U.S. patent application number 14/337940 was filed with the patent office on 2015-01-22 for prediction of fertility in males.
The applicant listed for this patent is WISCONSIN ALUMNI RESEARCH FOUNDATION. Invention is credited to John Parrish.
Application Number | 20150024385 14/337940 |
Document ID | / |
Family ID | 52343866 |
Filed Date | 2015-01-22 |
United States Patent
Application |
20150024385 |
Kind Code |
A1 |
Parrish; John |
January 22, 2015 |
PREDICTION OF FERTILITY IN MALES
Abstract
A method for evaluating sperm fertility. The method includes the
steps of obtaining a sample of sperm from an animal of a species;
staining the sample with a fluorescent DNA-binding dye; collecting
at least one image of the stained sample; determining an edge of a
nucleus of at least one sperm within the stained sample in the at
least one image; measuring an intensity of the DNA-binding dye
within an area defined by the edge of the nucleus of the at least
one sperm; determining an average intensity per unit area of the
area defined by the edge of the nucleus of the at least one sperm;
comparing the average intensity per unit area to an average
intensity per unit area for high-fertility sperm and low-fertility
sperm of the same species to determine if the sample has high or
low fertility.
Inventors: |
Parrish; John; (Mount Horeb,
WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WISCONSIN ALUMNI RESEARCH FOUNDATION |
Madison |
WI |
US |
|
|
Family ID: |
52343866 |
Appl. No.: |
14/337940 |
Filed: |
July 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61856828 |
Jul 22, 2013 |
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Current U.S.
Class: |
435/6.1 |
Current CPC
Class: |
G01N 2015/1472 20130101;
G01N 33/5091 20130101; G01N 15/147 20130101; G01N 2015/1006
20130101; G01N 2015/1497 20130101; G01N 2800/367 20130101; G01N
15/1475 20130101; C12N 5/061 20130101 |
Class at
Publication: |
435/6.1 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 15/14 20060101 G01N015/14 |
Claims
1. A method for evaluating sperm fertility, comprising the steps
of: obtaining a sample of sperm from an animal of a species;
staining the sample with a fluorescent DNA-binding dye; collecting
at least one image of the stained sample; determining an edge of a
nucleus of at least one sperm within the stained sample in the at
least one image; measuring an intensity of the DNA-binding dye
within an area defined by the edge of the nucleus of the at least
one sperm; determining an average intensity per unit area of the
area defined by the edge of the nucleus of the at least one sperm;
and comparing the average intensity per unit area to average
intensities per unit area for high-fertility sperm and
low-fertility sperm of the same species to determine if the sample
has high or low fertility.
2. The method of claim 1, wherein staining the sample with a
fluorescent DNA-binding dye comprises staining the sample with
Hoechst 33342.
3. The method of claim 1, wherein staining the sample with a
fluorescent DNA-binding dye further comprises applying the stained
sample to a substrate.
4. The method of claim 3, wherein the nucleus of the at least one
sperm within the stained sample comprises a flattened oval and
wherein the flattened portion is adjacent to the substrate.
5. The method of claim 1, wherein obtaining a sample of sperm from
an animal of a species comprises obtaining a sample of sperm from a
bull, a boar, a human, a horse, a ram, or a dog.
6. The method of claim 1, wherein collecting at least one image of
the stained sample comprises deconvolving the at least one image to
remove out of focus information.
7. The method of claim 1, wherein the high-fertility sperm has a
lower intensity per unit area than the low-fertility sperm.
8. A method for evaluating sperm fertility, comprising the steps
of: obtaining a sample of sperm from an animal of a species;
staining the sample with a fluorescent DNA-binding dye; obtaining
fluorescent intensity measurements from a plurality of sperm in the
stained sample; determining an average intensity of the fluorescent
intensity measurements obtained from the plurality of sperm in the
stained sample; and comparing the average intensity to average
intensities for high-fertility sperm and low-fertility sperm of the
same species to determine if the sample has high or low
fertility.
9. The method of claim 8, wherein obtaining fluorescent intensity
measurements from a plurality of sperm in the stained sample
further comprises collecting at least one image of the stained
sample.
10. The method of claim 9, wherein determining an average intensity
of the fluorescent intensity measurements obtained from the
plurality of sperm in the stained sample further comprises
determining an edge of a nucleus of at least one sperm within the
stained sample in the at least one image, measuring an intensity of
the DNA-binding dye within an area defined by the edge of the
nucleus of the at least one sperm, and determining an average
intensity per unit area of the area defined by the edge of the
nucleus of the at least one sperm.
11. The method of claim 10, wherein collecting at least one image
of the stained sample comprises deconvolving the at least one image
to remove out of focus information.
12. The method of claim 11, wherein staining the sample with a
fluorescent DNA-binding dye further comprises applying the stained
sample to a substrate.
13. The method of claim 12, wherein the nucleus of the at least one
sperm within the stained sample comprises a flattened oval and
wherein the flattened portion is adjacent to the substrate.
14. The method of claim 8, wherein obtaining fluorescent intensity
measurements from a plurality of sperm in a sample comprises
obtaining fluorescent intensity measurements using flow
cytometry.
15. The method of claim 14, wherein obtaining fluorescent intensity
measurements using flow cytometry comprises obtaining fluorescent
intensity measurements using flow cytometry with an orienting
nozzle.
16. The method of claim 8, wherein obtaining a sample of sperm from
an animal of a species comprises obtaining a sample of sperm from a
bull, a boar, a human, a horse, a ram, or a dog.
17. The method of claim 8, wherein staining the sample with a
fluorescent DNA-binding dye comprises staining the sample with
Hoechst 33342.
18. The method of claim 8, wherein the high-fertility sperm has a
lower average intensity than the low-fertility sperm.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to provisional application
No. 61/856,828, filed Jul. 22, 2013, which is incorporated herein
by reference in its entirety.
BACKGROUND
[0002] The present invention relates to sperm fertility and in
particular to prediction of fertility from DNA staining
[0003] Semen quality examinations are a central role of the
semen-processing laboratory. Many semen quality exams exist to
evaluate semen. However these tests are often flawed because they
are designed to find higher-rather than lower-fertility males, or
the approaches reward extreme values rather than those that pass a
minimum threshold (Parrish et al., 1998; 2006). In addition,
fertility of bulls used in commercial artificial insemination of
dairy cattle is likely most dependent on non-compensable semen
traits, i.e. traits that cannot be overcome by increasing the
number of sperm inseminated. Many semen quality exams, however,
target the evaluation of compensable semen traits such as the
percentage of motile, live or acrosome-intact sperm.
[0004] Research has been directed to potential non-compensable
defects in sperm of lower-fertility bulls that alter events during
the first cell cycle of the zygote and result in changes to the
timing of cell divisions and success of embryo development (Eid et
al., 1994; Parrish and Eid, 1994; Parrish et al., 2006). It has
been determined that defects or damage in sperm DNA are responsible
for these effects. Since a significant portion of the sperm nucleus
consists of DNA, it has been hypothesized that subtle changes in
sperm DNA might be reflected in physical properties such as sperm
nuclear shape. As a result of research in this area, it has been
demonstrated that careful measurements of sperm head morphology can
be used to predict fertility (Parrish et al., 1998, 2006, 2012).
Nevertheless, there is an ongoing need for additional methods to
assess fertility.
SUMMARY
[0005] Accordingly, disclosed herein are methods for predicting
fertility of sperm samples based on the intensity of DNA staining
of the samples, based on the surprising observation that brighter
DNA staining of sperm heads has a positive correlation with
decreased fertility rates.
