33342. In other embodiments,
the staining media may be formed by using one or more UV or visible
light excitable, DNA-selective dyes as previously described in U.S.
Pat. No. 5,135,759 and WO 02/41906, the contents of each of which
are hereby incorporated by reference. Exemplary UV light excitable,
selective dyes include Hoechst 33342 and Hoechst 33258.
[0066] Additionally, an aggregation-reducing compound may be added
to prevent aggregation of sperm cells or sperm nuclei, as well as
aggregation of midpieces and tails. Examples of
aggregation-reducing compounds suitable for use in the invention
include but are not limited to egg yolk, iodixanol, lecithin,
bovine serum albumin, gelatin, collagen or hydrolyzed collagen,
macromolecules such as arabinogalactan, and chemically defined
polyethylene or polypropylene glycols. In a particular embodiment
of the invention, the staining media can comprise 0.4% or more egg
yolk. In another embodiment, the staining media can comprise
between 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-3%, 1-2%, or 0.2-1%,
egg yolk.
[0067] The staining media, or its separate components, can then be
combined with a sperm cell sample to create a sperm cell mixture.
The sperm cell mixture is then sonicated in order to remove
midpieces and tails from the sperm heads to create sperm nuclei,
and to facilitate staining of the DNA within the sperm nuclei. In a
particular embodiment of the invention, the sperm cell mixture is
sonicated at a sufficient amplitude, frequency or duration to raise
the temperature of the sperm cell mixture to more than 30, 40, 50,
60 or 70.degree. C. in order to facilitate staining. In a
particular embodiment, the temperature of the sperm cell mixture is
raised to more than 50, 60 or 70.degree. C. during sonication. In
another embodiment, the temperature of the sperm cell mixture is
raised to at least approximately 60.degree. C. during sonication.
In other embodiments, the target temperature during sonication can
be 45.degree. C., or 70.degree. C., or can be in the range between
30-70.degree. C., or 55-65.degree. C. In another embodiment,
sonication can be carried out on the sperm cell mixture for a
particular duration to facilitate staining of sperm nuclei DNA. In
one embodiment, the sperm cell mixture can be sonicated for greater
than 1, 2, 3, 4 or 5 minutes. In another embodiment, the sperm cell
mixture can be sonicated for a total of 1-5 minutes or in an even
more particular embodiment, approximately 2-3 minutes. In a further
embodiment of the invention, once the sperm cell mixture is
sonicated, the tails and midpieces are removed from the mixture via
any known method know in the art, including but not limited to
filtration or centrifugation. Any suitable sonicator can be used to
make the sperm nuclei. In a particular embodiment, a sonicator with
a 20 mhz frequency can be used to make the sperm nuclei, such as
Fisher Scientific Model FB120. In a more particular embodiment, the
sonicator is set to an amplitude of 70%. Finally, one can check
whether sonication was successful by examining the sonicated sperm
cell mixture by microscope--the sonicated sperm cell mixture should
substantially comprise sperm heads, with midpieces and tails
removed and should be substantially be free of intact sperm
cells.
[0068] In a particular embodiment, the staining media is made by
combining 98.0 ml of TRIS-based media, comprising 2% egg yolk, with
2.0 ml of Hoechst 33342 (8.1 mM of Hoechst 33342) to yield a final
concentration of Hoechst 33342 of 160 .mu.M in the staining media.
1.5 ml of this staining media is then combined with a sperm cell
sample of 400 million sperm (extended or raw ejaculate) to create a
sperm cell mixture. The sperm cell mixture is then sonicated to for
about 2 minutes or until it reaches a temperature of about
60.degree. C. This sonication step acts to remove the midpieces and
tails from the sperm cells to create sperm nuclei (i.e., sperm
heads devoid of midpieces and tails) and to facilitate entry and
binding of the DNA-selective dye (in this case, Hoechst 33342) to
the DNA within the sperm nuclei.
Use of an Internal Standard with the Invention
[0069] In some embodiments, it is contemplated that an internal
standard is mixed with the stained sperm cells or stained sperm
nuclei before analysis or mixed with each of the parsed sorted
subpopulations prior to analysis. The internal standard may be
created from sperm of a different species from the parse sorted
subpopulation or the sperm of interest that is to be analyzed. The
internal standard can comprise sperm nuclei, in which case, the
internal standard can be made in accordance with the above
disclosure regarding making stained sperm nuclei.
[0070] The internal standard is combined with a sperm cell or
nuclei sample or a parse sorted sperm or nuclei subpopulation prior
to flow cytometric analysis. One aspect of the invention involves
determining the presence or the absence of a DNA aberration based
on a comparison of histograms of fluorescence intensity of the
native (i.e., unsorted) sperm cells or sperm nuclei, or parse
sorted subpopulations, with the internal standard. Generally, when
the histograms of the native or parse sorted subpopulations have a
peak to valley ratio that is less than the peak to valley ratio of
the internal standard, a DNA aberration is present. Generally, the
Gaussian distribution of modes in the internal standard DNA from
the sperm cells or sperm nuclei of the second mammalian species and
the Gaussian distribution of modes in the normal sample from DNA of
the reference species represent a combination of standards which
can then be compared to the Gaussian distribution of unknown
samples to identify a DNA aberration.