[0006] In one embodiment, the invention provides a method for
evaluating sperm fertility. The method includes the steps of
obtaining a sample of sperm from an animal of a species; staining
the sample with a fluorescent DNA-binding dye; collecting at least
one image of the stained sample; determining an edge of a nucleus
of at least one sperm within the stained sample in the at least one
image; measuring an intensity of the DNA-binding dye within an area
defined by the edge of the nucleus of the at least one sperm;
determining an average intensity per unit area of the area defined
by the edge of the nucleus of the at least one sperm; comparing the
average intensity per unit area to an average intensity per unit
area for high-fertility sperm and low-fertility sperm of the same
species to determine if the sample has high or low fertility.
[0007] In another embodiment, the invention provides a method for
evaluating sperm fertility. The method includes the steps of
obtaining a sample of sperm from an animal of a species, staining
the sample with a fluorescent DNA-binding dye, obtaining
fluorescent intensity measurements from a plurality of sperm in the
stained sample, determining an average intensity of the fluorescent
intensity measurements obtained from the plurality of sperm in the
stained sample, and comparing the average intensity to average
intensities for high-fertility sperm and low-fertility sperm of the
same species to determine if the sample has high or low
fertility.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows a fluorescent image of bovine sperm stained
with Hoechst 33342 with a line surrounding most of the sperm heads
showing the results of the automated edge detection procedure that
was used to obtain the outline of the sperm nucleus. Sperm nuclei
that touch another sperm, the edge of the image or have a
significant distortion were deleted from the analysis and do not
have an outline around the sperm nucleus.
[0009] FIG. 2 shows a phase contrast image of the bovine sperm
shown in FIG. 1, where the outline of the sperm nucleus was
obtained from the Hoechst 33342 fluorescent image and transferred
to this image.
[0010] FIG. 3 shows a phase contrast image of bull sperm.
[0011] FIG. 4 shows a fluorescent microscope image of Hoechst 33342
staining for the bull sperm of FIG. 3.
[0012] FIG. 5 shows the sperm heads identified from the images of
FIGS. 3 and 4.
[0013] FIG. 6 shows the fluorescently-stained sperm sample of FIG.
4 after deconvolution of the image.
[0014] FIG. 7 shows flow cytometer output for 1 bull, demonstrating
that sperm stained with Hoechst 33342 have 2 peak intensities;
these peaks relate to orientation of sperm passing through the
detectors; the mode is indicated for peak 1 and peak 2.
DETAILED DESCRIPTION
[0015] Before any embodiments of the invention are explained in
detail, it is to be understood that the invention is not limited in
its application to the details of construction and the arrangement
of components set forth in the following description or illustrated
in the following drawings. The invention is capable of other
embodiments and of being practiced or of being carried out in
various ways.
[0016] Fertility of males is critical to success of animal
agriculture as efficient production of the next generation is the
single biggest factor to profit or loss. In humans, predicting
fertility is important for selecting appropriate assisted
reproductive technology of infertile couples. In the pet industry,
including dogs and stallions, the high cost of a single
insemination dose creates a desire to select males with good
fertility or to eliminate purchase of poor fertility males. Semen
quality examinations include the collection of methods that a
semen-processing laboratory uses to ensure that high quality and
fertile semen is shipped to customers whether this is livestock,
humans, or pets. Many semen quality exams exist to evaluate semen
but few target defects in sperm that would be associated with
non-compensable semen defects. We have discovered that low
fertility bull sperm or boar sperm suffering summer infertility
have increased fluorescence of the sperm nuclei when exposed to a
DNA-binding and fluorescent dye such as Hoechst 33342.
[0017] The invention relates to sperm fertility and in particular
to prediction of fertility from DNA staining. The method relates to
the observation that increased DNA staining of sperm relates to
decreased fertility. This provides a relatively straightforward
method for assessing fertility of sperm samples that can be used,
among other things, by commercial animal breeding facilities to
improve success of artificial insemination. The method also detects
seasonal infertility when increased DNA staining exists.
[0018] As disclosed herein, the methodology includes the steps of
obtaining a sample of sperm from an individual of a species;
staining the sample with a fluorescent DNA-binding dye; collecting
images of the stained sample; determining an edge of the each
sperm's nucleus; measuring an intensity of the DNA-binding dye
within the sperm nucleus; determining an average intensity per unit
area; comparing the average intensity per unit area to an average
intensity per unit area for high-fertility and low-fertility sperm
of the same species to determine if the sample has high or low
fertility. To detect changes due to seasonal infertility the sample
is compared to semen collected before or during the periods of
seasonal infertility.
[0019] In the process of analyzing bull and boar sperm having
varying levels of fertility (e.g. due to male to male variation and
seasonal variations as well as due to heat stress) it was observed
that samples with lower fertility have brighter DNA staining
compared to samples with higher fertility, providing a relatively
straightforward method for assessing fertility of sperm samples
that can be used, among other things, by commercial animal breeding
facilities to improve success rates of artificial insemination.
[0020] In various embodiments, a fresh or frozen sperm sample is
exposed to a DNA-binding fluorescent dye (e.g. Hoechst 33342),
attached to a slide, and imaged using a fluorescence microscope.
The fluorescence per unit area of the sperm head is determined and
averaged together with like measurements obtained from the same
sample. The average sperm head DNA brightness values are then
compared between different samples, where the samples may be
obtained from different animals and/or from the same animal on
different occasions. The samples are then placed into groups based
on whether they are low- or high-fertility samples and the average
brightness values for the samples from each group are averaged
together.
[0021] The results of this procedure can then be used to predict
whether an unknown sperm sample will have low or high fertility
based on whether the average sperm head brightness of the unknown
sample is closer to the average brightness for the low-fertility or
the high-fertility samples.
[0022] The sperm head often has an oval shape which in some species
is slightly flattened. Therefore, in order to standardize the
fluorescence measurements, in various embodiments sperm heads are
selected for imaging and quantitative analysis based on sperm that
lay flat on the slide. Sperm which are not flat may have variations
in fluorescent intensity due to their particular orientation to the
light beam in the microscope.
[0023] The fluorescence signal of the selected sperm heads is then
determined on a per unit area basis, for example per pixel or per
square micrometer. The location of the edge of the sperm head
cannot always be determined with complete certainty. However, where
the edge of the head is drawn can affect the final value of the
average brightness of DNA for the head. Therefore, in some
embodiments an automated edge-detection scheme is used in order to
standardize the determination of the border of the sperm head
(FIGS. 1, 2). Even if the automated routine consistently over- or
under-estimates the size of the sperm heads, as long as this is
consistent across samples the over- or under-estimating effect is
expected to cancel out since the results are used for comparison
purposes. In one embodiment a series of commands are executed using
the NIH ImageJ software package, although other procedures and
software packages can be used as well. Other conventional
image-processing steps such as background subtraction may also be
used provided the steps are used consistently for all samples.
[0024] Once the outlines of sperm heads have been determined, the
total fluorescence within the outlined area is determined by
summing the light intensity of the pixels within the outlined area
(taking into account any steps such as background subtraction or
other processing steps) and the total intensity is then normalized
to a unit area such as per pixel or per square micrometer. For
comparison across samples, all of the samples should be normalized
to the same unit of area.
[0025] While the Examples below pertain to sperm from bulls and
boars, it is expected that the procedures are equally applicable to
evaluate the fertility of sperm samples from other animals
including, without limitation, humans, horses, sheep (ram), and
dogs.
[0026] Furthermore, while Hoechst 33342 is used in the Examples
below as the DNA-binding fluorescent dye, in various embodiments
other DNA dyes could be used. Hoechst 33342 has been used because
it can stain both live and dead sperm, particularly if incubation
is at 37-39.degree. C., and has a very high binding affinity and
bright fluorescence. In some cases the sperm need to be
permeabilized with a detergent (e.g. Triton X-100) prior to
application or along with the DNA dye to allow the dye to contact
the DNA. While Hoechst 33342 can pass through the lipid membrane,
other dyes cannot and therefore need detergent to produce holes in
the membrane. Even though the sperm cells are fixed before
staining, the paraformaldehyde that is generally used for fixation
does not produce large holes in the membrane, at least not
reliably. Other fixatives such as ethanol or glutaraldehyde could
potentially produce holes in the membrane and be useful approaches
to prepare sperm. Such other fixatives are included in this
embodiment. There are also other possible dyes including DAPI,
YOYO-1, DRAQ5, DRAQ7 and propidium iodide, and still other DNA dyes
are also possible. Other DNA dyes may be tested with known samples,
for example with semen from 10 high- and 10 low-fertility bulls.
Semen will be processed as for Hoechst 33342 except preliminary
experiments will establish optimal dye concentration levels and
sperm permeability treatments that produce sufficient fluorescence
for image analysis. Some of these dyes have higher increases in
fluorescence than Hoechst 33342 when binding to DNA but often have
lower binding affinity for the DNA as well. In view of the lower
binding affinity, one possible adaptation may be to not remove
unbound dye as is done in current procedures with Hoechst 33342.
Thus, optimization of processing conditions for each dye will be
investigated. It is predicted that at least some of the DNA dyes
listed herein will show an increase in DNA staining intensity that
is predictive of infertility as with Hoechst 33342 staining.
[0027] In the bull and boar sperm that are analyzed herein, the
nucleus makes up most of the bulk of the sperm head such that DNA
staining, which strictly speaking is limited to the nucleus, is an
effective measurement of the sperm head. As seen in FIG. 2, the
perimeter of each sperm nucleus, each of which is determined based
on Hoechst 33342 DNA staining, matches the outline of the
respective sperm head as seen in phase contrast microscopy.
[0028] A surprising finding was that the mean intensity of sperm
from low fertility bulls was increased along with increased
measures of dispersion among the intensity values. At this time it
is unclear why the two fertility groups are so different in the
various measures of intensity. Without being limited as to theory,
this may be due to nuclear condensation during spermatogenesis or
the further condensation of nuclei that occurs during passage of
sperm through the epididymis.
[0029] Calibration of Microscope/Camera System for Fluorescent
Intensity Measurements
[0030] Given that embodiments of the present invention relies on
fluorescent intensity of samples, it is helpful to have a method
for standardizing intensity measurements between samples and
between data collection systems (including microscopy setups).
Quantification of fluorescence via microscopy has an inherent
problem in that it is dependent on the fluorescent light intensity
delivered to the object (which is based on factors such as the
brightness of the light source and the transmission properties of
the optical system) and sensitivity of the detecting camera. One
factor which may vary even for the same microscopy system is the
intensity of the light source, for example a fluorescent bulb's
intensity often decreases as the bulb ages; light intensity also
varies between microscopes and camera systems.
[0031] Thus, utilization of the disclosed methods will be improved
in certain embodiments if the implementation also includes steps to
standardize conditions (e.g. to obtain consistent lighting on the
sample) and/or to adjust/calibrate the resulting data for the
particular conditions. To calibrate brightness results so that the
results can be more readily compared from one system to another and
for the same system over time, calibration curves may be obtained
by gathering data from fluorescent standards such as quantum dots
or standardized fluorescent microspheres.
[0032] Quantum dots or slides containing microsphere standards can
be obtained which emit consistent amounts of light in various
spectral regions when excited with light having a specific
intensity and wavelength. One can quantify how much fluorescence is
being produced with this technology and use standard curves to
adjust sperm fluorescence intensity on any collection system (e.g.
fluorescent microscope setup).
[0033] In some embodiments, the calibration data can be used to
compare data collected with varying exposure times. On the
assumption that the observed light intensity is a linear function
of the exposure time, one can alter the exposure times of dots and
sperm samples relative to a given set of data collection
conditions. If one currently uses a 125 msec exposure time for boar
sperm images, this can be increased or decreased by specific
amounts and this will deliver proportionally more or less light.
For example, doubling exposure will deliver 2.times. the amount of
light and fluorescence intensity. By examining how variation in
exposure changes intensity of quantum dots and sperm, one can
establish a calibration curve to adjust observed sperm intensity to
what a given microscope system delivers using a reference standard
such as microspheres or quantum dots and a particular exposure time
(e.g. 125 msec). Other exposure settings for other species of sperm
can also be calculated using a similar approach.
[0034] Thus, in one embodiment, calibration was performed using a
slide containing microspheres (Molecular Probes ref: F36914 "Focal
Check fluorescence microscope test slide #3"; hereinafter referred
to as "FCF test slide"; Life Technologies, Thermo Fisher
Scientific). The slide contains fluorescent microspheres which emit
light at different colors/wavelength ranges. The blue (440 nm
emission) microspheres are chosen because their excitation and
emission wavelengths are similar to those of Hoechst 33342, the dye
used to obtain fluorescent intensity data on sperm.
[0035] The FCF test slide has both bright and dim dot options; the
dim dots were chosen and specifically the blue ones were imaged. A
series of images were taken on a Nikon Microphot microscope with
the setup as described above for either bovine or porcine
fluorescent sperm imaging. Images were taken at 250, 500, 625 and
1000 msec exposure times. Images were viewed in ImageJ and dots
were thresholded manually using ImageJ tools. The mean intensity of
only those spheres that were determined to be in focus in a given
image were analyzed. A regression line was then obtained of average
intensity (y) vs. exposure time (x) with the intercept going
through 0. For one particular set of conditions, the resulting
equation will be of the form:
y=0.128(x)
[0036] To calibrate the intensity measurements from a second
optical system and compare them to a known system (such as the
system described herein) one would image the same fluorescent
spheres on the FCF test slide at a range of exposure times using
the second optical system and then calculate the equation of a
second line that is obtained from the data on the second optical
system. One would follow the same protocol used for establishing
the standardization line from the known system, including imaging
fluorescent microspheres followed by thresholding with ImageJ
software and measuring average intensity on only those microspheres
that are in focus in a given image.
[0037] The equation of the second line will be calculated with the
y-intercept also going through 0. The resulting equation will be of
the form:
y2=m2(x)
[0038] Next, a calibration coefficient will be calculated by taking
a ratio of the slope 0.128 of the original system and the slope m2
determined with the new optical system: c (coefficient)=0.128/m2.
All of the intensity values collected on the second imaging system
will be multiplied by the coefficient c to allow the intensity
values that are obtained on the second system to be compared to the
intensity values that would be obtained on the known "standard"
system such as that described herein.
[0039] Deconvolution
[0040] In some embodiments, fluorescence microscopy images of sperm
head DNA staining are processed to minimize or remove out of focus
information (e.g. out of focus `blue`), which in one particular
embodiment is performed using deconvolution techniques.