[0071] In one particular embodiment, the internal standard is
created using a staining media made by combining 49.0 ml of
TRIS-based media comprising 20% egg yolk with 432 .mu.l of Hoechst
33342 stain. 1.66 ml of sperm or sperm nuclei (at a concentration
of 200 million/ml) are then added to the staining media. This
mixture can then be sonicated to create stained sperm nuclei.
Detecting the Presence or Absence of DNA Aberrations
[0072] One aspect of the invention comprises analyzing stained
sperm cells or sperm nuclei via flow cytometry in order to detect
the presence or absence of DNA aberrations, such as chromosomal
translocations. As noted above, in certain embodiments of the
invention, sperm cells or sperm nuclei analyzed by flow cytometry
are also optionally sorted based on falling within a center or
flanking sort region in the context of the parse sorting method for
detection of DNA aberrations. Commonly used and well known sperm
cell analysis and sorting methods via flow cytometry are
exemplified by and described in U.S. Pat. Nos. 5,135,759,
5,985,216, 6,071,689, 6,149,867, and 6,263,745; International
Patent Publications WO 99/33956 and WO 01/37655; and U.S. patent
application Ser. No. 10/812,351 (corresponding International Patent
Publication WO 2004/088283), the content of each of which is hereby
incorporated herein by reference.
[0073] In certain embodiments of the invention, analysis of sperm
cells may be accomplished using any process or device known in the
art for cell analysis including but not limited to use of a flow
cytometer (including the use of a microfluidic chip), and
optionally encompasses techniques for physically separating sperm
from each other, as with droplet sorting and fluid switching
sorting, and techniques in which sperm bearing an undesired
characteristics are killed, immobilized, or otherwise rendered
infertile, such as by use of laser ablation/photo-damage
techniques. Based on the fluorescence emitted by a DNA selective
dye upon exposure to a light source such as a high intensity laser
beam, a flow cytometer (including a microfluidic device) is able to
measure or quantify the amount of DNA present in each cell stained
with the DNA selective dye.
[0074] A sperm cell or nuclei sample to by analyzed via a flow
cytometer (including a microfluidic device) is contained in a
sample fluid. A sheath fluid is generally used in a flow cytometer
or microfluidic device to hydrodynamically focus, entrain or orient
sperm or nuclei in the sample fluid. Generally, the sheath fluid is
introduced into a nozzle of a flow cytometer or into a microfluidic
device using pressurized gas or by a syringe pump. The pressurized
gas is often high quality compressed air. In certain embodiments of
the invention, a stream containing sperm cells or nuclei to be
analyzed may be comprised of a sample fluid and a sheath fluid, or
a sample fluid alone. Optionally, the sample fluid or sheath fluid
may also contain an additive, such as, one or more antioxidants, an
antibiotic or a growth factor, as discussed above with respect to
sperm sample collection. Each of these additives may be added to
either fluid in accordance therewith.
[0075] FIG. 2 illustrates, in schematic form, part of a flow
cytometer used to analyze and then sort a sperm or nuclei
composition to form one or more subpopulations, the flow cytometer
being generally referenced as 10. The flow cytometer 10 of FIG. 2
can be programmed by an operator to generate two charged droplet
streams, one containing cells or nuclei within a center sort region
charged positively 12, for example, one containing cells or nuclei
within a flanking sort region charged negatively 13 for example,
while an uncharged undeflected stream of indeterminate or undesired
cells or nuclei 14 simply goes to waste, each stream collected in
receptacles 28, 29, and 30, respectively.
[0076] Initially, a stream of sperm cells or nuclei under pressure,
is deposited into the nozzle 15 from the sperm cell or nuclei
source 11 in a manner such that they are able to be coaxially
surrounded by a sheath fluid supplied to the nozzle 15 under
pressure from a sheath fluid source 16. An oscillator 17 which may
be present can be very precisely controlled via an oscillator
control mechanism 18, creating pressure waves within the nozzle 15
which are transmitted to the coaxially surrounded sperm cell or
nuclei stream as it leaves the nozzle orifice 19. As a result, the
exiting coaxially surrounded sperm cell or nuclei stream 20 could
eventually and regularly form droplets 21.
[0077] The charging of the respective droplet streams is made
possible by the cell sensing system 22 which includes a laser 23
which illuminates the nozzle exiting stream 20, and the light
emission of the fluorescing stream is detected by a sensor 24. The
information received by the sensor 24 is fed to a sorter
discrimination system 25 which very rapidly makes the decision as
to whether to charge a forming droplet and if so which charge to
provide the forming drop and then charges the droplet 21
accordingly.
[0078] A characteristic of X chromosome bearing sperm cells or
nuclei is that they absorb more fluorochrome dye than Y chromosome
bearing sperm cells or nuclei because of the presence of more DNA,
and as such, the amount of light emitted by the laser excited
absorbed dye in the X chromosome bearing sperm cell or nuclei
differs from that of the Y chromosome bearing sperm cells or
nuclei. One of the difficulties in accurate quantification of sperm
DNA using fluorescence is the geometry of the sperm head, which is
shaped like a paddle in most species. Generally, the intensity of
fluorescence is lowest when the flat face of the sperm is oriented
toward a fluorescence detector. This flat orientation actually
results in the most accurate measure of DNA content within a cell
and thus, in sex sorting applications, the best discrimination
between X and Y chromosome bearing sperm subpopulations. It is
therefore desirable that only properly oriented sperm or sperm
nuclei are considered in determining the presence or absence of a
chromosomal aberrations. There are many techniques known in the art
used to orient sperm using various forces generated by the flow
cytometer and/or microfluidic device, all of which are contemplated
for use with the invention. One way in which orientation can be
accomplished in a flow cytometer is by using an orienting nozzle
such as described in U.S. Pat. No. 6,357,307, which is hereby
incorporated by reference in its entirety. In one embodiment of the
invention, two detectors are used for detecting fluorescence
emitted by sperm cells or nuclei. One of the detectors is oriented
at 0.degree. relative to the laser beam or other source of
electromagnetic radiation and is used to measure forward
fluorescence, which corresponds to cell DNA content. The second
detector is oriented 90.degree. relative to the laser beam and is
used to measure side fluorescence, which corresponds to the
orientation of the sperm cell or nuclei. Since the fluorescence
signal is highest for sperm cells or nuclei oriented with their
paddle edge toward the side fluorescence detector, only the sperm
cells or nuclei that emit peak fluorescence to the side
fluorescence detector are considered oriented by the flow
cytometer.
[0079] The charged or uncharged droplet streams pass between a pair
of electrostatically charged plates 26, which cause them to be
deflected either one way or the other or not at all depending on
their charge into respective collection vessels 28 and 29 to form a
subpopulation of sperm cells or sperm nuclei that fell within the
center sort region and a subpopulation of cells or nuclei that fell
within the flanking sort region, respectively. The uncharged
non-deflected sub-population stream containing undesired or
indeterminate cells or nuclei go to the waste container 30.
[0080] Turning now to FIG. 3, an alternative particle sorting
instrument or flow cytometer is partially illustrated in the form
of a microfluidic chip (60). The microfluidic chip (60) may include
a sample inlet (62) for introducing sample containing particles or
cells into a fluid chamber (64) and through an inspection zone
(66). Sample introduced through the sample inlet (62) may be
insulated from interior channel walls and/or hydrodynamically
focused with a sheath fluid introduced through a sheath inlet (68).
Sample may be interrogated at the inspection zone (66) with an
electromagnetic radiation source (not shown), such as a laser, arc
lamp, or other source of electromagnetic electricity. Resulting
emitted or reflected light may be detected by a sensor (not shown)
and analyzed with an analyzer (not shown). Each of the sheath
pressure, sample pressure, sheath flow rate, and sample flow rate
in the microfluidic chip may be manipulated in a manner similar to
a jet-in-air flow cytometer, by either automatic adjustments
performed by the execution of written instructions in the analyzer
or by manual adjustments performed by an operator.
[0081] In certain embodiments of the invention, once inspected,
particles or cells in the fluid chamber (64) may be mechanically
diverted from a first flow path (70) to a second flow path (72)
with a separator (74), for altering fluid pressure or diverting
fluid flow. The particles or cells may also be permitted to
continue flowing along the first flow path (70) for collection. The
illustrated separator (74) comprises a membrane which, when
depressed, may divert particles into the second flow path (72).
Other mechanical or electro-mechanical switching devices such as
transducers and switches may also be used to divert particle
flow.
[0082] Flow cytometry data analysis is based on the principle of
gating. Typically, gates and regions are created around populations
of cells with common characteristics. In the context of the
invention, these characteristics can include forward fluorescence
and side fluorescence.
[0083] Generally, the first step in gating when flow cytometrically
analyzing sperm cell or sperm nuclei to determine the presence or
absence of a DNA aberration is distinguishing populations of sperm
cells or sperm nuclei based on their forward and side fluorescence
properties. As noted above, forward and side fluorescence provide
an estimate of the DNA content of the cells or nuclei and their
orientation, respectively. Unoriented sperm cells or nuclei will
generate events having a lower level of side fluorescence, as noted
above, and are not resolvable or are low resolution. These events
can be removed by gating on the population of interest only (i.e.,
oriented sperm cells or nuclei).
[0084] Gates can be applied to density plots or contour maps to
exclude certain populations (e.g. unoriented sperm cell or nuclei)
or to positively select populations for further analysis,
processing or examination. Using analytical software, measurements
and statistics can be obtained for various parameters in addition
to the number of cells or nuclei and percentage of cells or nuclei
within a gate. This can include such measurements as median and
mean fluorescence intensity.