Deconvolution involves mathematically correcting for the
imperfections in an image that arise from the microscopy system. To
evaluate the imperfections in the system, an image of a point
source of light (e.g. using a microsphere or quantum dot) is
collected as a reference to show the distortions caused by the
imaging system. The image or collection of images of the dot, which
is referred to as a point spread function or PSF, is used in a
mathematical deconvolution procedure to undo the distorting effects
of the imaging system. In some embodiments a
mathematically-determined PSF may be used instead of the images of
the microsphere or quantum dot. The deconvolved images of sperm
heads are then subjected to further intensity staining analyses as
disclosed herein.
[0041] The specific process followed has been to obtain a PSF from
an average of 8 quantum dot images. The PS-Speck Microscope Point
Source Kit (molecular probes product #P7220) was used to obtain
solutions of the quantum dots which are then placed under a cover
slip. Images of dots were obtained with ImageJ and 8 dots were
averaged. The averaged dot image was then centered on a
1024.times.1024 pixel image with a black background (although in
some embodiments rectangular image sizes also worked). The plugin
from ImageJ FIJI release "Parallel Iterative Deconvolution" was
used in two dimensions (2d). The approach is to select the Hoechst
image as the blurred image, and then use the square PSF (e.g.
1024.times.1024 pixels, although other dimensions can be used) as
the PSF image, use the Preconditioner as WPL (Weiner Filter),
Boundary as Reflexive, Resizing as Auto, Output as Same as source,
Precision Type as single, Max number of iterations as 5, Max number
of threads as 8. The deconvolved image then has edges of sperm
nuclei identified using the same object edges identified in the
original Hoechst image as described above. The intensity measures
can now be determined on the deconvolved sperm or various measures
of fluorescence (e.g. DNA since the DNA-binding dye Hoechst 33342
is used to label the sperm) distributions. Additionally, these
sperm can be evaluated with the ImageJ "Texture Analyzer" plugin to
produce 5 different texture measures, e.g. as defined by Haralick
(1973), namely angular second moment, contrast, correlation,
inverse difference moment, and entropy. It is important to note
that "texture" here refers to the distribution of pixel intensity
within an object, in this case a sperm head. Images from the
standard intensity analysis (phase, FIG. 3; Hoechst staining, FIG.
4; and identified sperm nuclei, FIG. 5) as well as the
deconvolution approach (decon) are shown for bull sperm. The
deconvolved image has the out of focus fluorescence removed (FIG.
6). Table 1 shows the results from analysis of the 16 sperm
identified in FIGS. 3-6. The mean intensity and STD come from the
standard analysis on image intensity as disclosed herein. The other
measures (texture measures) are derived from the deconvolution
analysis. The deconvolution analysis reveals differences from the
standard analysis of mean intensity. For example the Contrast value
generally increases with increasing mean intensity (see sperm 1 and
2) but sperm 9 has a greater mean intensity than sperm 2 while the
contrast of sperm 9 is less than sperm 2. In some embodiments,
variations in sperm nuclear texture are expected to be related to
male fertility as reflected in one or more of the texture measures
shown in Table 1.
TABLE-US-00001 TABLE 1 The results of deconvolution analysis for 16
bull sperm also evaluated for mean intensity and standard deviation
(STD). Mean Angular Inverse Inten- Second Correla- Difference Sperm
sity STD Moment Contrast tion Moment Entropy 1 134 33 0.102 25
0.00079 0.494 5.446 2 180 45 0.030 95 0.00046 0.308 6.790 3 133 38
0.033 21 0.00078 0.426 6.278 4 154 37 0.098 44 0.00064 0.472 5.655
5 139 33 0.033 44 0.00078 0.348 6.565 6 128 30 0.080 21 0.00087
0.481 5.653 7 152 38 0.118 41 0.00061 0.498 5.377 8 167 40 0.128 51
0.00046 0.520 5.232 9 188 50 0.068 89 0.00040 0.384 6.224 10 161 41
0.112 53 0.00048 0.471 5.649 11 167 44 0.118 45 0.00049 0.507 5.369
12 132 36 0.094 28 0.00085 0.464 5.613 13 173 41 0.024 85 0.00052
0.308 6.692 14 179 43 0.081 56 0.00044 0.415 6.074 15 142 30 0.067
27 0.00072 0.433 5.996 16 175 44 0.090 61 0.00050 0.430 5.882
[0042] The following non-limiting Examples are intended to be
purely illustrative, and show specific experiments that were
carried out in accordance with embodiments of the invention.
EXAMPLES
Example 1
Bull Sperm Fertility and Brightness Determination Procedures
[0043] Materials and Methods
[0044] Frozen semen samples and fertility data from 107 bulls with
varying fertility were provided by Alta Genetics Inc. (Watertown,
Wis.). All semen was frozen as per Alta Genetics commercial
protocol using egg-yolk Tris extender.
[0045] Fertility Prediction and Bull Selection
[0046] The fertility data were obtained from Alta Genetics progeny
testing. The program consists of more than 180 well-managed dairy
farms located in different geographical regions across the United
States. Evaluation of fertility of bulls in the program includes
DNA verification of paternity and confirmation of pregnancies by
rectal palpation or ultrasonographic exam. The outcome of each
breeding event was registered into farm management software (DC305,
Valley Ag), and the data was collected from partnering farms every
three months. The fertility of each bull was predicted using the
statistical methods developed by Zwald et al. (2004a,b). The model
takes into account the breeding event as well as environmental and
herd management factors that influence fertility performance of
sires (i.e. effects of herd/year/month, parity, cow, days in milk,
sire proven status) as described by Peddinti et al. (2008).
Fertility prediction of the sires was expressed as the percentage
deviation of its conception rate from the average conception of all
bulls. For the present study, 107 bulls with a minimum of 400
breeding records and at least one standard deviation (SD) below or
above the average were selected. The respective low- and
high-fertility groups thus represented two standard deviations of
fertility difference between groups.
[0047] Media Required
[0048] The following media are required in the preparation of
samples: 2.9% Sodium Citrate Buffer (2.9 gm sodium citrate
dihydrate, 90 ml distilled water, adjust pH to 7.4, adjust final
volume to 100 ml); Hepes buffered saline (0.238 gm Hepes free acid,
0.9 gm NaCl, 90 ml distilled water, adjust pH to 7.4, adjust final
volume to 100 ml with distilled water); DABCO mounting media (25 mg
1,4-Diazabicyclo[2.2.2.]octane Triethylenediamine, DABCO, 100 .mu.l
Hepes buffered saline, mix until dissolved, 900 .mu.l glycerol,
store in dark or foil-wrapped tube); Paraformaldehyde stock
solution (4%, 4 gm paraformaldehyde, 50 ml water, mix, add NaOH
pellets with mixing until paraformaldehyde dissolves, add 0.238 gm
Hepes, adjust pH to 7.4, adjust to 100 ml with water); Parrish
citrate fixative (10 ml Paraformaldehyde stock, fill to 100 ml with
2.9% sodium citrate buffer); Hoechst 33342 stain solution (5 mg/ml
in water made fresh daily).