[0085] Generally, two-parameter density plots (i.e., bivariate
plots) display two measurement parameters, one on the x-axis and
one on the y-axis and the events as a density (or dot) plot. The
parameters can include forward florescence intensity, side
fluorescence intensity and an integral of forward florescence
intensity.
[0086] FIG. 4 illustrates a univariate plot in the form of a
histogram that may be produced by the analyzer (36) and generated
into a graphical presentation for an operator. The data illustrated
in FIG. 4 may represent the number of occurrences of peak signal
intensities from the side or forward fluoresce within a certain
period. In the case of sperm cells or sperm nuclei, X chromosome
bearing sperm cells or nuclei and Y chromosome bearing sperm cells
or nuclei tend to have peak intensities that vary by between 2 and
5%, depending on the species, and this difference is reflected in
the bimodal distribution of peak intensities. Because X chromosome
bearing sperm cells or nuclei and Y chromosome bearing sperm cells
or nuclei tend to have differing fluorescence values, each of the
peaks represents either X chromosome bearing sperm cells or nuclei
or Y chromosome bearing sperm cells or nuclei. FIG. 4 further
illustrates the concept of the peak to valley ratio, which is
derived from a relative intensity measurement at the lowest point
between the two groups, the valley, which may be considered a value
V, and a second relative intensity measurement at the peak or peaks
of the histogram at P.
Example 1
[0087] Frozen thawed sperm cell samples of seven different boars
were centrifuged and then gently resuspended in TRIS A, an isotonic
salt solution comprising a TRIS CITRATE buffer with FRUCTOSE and
20% v/v clarified Egg Yolk pH 6.80 and 300 mOsm. A 3-5 ml sample of
suspended sperm cells (concentrations in the range of 200-300
million sperm per milliliter) was combined with Hoechst 333342 in
ratio of 500-700 nanomoles of stain per 1 million sperm. The
mixture was sonicated for 2-5 minutes until the temperature of the
mixture reached 60.degree. C., thereby creating stained sperm
nuclei.
[0088] In the parse sorting method, one sort region is used to
collect cells near the center (center sort region) while the other
sort region is used to collect the remaining cells adjacent to the
center sort region on both sides (flanking sort region). The
combined percentages of both sort regions was typically about 90%
of the cells. Generally, the percentage in the center sort region
was somewhat less than the percentage in the flanking gate. The
event rate during parse sorting of the native sample were typically
in the range of 10,000-25,000 cells per second. The actual event
rate chosen for each sample is determined by the operator and may
change during the sorting.
[0089] For each of the seven boars exemplified here, the stained
sperm nuclei samples were flow cytometrically sorted for the center
sort region and flanking sort region into a collection tube. The
sorted cells were centrifuged and decanted to remove most of the
fluid. The cells were resuspended in approximately 300 microliters
of the same buffer used for pre-sort staining and sonicating
supplemented with about 100 nanomoles of Hoechst 33342 per million
sperm and sonicated for 60 seconds before analysis. Since the
purpose of the analysis is to have high quality resolution, and
since slower event rates give higher quality resolution due to much
smaller core stream diameters, event rates for analysis were
typically 50-200 cells per second. The actual event rate chosen for
each sample is determined by the operator and may change during the
analysis.
[0090] Depending upon whether the boars were normal or had a DNA
aberration, the number of peaks in the monovariate histogram were
between 2 and 6 and the quality (peak to valley ratios) and peak
clarity (co-efficient of variation) in the samples varied. Although
the different "native samples" had different numbers of peaks, the
distribution of peaks was typically symmetric around a central
point. In normal boars showing only two peaks (X chromosome and Y
chromosome bearing sperm population) the center was between those
two peaks. In some boars with DNA aberrations there only appeared
one poor quality peak and in some abnormal boars there appeared the
typical X and Y peaks near the center as well as additional clearly
defined peaks outside the two central peaks.
[0091] FIGS. 5 through 11 (each Figure corresponding to one of the
seven boars, respectively) encompass 5 different categories of
stable chromosomal translocation in found in boars, and the effect
the size of translocated DNA in each of those categories has on the
distribution of peaks (modes) in monovariate histograms of the
sperm populations when the total DNA content is measured by the
parse-sorted method. In each figure, the NATIVE histogram
represents the data that was used for sorting (parse-sorted
method). The PARSED CENTER histogram represents the data from sperm
populations that were sorted by the CENTER SORT REGION and
reanalyzed on the sorter. The PARSED FLANK histogram represents the
data from sperm populations that were sorted by the FLANKING SORT
REGION and reanalyzed on the sorter. The labels representing the 6
peaks (modes) are: [0092] Y=Sperm cells or sperm nuclei that
comprise a completely normal chromosome content (normal autosomes)
and a Y chromosome. Sperm or sperm nuclei that comprise the short
translocation (deleted) and long translocation (insert) in
combination with other normal autosomes and a Y chromosome. NOTE:
This designated mode contains two genotypes (two different types of
chromosomal combinations) but the same amount of total DNA. [0093]
X=Sperm cells or sperm nuclei that comprise a completely normal
chromosome content (normal autosomes) and an X chromosome.
[0094] Sperm cells or sperm nuclei that comprise the short
translocation (deleted) and long translocation (insert) in
combination with other normal autosomes and an X chromosome. NOTE:
This designated mode contains two genotypes (two different types of
chromosomal combinations) but the same amount of total DNA. [0095]
YS=Sperm cells or sperm nuclei that contain the short translocation
(deletion) with all other normal autosomes and a Y chromosome.
[0096] XS=Sperm cells or sperm nuclei that contain the short
translocation (deletion) with all other normal autosomes and an X
chromosome. [0097] YL=Sperm cells or sperm nuclei that contain the
long translocation (insert) with all other normal autosomes and a Y
chromosome. [0098] XL=Sperm cells or sperm nuclei that contain the
long translocation (insert) with all other normal autosomes and an
X chromosome.
[0099] Based on a convention comparing the amount of DNA in the
stable translocation to the amount of DNA difference between X and
Y chromosome, there are 4 categories of visible (measurable)
aberrant DNA and the category of normal.
[0100] FIG. 5 depicts the parse-sorted analysis of a Boar with a
Mos t(7-9) translocation that is typical of a Category I
Translocation. In this case, since the number of base pairs in the
translocated element is enough greater than the number of base
pairs in the difference between an X chromosome and a Y chromosome,
not only does the YS mode contain less DNA than the Y mode, the XS
mode also contains less. Due to that, there are two modes visible
below the Y mode. Conversely, there are also two modes larger than
the X mode and even in the NATIVE histogram, it is possible to see
6 separate peaks. In the analysis of Category I PARSED CENTER and
PARSED FLANK subpopulations that have been parse sorted and
reanalyzed separately, the PARSED CENTER histogram is similar to a
NORMAL XY distribution, but the PARSED FLANK histogram shows all 6
peaks very clearly. In this category, the aberrant DNA structure of
the translocation can already be seen in the NATIVE histogram and
is more clearly confirmed in the reanalyzed samples.
[0101] FIG. 6 depicts the parse-sorted analysis of a Boar with an
RCP (3-6)2 translocation that is typical of a Category II
Translocation. In this case, since the number of base pairs in the
translocated element is about the same as the number of base pairs
in the difference between an X chromosome and a Y chromosome, the
XS mode tends to overlap well with the Y mode and the YL mode tends
to overlap well with the X mode. In this case, both the NATIVE and
the PARSED FLANK histograms show 4 peaks (modes). In this category,
the aberrant DNA structure of the translocation can already be seen
in the NATIVE histogram and is more clearly confirmed in the
reanalyzed samples.
[0102] FIG. 7 depicts the parse-sorted analysis of a Boar with an
RCP (2q13, 15q24) translocation that is typical of a Category III
Translocation. In this case, since the number of base pairs in the
translocated element is somewhat less than the number of base pairs
in the difference between an X chromosome and a Y chromosome, the
4-6 peaks are poorly separated in the NATIVE histogram, but more
clearly separated in the PARSED FLANK histogram. The PARSED CENTER
histogram may still show two peaks (modes) that significantly
overlap, or even show as one peak. A distinct feature of the
Category III Translocation is that the PARSED FLANK histogram shows
4 peaks (modes).
[0103] FIG. 8 depicts the parse-sorted analysis of a Boar with an
RCP (5-12) translocation that is typical of a Category IV
Translocation. In this case, since the number of base pairs in the
translocated element is very small, the X modes (XS, X, XL) are all
overlapping in the NATIVE histogram, while the Y modes (YS, Y, YL)
are also overlapping in the NATIVE histogram and the NATIVE
histogram looks similar to the NATIVE histogram of a NORMAL
distribution (a boar with no large aberrant DNA). The Category IV
Translocation still shows significant differences in the PARSED
CENTER and PARSED FLANK analysis, but if the DNA element is very
small, the only difference may be different CV (or PVR) between
them.
[0104] FIG. 9 depicts the parse-sorted analysis of a boar with an
RCP (5-12) translocation that is typical of a Category IV
Translocation where the reanalysis of the PARSED CENTER and PARSED
FLANK populations is done with normal sperm nuclei from cattle
(bovine, Jersey breeds) included in the analysis. The bovine sperm
nuclei contain more total DNA than the boar DNA, so the bovine
sperm nuclei create a typical two-mode split seen in sex sorting
but shifted to the right. Since both types of sperm nuclei are
being analyzed together, the quality of the split (separation of
modes) in the bovine nuclei is used to optimize the analysis and
prove to the operator that the best possible separation has been
created. That means that the best possible separation has also been
created in the boar sperm nuclei test sample.
[0105] FIG. 10 depicts the parse-sorted analysis of a Boar with
NORMAL DNA content. In this case, the designation of NORMAL has
been created by the observation that the boar, when analyzed by
Karyotype method, shows no recognizable abnormality in chromosome
sizes. In this case, the NATIVE, PARSE CENTER and PARSE FLANK
analysis all show 2 distinct peaks. The somewhat improved
separations in the PARSE CENTER and PARSE FLANK are created because
the reanalysis runs at a slow Event Rate (cell analysis rate) which
typically gives better separation.