[0049] Slide Preparation
[0050] Straws containing sperm samples (0.25 ml or 0.5 ml) were
thawed at 37.degree. C. for 60 sec and the contents were expelled
into 1.5 ml microcentrifuge tubes. The sperm were diluted 1:1 in
2.9% Sodium Citrate dihydrate solution to a final volume of 500 or
1000 .mu.l respectively. If the volume was 1000 .mu.l, then 500
.mu.l of the diluted sperm sample was placed in a new tube for
staining of sperm. The sperm cells were stained by adding 2.5 .mu.l
of Hoechst-33342 stain solution and incubated at 37.degree. C. for
30 min. After incubation, 250 .mu.l of 2.9% Sodium Citrate solution
was added to each tube, each of which was then centrifuged at
6,000.times.g for 15 sec. The supernatant containing excess stain
and extender was removed by aspiration. The sperm pellets were
resuspended with 650 .mu.l of Parrish citrate fixative and
incubated for 3-5 min at room temperature. The fixed samples were
briefly vortexed and centrifuged as above, the supernatant removed,
and the sperm pellet resuspended with 750 .mu.l of water. The sperm
pellet was washed a second time with 750 .mu.l water and
centrifuged as above, and then finally resuspended with 500 .mu.l
water and vortexed. Then, a 10 .mu.l of sample was placed onto a
microscope slide and gently spread out to make homogenous sperm
distribution, and allowed to air dry completely on a slide warmer
at 37-39.degree. C. Next, a 3.5 .mu.l drop of the DABCO mounting
solution was placed on top of the sample to prevent fluorescent
fading. An 18.times.18 mm coverslip was added on top and the edges
were sealed with clear fingernail polish.
[0051] The procedures for using fresh bovine sperm are the same as
for frozen-thawed semen as described above except for sperm
dilution to start. Sperm are diluted to either an insemination dose
or 40.times.10.sup.6 sperm/ml in citrate buffer with BSA (Fraction
V BSA at 3 mg/ml in standard citrate buffer). Semen can also be
diluted with egg yolk- or milk-based extenders, instead of BSA and
then processed as described for frozen-thawed semen citrate
buffer.
[0052] Image Collection
[0053] Sperm cells were imaged on a Nikon Microphot with phase
contrast and epifluorescent microscopy (excitation 365.+-.20 nm,
dichromatic mirror 400 nm, emission >400 nm); images were
collected using a 40.times. objective, 1.25.times. magnifier. A
QIClick monochrome camera operating in 8 bit mode and using an
exposure setting of 62.5 msec was used to collect a tiff format
image that was then saved for further image analysis.
[0054] Image Analysis Procedures for Bovine Sperm
[0055] Images were analyzed with NIH ImageJ version 1.47m using
custom macros to implement the procedures described below. The
following describes how an image pair is analyzed, where the image
pair includes a phase (p) image and a Hoechst 33342 (h) intensity
image.
[0056] 1. Thresholding is applied to the Hoechst image and sperm
nuclear object identified. [0057] a. Duplicate the image and rename
it (e.g. `h-1`). [0058] b. Apply `Unsharp Mask` with radius=20 and
mask=0.60 to duplicate image. [0059] c. Apply `Autothreshold` to
the duplicate image selecting the `IsoData` method with the `Dark
background` box checked. [0060] d. Apply `Convert to Mask`,
followed by `Dilate,` followed by `Erode.` [0061] e. Use `Analyze
Particles` with the following settings: size=700-4500;
circularity=0.5-1.0; show=Masks; and with the `Exclude . . . ,`
`Clear . . . ,` and `Add . . . ` boxes checked. [0062] f. Rename
resulting image (e.g. `hmask`) and run the `Fill Holes` routine on
the resulting image. [0063] g. Delete any sperm nuclei that do not
appear to be thresholded correctly. Indications that a given sperm
nucleus is not thresholded correctly can include holes in the edge
of the sperm head, additional area in a part of the sperm head due
to overlapping of another sperm head or tail in the image, presence
of fluorescent debris, or an object that has a shape not consistent
with the sperm head of the species under investigation. [0064] h.
Use `Create Selection` command to make the perimeter of all sperm
nuclei in the image from step f (hmask') an object. Lay this object
on the p image to check if objects are the same approximate size
and shape of the sperm head in the p image. If any of the objects
are not similar between the p and hmask image, then show the
`hmask` file and delete any non-correct sperm nuclei. Rename the
`hmask` image to `all`. [0065] i. Save the `p`, `h`, and `all`
images to a subfolder (e.g. called `mfiles`) as separate images
(e.g. 2001.tiff, 2002.tiff, 2003.tiff, respectively) and extending
the naming convention with more image sets evaluated.
[0066] 2. Obtaining intensity values and obtaining mean values:
[0067] a. Access the appropriate `mfiles` folder for a particular
sample. [0068] b. Set `Measurements` for mean. Additional measures
of the intensity of the object (nucleus) and its dispersion can
also be selected as shown in Table 2 and include median, standard
deviation, skewness, and kurtosis. It is also possible to select
other measures that describe the object such as perimeter and
ellipse. The perimeter is simply the outside boundary of the
selected sperm head. The ellipse option fits an ellipse to the
sperm head and the major axis of the ellipse is the length and
minor axis the width. The perimeter, length and width may be
expressed in .mu.m or other suitable units. [0069] c. Select the
first `all` image (e.g. 2003.tiff), use `Analyze Particles` with:
size=0-Infinity, circularity=0.00-1.00, show=Nothing, and with the
`Display . . . `, `Exclude . . . `, `Include . . . `, and `Add . .
. ` boxes checked. [0070] d. Data per sperm is saved in a results
table. [0071] e. The next `all` image (e.g. 2006.tiff) is opened
and the above steps are repeated. This continues until no more
`all` images exist. The results are saved and evaluated within
Statistical Analysis System (SAS Inc.). [0072] f. Within SAS, 100
randomly-selected sperm from those evaluated are selected and means
from those selected sperm are obtained for the various
measurements.
[0073] Bull Sperm Measurement Results
[0074] This Example involves evaluating sperm from two populations
of bulls that represent extremes of fertility, with a difference of
8.8% fertility between the two populations. This is the largest
difference that can be obtained on a population of bulls used for
commercial insemination. There were 53 bulls in the high fertility
group and 54 in the low fertility group. The mean.+-.sem number of
breedings used to determine fertility for the high and low
fertility groups was 2368.+-.324 and 1124.+-.137, respectively.
[0075] Samples were collected and analyzed as described above.
Results comparing mean intensity of Hoechst 33342 staining in the
sperm head and other parameters measured on the `h` image are shown
in Table 2. There were differences in mean intensity, standard
deviation of intensity, skewness of intensity, kurtosis of
intensity, median of intensity, area of the sperm head, perimeter,
and width between sperm from the two fertility groups (p<0.05).
Surprisingly, the low fertility bulls have an increased intensity
and variation between sperm heads. The low fertility bulls also
have sperm with a smaller area, perimeter, and width.
TABLE-US-00002 TABLE 2 The mean .+-. SEM for bulls in the two
fertility groups for mean intensity (INT, in arbitrary fluorescence
units) and other measures of sperm head characteristics determined
directly from ImageJ. Fert1(high) Fert2(low) p value Criteria N =
53 N = 54 (ANOVA)c Fertlity Group 4.1 .+-. 0.1 -4.7 .+-. 0.3 --
Mean INT 99 .+-. 3 109 .+-. 3 0.0078 Std of Mean 19.0 .+-. 0.6 22.2
.+-. 0.6 0.0002 INT Skewness of 0.0088 .+-. 0.0316 0.1741 .+-.
0.0278 0.0001 INT Kurtosis of -0.2681 .+-. 0.0483 -0.0879 .+-.
0.0484 0.0097 INT Median of INT 100 .+-. 3 110 .+-. 3 0.0138 Area
(microns) 31.3 .+-. .2 30.4 .+-. 0.2 0.0017 Perimeter 23.08 .+-.