[0106] FIG. 11 depicts the parse-sorted analysis of a boar with
NORMAL DNA content where the reanalysis includes bovine nuclei as
described in FIG. 9. The parse-sorted method on NORMAL samples
establishes a threshold of NORMAL vs false positive (falsely
described as abnormal when there is no aberrant DNA content) vs
false negative (falsely described as normal when there is an
aberrant DNA content)
Example 2
[0107] STAINING/SORTING MEDIA (SSM) used in Example 2--One liter
comprises 23.00 grams tris(hydroxymethyl)aminomethane (TRIS BASE),
9.56 grams anhydrous fructose, 13.28 grams citric acid monohydrate,
200 mL chicken egg yolk, 100 milligrams Hoechst 33342,
Trihydrochloride, Trihydrate and approximately 800 mL of deionized
water with a final pH about 6.72.
[0108] SORT REANALYSIS MEDIA (SRM) used in Example 2--One liter
comprises: 25.88 grams tris(hydroxymethyl)aminomethane, 10.75 grams
anhydrous fructose, 14.94 grams citric acid monohydrate, 100 mL
chicken egg yolk, 21.5 milligrams Hoechst 33342, Trihydrochloride,
Trihydrate and approximately 900 ml of deionized water with a final
pH about 6.72. If bovine nuclei are included as internal standard,
6.70 billion Bovine Sperm Nuclei (BSN) where BSN are azide
stabilized purified sperm heads that have all midpieces and tail
fragments removed and which have been pre-stained with Hoechst
33342.
[0109] Ejaculate preparation: Frozen ejaculates from 7 boars were
thawed at 34.degree. C. and each ejaculate then mixed well using a
5 ml pipette. 7 ml of each ejaculate was placed into a 15 ml
conical tube. 7 ml of TrisWS300 was placed into each sample and
vortexed. Samples were then centrifuged at 950 G for 5 minutes, and
all supernatant was then decanted leaving the pellet undisturbed. 4
ml of TrisWS300 was added to each sample and the pellet was broken
up using a pipette. Each sample was then mixed by vortexing. Cell
concentrations were determined using NucleoCounter.
[0110] Sample preparation using SSM media: 615 .mu.l of each of the
prepared ejaculates was then placed in a 5 ml tube (assuming the
sample concentration was 650 million sperm per ml). Using a P-1000
pipette, 400 million sperm cells from each sample were dispensed
into a 5 ml sample tube. Using a P-1000 pipette, 1000 .mu.l of SSM
(prepared as above) was dispensed into the same tube. Each sample
was then sonicated for 2 minutes, taking the samples to
approximately 60.degree. C. 500 ul of Tris A was then added to each
sample and then each sample was sonicated again for .about.30
seconds.
[0111] Each of the 7 stained sperm nuclei samples was then parse
sorted by placing 5.0 million sperm nuclei from each sample into a
5 ml catch tube and sorting them on a flow cytometer. A CENTER sort
region and a FLANK sort region were placed on the bivariate plot of
forward fluorescence peak vs forward fluorescence integrated. The
sorted samples (2 for each bull) were then centrifuged at 950 G for
5 minutes, and all supernatant was decanted leaving the pellet
undisturbed. 500 .mu.l of SRM (as prepared above) was then added to
each sample and the pellet was broken up with a sonicator for 1
minute. Each sorted sample was then analyzed on a flow cytometer at
an event rate of between 40-100 events per second.
[0112] FIGS. 12 through 18 comprise the 7 examples of using the SSM
staining media, sorting of SSM stained boar nuclei with the parse
sort method, and reanalysis of the PARSED CENTER and PARSED FLANK
populations using the SRM media, as indicated above. In all cases,
the upper left side of the image shows two dot plots: the first
shows an oriented gate applied to SSM stained sperm in a bivariate
plot of forward fluorescence (FF) vs side fluorescence (SFF) and
the second shows a set of CENTER and FLANK region polygons that
define two separate sort regions on a bivariate plot of forward
fluorescence (FF) vs forward fluorescence integrated (FFI, Area).
The first monovariate histogram is the NATIVE histogram used for
parsed sorting, the second monovariate histogram is the PARSED
CENTER sorted sperm with bovine stained nuclei added and the third
monovariate histogram is the PARSED FLANK sorted sperm with bovine
stained nuclei added.
[0113] FIG. 12 depicts the parse-sorted analysis of a Boar with
NORMAL DNA content where the reanalysis includes bovine stained
nuclei as described above for FIG. 9.
[0114] FIG. 13 depicts the parse-sorted analysis of a Boar with
NORMAL DNA content where the reanalysis includes bovine stained
nuclei as described above for FIG. 9.
[0115] FIG. 14 depicts the parse-sorted analysis of a Boar with
NORMAL DNA content where the reanalysis includes bovine stained
nuclei as described above for FIG. 9.
[0116] FIG. 15 depicts the parse-sorted analysis of a Boar with a
Category IV Stable Reciprocal Translocation confirmed by Karyotype,
with aberrant DNA content where the reanalysis includes bovine
stained nuclei as described above for FIG. 9.
[0117] FIG. 16 depicts the parse-sorted analysis of a Boar with a
Category I Stable Reciprocal Translocation confirmed by Karyotype,
with aberrant DNA content where the reanalysis includes bovine
stained nuclei as described above for FIG. 9.
[0118] FIG. 17 depicts the parse-sorted analysis of a Boar with a
Category III Stable Reciprocal Translocation confirmed by
Karyotype, with aberrant DNA content where the reanalysis includes
bovine stained nuclei as described above for FIG. 9.
[0119] FIG. 18 depicts the parse-sorted analysis of a Boar with
NORMAL DNA content where the reanalysis includes bovine stained
nuclei as described above for FIG. 9.
Example 3
[0120] This example demonstrates the use of logical gating method
on boar sperm. After collection, 400 million boar sperm cells from
each of four sperm cell samples was mixed with 1 ml of a TRIS-based
media comprising egg yolk and Hoechst 33342. Each sperm cell
mixture was then sonicated until reaching 60.degree. C. to create
stained sperm nuclei. Each stained sperm nuclei sample was then
analyzed on a flow cytometer having a forward and side fluorescence
detector. Events representing oriented sperm nuclei were first
gated on a multivariate plot. A histogram of fluorescence intensity
for all of these gated events was generated ("native" histogram). A
second multivariate plot, with forward fluorescence intensity on
the y-axis and an integral of forward fluorescent intensity on the
x-axis, was generated based on these gated events. A center gate
and a flanking gate were placed on the second multivariate plot.
Histograms of fluorescence intensities of events for each of the
center gate and the flanking gate were generated. The results are
shown in FIGS. 19-22.
[0121] FIG. 19 represents the above-referenced histograms (native,
center gate and flanking gate) for sperm nuclei obtained from a
boar having a stable chromosomal translocation where the quantity
of translocated DNA is greater than the DNA quantity difference
between X and Y chromosomes. While the center gate shows two peaks,
which is normal, the native and flanking gate histograms show six
clearly separated peaks, indicative of a chromosomal
aberration.
[0122] FIG. 20 represents the above-referenced histograms (native,
center gate and flanking gate) for sperm nuclei obtained from a
boar having a stable chromosomal translocation where the quantity
of translocated DNA is about the same as the DNA quantity
difference between X and Y chromosomes. While the center gate shows
two peaks, which is normal, the histograms for the native and
flanking gate show four clearly separated peaks, indicative of a
DNA aberration.
[0123] FIG. 21 represents the above-referenced histograms (native,
center gate and flanking gate) for sperm nuclei obtained from a
boar having a stable chromosomal translocation where the quantity
of translocated DNA is less than the DNA quantity difference
between X and Y chromosomes. The histograms for the native and the
center gate show four poorly separated peaks and the flanking gate
shows four separated peaks, all indicative of a DNA aberration.
[0124] FIG. 22 represents the above-referenced histograms (native,
center and flanking) for sperm nuclei obtained from a boar having a
stable chromosomal translocation where the quantity of translocated
DNA is much less than the DNA quantity difference between X and Y
chromosomes. The histograms for the native and center gate show two
poorly separated peaks, including having peak to valley ratios of
50% or less, which is indicative of a DNA aberration.
Example 4
[0125] After collection, 400 million boar sperm cells from each of
eleven sperm cell samples was mixed with 1 ml of a TRIS-based media
comprising egg yolk and Hoechst 33342. Each sperm cell mixture was
then sonicated until reaching 60.degree. C. to create stained sperm
nuclei. Each stained sperm nuclei sample was then parse sorted on a
flow cytometer having a forward and side fluorescence detector.
Events representing oriented sperm nuclei were first gated on a
multivariate plot. A histogram of fluorescence intensity for all of
these gated events was generated ("native" histogram). A second
multivariate plot, with forward fluorescence intensity on the
y-axis and an integral of forward fluorescent intensity on the
x-axis, was generated based on these gated events. A center sort
region and a flanking sort region were provided on the second
multivariate plot. 2 million cells that generated events in the
center sort region and 2 million cells that generated events in the
flanking sort region were then separated and collected. Each
subpopulation was then analyzed separately on a flow cytometer.
[0126] For six of the sperm nuclei samples (each sample
corresponding to FIGS. 23, 29, 30, 31, 32 and 33, respectively),
each collected subpopulation was first centrifuged at 950 g for 5
minutes and decanted. 1 ml of TRIS-based media comprising egg yolk,
Hoechst 33342 and an internal standard comprising bovine stained
sperm nuclei was added to each of the collected subpopulations from
these six sperm nuclei samples. These mixtures were then sonicated
and separately analyzed on a flow cytometer.