0.07 22.79 .+-. 0.09 0.0151 (microns) Length 8.96 .+-. 0.03 8.85
.+-. 0.05 0.0611 (microns) Width 4.45 .+-. 0.02 4.37 .+-. 0.02
0.0041 (microns)
Example 2
Sperm Nuclear Structure of Boars is Impacted by the Summer
Environment
[0076] The experiments in this Example involved the evaluation of
boar semen collected over the summer of 2012. During the summer, it
is known that boar semen declines in fertility in response to
increases in summer temperatures (Flowers, 1997). The summer of
2012 was extreme in Wisconsin with daily high temperatures during
the period of our study exceeding 90.degree. F. on 38 days compared
to an average of 12 days in a normal year. In Table 3, it can be
observed that boar sperm nuclei change in their ability to have
their DNA stain with Hoechst 33342 that includes an increase in
mean fluorescent intensity as well as increases in the length and
width of the sperm heads.
[0077] The data from boars is similar to the bull data, except that
the bull sperm data was correlated with known female fertility
information. For boars, on the other hand, fertility is inferred
from a known seasonal decline in fertility. As shown above, low
fertility bulls had high mean fluorescent intensity of their sperm
nuclei which is what is demonstrated below to occur to boar sperm
nuclei as the period of summer infertility occurred. In contrast to
bulls, in which higher fertility sperm have larger heads that those
of low fertility groups, boar sperm nuclei increased in length and
width during a period in which it is expected that fertility
decreases. Low fertility bull sperm decreased in length and width
of the heads. This may be due to the differences in geometry of the
boar sperm as they are more tubular than bull sperm. The reason for
the changes in sperm nuclear parameters of bulls and boars is
unclear at the present time. This may be due to nuclear
condensation during spermatogenesis or changes to condensation of
nuclei that occurs during passage of sperm through the epididymis.
The differences in sperm nuclear intensity, length, and width
provide the means however to identify bulls of different fertility.
For boar ejaculates, the differences in nuclear intensity, length,
and width provide the means to identify a male suffering from
summer heat stress and, by correlation with the known decline in
fertility over the course of the summer, the means to predict lower
fertility.
[0078] Materials and Methods
[0079] Semen was collected at a commercial boar stud in Southern
Wisconsin from Jun. 18, 2012 to Nov. 2, 2012. The number of boars
collected each week and the Wednesday date for a particular week
are listed in Table 3. The number of boars from which samples were
collected/week ranged from 45-60 over the course of the summer of
2012. Boars were only those that were used for single sire
insemination. It is known that fertility of boars declines over the
course of the summer with peak declines occurring from July-August.
Thus the samples represent the period of time when fertility of
these specific boars are expected to decline.
[0080] Media
[0081] The following media are required in the preparation of
samples: 2.9% Sodium Citrate Buffer (2.9 gm sodium citrate
dihydrate, 90 ml distilled water, adjust pH to 7.4, adjust final
volume to 100 ml); Hepes buffered saline (0.238 gm Hepes free acid,
0.9 gm NaCl, 90 ml distilled water, adjust pH to 7.4, adjust final
volume to 100 ml with distilled water); DABCO mounting media (25 mg
1,4-Diazabicyclo[2.2.2.]octane Triethylenediamine (DABCO), 100
.mu.l Hepes buffered saline, mix until dissolved, combine with 900
.mu.l glycerol); Paraformaldehyde stock solution (4%; 4 gm
paraformaldehyde, 50 ml water, mix, add NaOH pellets with mixing
until paraformaldehyde dissolves, add 0.238 gm Hepes, adjust pH to
7.4, adjust to 100 ml with water); Parrish boar citrate fixative
(6.25 ml Paraformaldehyde stock, fill to 100 ml with 2.9% sodium
citrate buffer, add 300 mg Bovine Serum Albumin, pass through a
0.22 .mu.m filter to sterilize); Hoechst 33342 stain solution (1
mg/ml in water made fresh daily).
[0082] Sample and Slide Preparation
[0083] Following semen collection, 0.25 ml semen is added to 0.75
ml of the Parrish boar citrate fixative, mixed and shipped to the
lab for further analysis. Upon arrival at the lab, fixed semen is
stored at 5.degree. C. until further analysis. Concentration of the
semen sample in the fixative is determined using a
spectrophotometer at 650 nm. To a 1 ml cuvette, add 0.9 ml of the
sodium citrate buffer, zero cuvette, add 0.1 ml of the fixed semen
sample, measure absorbance. Adjust the sample to 0.2 absorbance,
using the Parrish boar citrate fixative. This will yield a sperm
concentration approximately 40.times.10.sup.6 sperm/ml.
[0084] To stain sperm, place 500 .mu.l of the 40.times.10.sup.6
sperm/ml semen in a 1.5 ml microcentrifuge tube, add 2.5 .mu.l of
Hoechst stain, vortex briefly and incubate at 35-37.degree. C. for
30 minutes. Centrifuge the stained sample in microcentrifuge at
maximum at 6000 g for 15 seconds. Remove supernatant, vortex for
1-2 seconds, and suspend the sperm pellet in 750 .mu.l of the
Parrish boar citrate fixative. Repeat the centrifugation, remove
supernatant, suspend with 750 .mu.l water. Place 10 .mu.l of sample
on a slide and dry on a stage warmer. When dry, sample can be
stored if desired. To continue on, add 3.5 .mu.l of the DABCO
mounting media over the sample, add a 18 mm.times.18 mm, #1 or #1.5
coverslip. After mounting media reaches the edge of coverslip, seal
with clear finger nail polish. When the finger nail polish dries,
repeat to ensure a complete seal.
[0085] Image Analysis
[0086] Sperm cells were imaged on a Nikon Microphot with phase
contrast and epifluorescent microscopy (excitation 365.+-.20 nm,
dichromatic mirror 400 nm, emission >400 nm), using a 40.times.
objective and 1.25.times. magnifier. A QIClick monochrome camera
operating in 8 bit mode and using an exposure setting of 125 msec
was used to collect a tiff format image that was then saved for
further image analysis.
[0087] Images were analyzed with ImageJ 1.47 m using a combination
of procedures available within ImageJ and custom designed macros.
The following is how an image pair is analyzed. The image pair is a
phase (p) image and a Hoechst (h) image of the same field of view.
Thresholding is applied to the Hoechst image and the sperm nuclear
object identified. The main difference from the bull procedure (see
above) is the thresholding approach. The `h` image is duplicated
and renamed as `h-1`, it is then smoothed, and laplacian edge
detection is done with a smoothing of 3 applied. The resulting
image has the contrast enhanced with a saturation=1 and
normalization applied. Now an autothreshold of MaxEntropy is
applied with threshold remaining dark. The resulting image is
dilated and then eroded.
[0088] The remaining steps described are the same as for the bull
sperm, as discussed above. The analyze particles routine in used
with size=700-4500, circularity=0.5-1.0, show=Masks, exclude,
clear, and add selected. The resulting image is renamed as `hmask`
and a fill holes routine run. The custom macros next allow the user
to delete any sperm nuclei that appear not thresholded correctly.
The create selection command is then used to make the perimeter of
all sperm nuclei in `hmask` an object that is then overlaid on the
`p` image to check if objects are correct. If any are not correct,
then the user has the option to delete any non-correct sperm
nuclei. Lastly the `hmask` image is renamed to `all`. The `p`, `h`
and `all` images are then saved to a subfolder called `unifies` as
image 2001.tiff, 2002.tiff, 2003.tiff respectively and extending
with more image sets evaluated.
[0089] To obtain the intensity values as well as the length and
width of the sperm heads, in the set measurements dialog box in
ImageJ the mean gray value and shape descriptors options should be
selected. Now select the first all image (for example, 2003.tiff)
use analyze particles command with size=0-Infinity,
circularity=0.00-1.00, show=Nothing, display, exclude, include, and
add checked. Data per sperm is then saved in a results table. The
next all image, 2006.tiff is opened and steps repeated. This
continues until no more all images exist. The results are saved as
means per sample generated within Statistical Analysis System (SAS
Inc.) or can be directly generated within ImageJ using the
summarize command. Within SAS it is possible to randomly select 100
sperm from those evaluated and then obtain the means from those
selected sperm.
[0090] Results
[0091] Semen was collected over a 20-week interval in the summer of
2012. Data is presented in Table 3 and is expressed as the
mean.+-.SEM among boars collected in a particular week. The listed
date for each week corresponds to the Wednesday date for the
particular week. The data collected included the mean fluorescent
intensity along with the length and width of the sperm nucleus as
determined from the fluorescent image of the Hoechst stained sperm,
where the data are presented as mean.+-.sem. Data in each week were
compared to the data in the Jun. 20, 2012 week, which was
considered the control representing sperm not yet expressing summer
heat stress. There were no differences, p>0.05, between data in
week Jun. 20, 2012 and Jun. 27, 2012 for all 3 measurements.
Beginning in week Jul. 4, 2012 and continuing through week Oct. 3,
2012 the mean intensity of sperm nuclei was greater than seen in
week Jun. 20, 2012, p<0.05. The other measures showed
differences as the summer progressed but required longer to return
to pre-heat stress measurement levels. Effects of heat stress on
boars requires >35 days for recovery (Gibbs et al., 2013). As
the last days of >90.degree. F. (heat stress temperatures)
occurred during the week of Sep. 5, 2012, it was predicted from the
Gibbs et al. (2013) data that recovery of any heat stress effects
would occur by the week of Oct. 10, 2012. This is indeed what was
observed. Over the course of the summer, there was an increase in
the mean intensity, length and width of sperm nuclei.
TABLE-US-00003 TABLE 3 Changes in boar sperm nuclear intensity,
length and width over the summer of 2012. Dates indicate the
Wednesday date for each specific week. Values shown are mean .+-.
sem and ANOVA was used for analysis. A * indicates a difference
greater than date Jun. 20, 2012, p < 0.05. Date Boars (#)
Intensity Length (.mu.m) Width (.mu.m) Jun. 20, 2012 45 104 .+-. 5
8.49 .+-. 0.04 4.16 .+-. 0.02 Jun. 27, 2012 46 106 .+-. 4 8.48 .+-.
0.03 4.15 .+-. 0.02 Jul. 04, 2012 53 115 .+-. 2* 8.50 .+-. 0.03
4.23 .+-. 0.01* Jul. 11, 2012 53 122 .+-. 3* 8.55 .+-. 0.03 4.24
.+-. 0.01* Jul. 18, 2012 48 137 .+-. 2* 8.53 .+-. 0.03 4.19 .+-.
0.01 Jul. 25, 2012 50 147 .+-. 3* 8.59 .+-. 0.04 4.24 .+-. 0.01*
Aug. 01, 2012 50 143 .+-. 2* 8.64 .+-. 0.03* 4.27 .+-. 0.01* Aug.
09, 2012 52 148 .+-. 2* 8.58 .+-. 0.04 4.24 .+-. 0.01* Aug. 15,
2012 52 158 .+-. 2* 8.66 .+-. 0.03* 4.22 .+-. 0.01* Aug. 22, 2012
49 128 .+-. 2* 8.66 .+-. 0.04* 4.23 .+-. 0.01* Aug. 29, 2012 60 139
.+-. 2* 8.71 .+-. 0.03* 4.26 .+-. 0.01* Sep. 05, 2012 54 145 .+-.
3* 8.73 .+-. 0.03* 4.27 .+-. 0.01* Sep. 12, 2012 56 141 .+-. 3*
8.72 .+-. 0.03* 4.25 .+-. 0.02* Sep. 19, 2012 52 150 .+-. 3* 8.70
.+-. 0.03* 4.22 .+-. 0.02* Sep. 26, 2012 55 144 .+-. 3* 8.71 .+-.
0.03* 4.24 .+-. 0.01* Oct. 3, 2012 55 115 .+-. 2* 8.69 .+-. 0.02*
4.26 .+-. 0.01* Oct. 10, 2012 56 98 .+-. 2 8.71 .+-. 0.02* 4.23
.+-. 0.01* Oct. 17, 2012 50 93 .+-. 3 8.68 .+-. 0.03* 4.25 .+-.
0.01* Oct. 24, 2012 56 111 .+-. 3 8.68 .+-. 0.03* 4.21 .+-. 0.01
Oct. 31, 2012 46 106 .+-. 3 8.62 .+-. 0.03 4.19 .+-. 0.01
Example 2a
Analysis of Sperm DNA Staining Intensity in Boars During Non-Heat
Stress Periods
[0092] In some embodiments, additional studies will be performed to
determine if there is a correlation between sperm head DNA staining
intensity in boars during non-heat stress periods. It is predicted
that the correlation between higher intensity and decreased
fertility that has been seen in heat-stressed boars as well as with
non-heat stress bulls will also be seen in non-heat stress boar
samples.
[0093] The time interval for seasonal fertility differences is
different for males and females. Females exhibit reduced fertility
.+-.2 weeks from a heat event while boars ejaculate defective sperm
21-35 days following the heat event. The last significant heat
events of the summer of 2012 occurred during August based on
available temperature records. The comparison of earlier data (e.g.
from September) and later collected data (e.g. from October and
November) thus provides the ability to remove the sow effect in
comparisons. Thus, semen and fertility records from
October-November 2012 may be used and then compared to data for
October-November of 2013 to compare boar fertility and fluorescent
intensity of sperm nuclei in a period of the year with no summer
infertility effects present for either the sow or boar. Boars will
be grouped as either being of high or low fertility and then sperm
fluorescent intensity will be compared between these two groups.
This will allow a similar comparison as was done for bulls of
differing fertility. Fertility data will include farrowing rate
(conception data) and pigs/litter born for matings to these boars.
In various embodiments, data will be collected from at least 50
matings to provide statistical significance. Accordingly, semen and
records from 2012 and 2013 will be used to ensure there is
sufficient data from boars to perform meaningful statistical
comparisons.
Example 3
Human Sperm Samples
[0094] In this Example, the relationship of sperm evaluation using
Fourier Harmonic Amplitudes (FHA; see Parrish et al. 2006) and DNA
staining intensity of human sperm to fertility measure by TI, IUI
and IVF (with or without intracytoplasmic sperm injection,
IVF.+-.ICSI) will be studied. Sperm nuclear shapes will be
described using FHA in a population of normal and infertile males
from couples seeking fertility treatment. In addition, canonical
discriminant analysis will be used to determine if FHA variables
are able to predict fertility of sperm samples as indicated by
pregnancy outcome following fertility treatment (TI, IUI, or
IVF.+-.ICSI). Further, DNA staining intensity will be described in
a population of normal and infertile males from couples seeking
fertility treatment. Finally, DNA staining intensity will be
predictive of male fertility treatment outcomes as indicated by
pregnancy outcome following fertility treatment (TI, IUI, or
IVF.+-.ICSI).