[0127] For each of the nine boars evaluated, multivariate plots and
histograms of forward fluorescence intensities were generated for
the unsorted sperm nuclei as well as for each of the separated
subpopulations that were subsequently analyzed. These results are
shown in FIGS. 23-33 for each boar, respectively, and show that the
evaluated boars all have DNA aberrations, either by showing more
than two separate peaks or peak to valley ratios of 50% or less on
the native, center and flanking histograms (FIGS. 23-30, 32 and
33), or by showing a peak to valley ratio markedly worse than that
generated for the internal standard (see, e.g., FIG. 31). Boars
whose results are shown in FIGS. 32 and 33 were actually karyotyped
as normal, which indicates that the invention is more sensitive in
the presence or absence of DNA aberrations than traditional
karyotyping methods.
Example 5
[0128] After collection, 400 million bull (bovine) sperm cells from
a sperm cell sample were mixed with 1 ml of a TRIS-based media
comprising egg yolk and Hoechst 33342. The mixture was then
sonicated until reaching 60.degree. C. to create stained sperm
nuclei. The sperm nuclei sample was then parse sorted on a flow
cytometer having a forward and side fluorescence detector. Events
representing oriented sperm nuclei were first gated on a
multivariate plot. A histogram of fluorescence intensity for all of
these gated events was generated ("native" histogram). A second
multivariate plot, with forward fluorescence intensity on the
y-axis and an integral of forward fluorescent intensity on the
x-axis, was generated based on these gated events. A center sort
region and a flanking sort region were provided on the second
multivariate plot. Sperm nuclei that generated events in the center
sort region and in the flanking sort region were then separated and
collected. Each subpopulation was then re-stained with Hoechst
33342 (each 1 million sperm nuclei subpopulation stained with 20
.mu.l of Hoechst 33342 at a concentration of 5 mg/ml) and analyzed
separately on a flow cytometer.
[0129] Multivariate plots and univariate histograms of forward
fluorescent intensities were generated for the unsorted sperm
nuclei as well as for each of the separated subpopulations that
were subsequently analyzed. These results are shown in FIG. 34, and
indicate that the evaluated bull has a Category IV
Translocation.
Example 6
[0130] After being collected in an extender, semen samples from 2
boars were brought to a sperm cell concentration of
200.times.10.sup.6 sperm cells/ml in test tubes. 8-10 .mu.l of
Hoechst 33342 (at a concentration of 5 mg/ml) was added for every
ml of extended semen in the samples. Yellow 6 quenching dye was
added at 3 .mu.l for every ml of extended semen in the samples to
identify dead or dying cells. The staining solutions were gently
swirled for 10 seconds and then placed in a water bath at 34.5 to
35.degree. C. for 60 minutes. The tubes were inverted every 15
minutes while the samples were incubating. Each staining solution
was then filtered through a CellTrics.RTM. filter into properly
labeled sample tubes and the stained sperm cell samples sorted via
flow cytometer.
[0131] The results for each of the boars' sperm cell samples can be
seen in FIGS. 35 and 36, respectively. FIG. 35 indicates that the
boar has a Category IV Translocation due to the bi-modal histogram
of forward fluorescence intensities having a low PVR (only 38%),
while FIG. 36 indicates that the boar has a Category III
Translocation based on the small side peaks present on the
histogram and low PVR (only 51%).
Example 7
[0132] After collection, a bull (bovine)'s semen was extended and
stained with Hoechst 33342 and a quenching dye using standard
techniques when sex-sorting viable sperm. The stained sperm cell
sample was then parse sorted on a flow cytometer having a forward
and side fluorescence detector. Events representing oriented sperm
nuclei were first gated on a multivariate plot, as can be seen in
the upper left portion of FIG. 37. A histogram of fluorescence
intensity for all of these gated events was generated as can also
be seen in the middle of FIG. 37, which indicates that the bull has
a Category IV Translocation due to the bi-modal histogram having a
low PVR. A second multivariate plot, with forward fluorescence
intensity on the y-axis and an integral of forward fluorescent
intensity on the x-axis, was generated based on these gated events
as can be seen on the lower left portion of FIG. 37. A center sort
region and a flanking sort region were provided on the second
multivariate plot as seen in FIG. 37. 1 million cells that
generated events in the center sort region and 1 million cells that
generated events in the flanking sort region were then separated
and collected. Each subpopulation was then re-stained with Hoechst
33342 (each 1 million sperm nuclei subpopulation stained with 20
.mu.l of Hoechst 33342 at a concentration of 5 mg/ml and 20 .mu.l
of TRIS-based media with 20% egg yolk), sonicated and then analyzed
separately on a flow cytometer.
[0133] The resulting univariate histograms of forward fluorescence
intensities for each subpopulation are shown in FIGS. 38 and 39
respectively, which indicate the presence of a Category IV
translocation (FIG. 38 shows a single mode (i.e. the absence of a
PVR), while FIG. 39 shows a bi-modal histogram with a low PVR).
* * * * *