[0095] It is estimated that of the 15% of human couples that are
infertile, half of those couples suffer from male infertility and
more than 50% of those men have idiopathic infertility. Male
subfertility or infertility also plays an important part in the
decrease in reproductive efficiency of agricultural species
(cattle, swine, sheep, horses). Predicting male fertility from
semen characteristics is important in both domestic animals and
humans. Diagnosis of male factor infertility is often based on
"abnormal" semen analysis even though it sometimes fails to
accurately predict a man's fertility. Therefore, there has been a
search for other tests to improve the evaluation of infertile
males. Treatment of idiopathic "unexplained" infertility consists
of ovulation induction with timed intercourse (OI/TI) and
intrauterine insemination (IUI), which has pregnancy success rates
of approximately 4-8% per cycle respectively. The pregnancy rate
increases to over 50% with in vitro fertilization (IVF) in patients
younger than 35. Current semen analysis criteria however do not
discriminate between patients who will benefit from IUI and those
that need IVF. This highlights the shortcoming of current fertility
evaluation, in particular SA.
[0096] Fertility in the male is a complex trait that can be
impacted by both compensable and non-compensable components.
Reduced fertility caused by compensable components, such as sperm
numbers, motility, morphology, and ability to undergo capacitation
and acrosome reaction, can be overcome by increasing the number of
sperm inseminated. Non-compensable components include traits
associated with binding of sperm to the oocyte plasma membrane, DNA
integrity, and genetic mutations. These traits are those that
impact the ability of the embryo or zygote to develop after oocyte
activation by the sperm. They pose a dilemma because these traits
are hard to evaluate and there are currently limited solutions
(Assisted Reproductive Technology) for increasing fertility if
these traits are found. A number of tests routinely used for semen
evaluation describe the compensable defects, however they lack the
ability to identify the non-compensable defects that result in
reduced fertility. In cattle, semen from different bulls, which
meet minimum quality requirements, can differ in fertility by
20-40%. The same is true for the clinical value of WHO criteria for
basic semen analysis (concentration, morphology and motility) in
the prediction of fecundity. Tests for non-compensable traits have
been developed and include hamster zona-free ovum test (HZFO),
sperm chromatin structure assay (SCSA) and a myriad of genetic
tests. However, a simple, objective test that could be incorporated
routinely in semen analysis in the clinic, and that is highly
correlated with fertility has yet to be identified.
[0097] Sperm cells are unique in that DNA accounts for 90% of the
total nuclear volume, is highly organized and condensed, and
transcriptionally inactive. Any aberration in the nuclear matrix or
chromatin packaging should result in a change in sperm nuclear
shape, although it would be minute. FHA is a procedure that uses
quantitative binding of fluorochromes (Hoescht 33352) to sperm
nuclear DNA as a method to accurately describe the curvature of the
perimeter of the sperm nucleus with computer aided image analysis.
Changes in the sperm nuclear shape determined with the FHA method
are not visible with the naked eye but are correlated with male
fertility in the species tested (bull, boar). Comparison of FHA to
SCSA shows that although the two tests are related, FHA is able to
describe alteration in chromatin structure within specific regions
of the nucleus that are critical to fertility. While FHA is a
powerful technique for evaluating sperm properties, there is a need
for other evaluation techniques which may complement FHA or which
may be used alone, particularly other techniques which may be more
straightforward to use in practice such as intensity-based
techniques.
[0098] As disclosed herein, it is possible to measure the average
intensity of fluorescence in sperm nuclei from a particular male.
The average fluorescence intensity correlates with bull and boar
fertility as disclosed herein. Bovine embryos produced by in vitro
fertilization (IVF) from bulls of lower fertility divide later to
two cells and have fewer cells at the blastocyst stage. Sperm from
bulls challenged with a heat event either experimentally induced
with testicular insulation for 48 hours, or from summer heat stress
in Wisconsin, have sperm with specific FHA profiles linked to lower
fertility in previous studies and also have IVF results similar to
lower fertility bulls. Analysis of sperm nuclear morphology with
FHA and DNA staining intensity, as disclosed herein, shows that
both correlate with fertility in bulls and boars. Accordingly,
these results will be extended to human samples.
[0099] Human semen samples will be obtained from discarded sperm
from semen analysis, IUI, or IVF.+-.ICSI from men with partners
undergoing fertility investigation (SA), or fertility treatment
(IUI or IVF.+-.ICSI). Samples may be obtained from up to 250 men
(with or without their partners, 250 adult women), 18 years old or
older who are partners in couples diagnosed with infertility. All
male partners of couples will be routinely evaluated with semen
analysis to diagnose male factor infertility. The samples will be
processed per standard laboratory procedures for semen analysis and
treatments with IUI or IVF.+-.ICSI. Sperm samples used for semen
analysis, or IVF.+-.ICSI will be materials that are either donated
(if consent was given by the patient) or discarded per standard
operating procedures. Only after the sperm necessary for the
clinical semen analysis, IUI, or IVF.+-.ICSI are removed will the
sample for research be obtained from the unused liquefied or
processed sample. All donated excess sperm samples will be diluted
1:3 into tubes of fixative (2.9% NaCitrate, 1.0% paraformaldehyde,
3 mg/ml BSA). The fixative will render the sperm non-viable and
preserved for nuclear morphology and DNA staining analysis. No more
than 2.0 ml of semen will be required (usually containing
10-40.times.10.sup.6 sperm). Tubes will be labeled with the IRB
protocol number, identification of contents (3.0 mL 1%
paraformaldehyde), and serial study number. If the patient
initiates a pregnancy, a link between the sample data and the
medical record number will be maintained for 9 months to ascertain
pregnancy outcomes in couples whose partners have consented to the
study.
[0100] While sperm may be collected from up to 250 males, based on
data gathered from sperm of other species as few as 10 samples in
both the normal and lower fertility populations may be required to
determine a statistical difference in FHA. To correlate FHA
criteria in humans and in relation to pregnancy outcome in
different fertility treatment groups however, it may be necessary
to collect data on a larger sample size since no there is no
existing data on FHA in humans and/or on FHA and pregnancy outcomes
in humans. It is presently unclear as to the means and variance in
the population, which may not be the same as in domestic animal
populations, which are somewhat selected for fertility. Thus, in
one embodiment 100 samples will be collected from subjects
undergoing semen analysis and 50 samples from each of the treatment
arms (IUI, IVF, ICSI), for a total of 250 patients.
[0101] Experimental Analysis
[0102] Data will be collected from image analysis of fluorescently
stained sperm dried onto slides or evaluated through flow
cytometery. The perimeter coordinates are converted in Statistical
Analysis Systems (SAS) software to Fourier functions and then
harmonic amplitudes of the functions determined. Discriminate
analysis will be used to determine the best method to separate the
fertile and subfertile/infertile males based on nuclear shape,
nuclear staining, sperm laboratory tests, and measures of TI, IUI,
IVF.+-.ICSI success. Diagnostic statistics will be used to
determine the best discriminate model for predicting the fertility
potential of a particular male.
[0103] Sample Preparation
[0104] Aliquots of the fixed sample will be stained with Hoescht
33352, with excess stain removed by centrifugation and the sperm
pellet resuspended in water. The stained sample will be dried onto
microscope slides, antifade agent used to mount a coverslip, and
sealed with fingernail polish to preserve the sample for imaging.
This procedure has been used successfully for imaging sperm from
multiple species (bull, boar, stallion, dog). The slides will be
imaged with phase contrast and epifluorescent microscopy as
disclosed herein. The images will be evaluated using a specifically
written ImageJ-based program for sperm head morphology (phase
contrast, epifluorescence), sperm head perimeter output (used for
FHA analysis), mean and variation in sperm head fluorescent
staining intensity (epifluorescence), sperm head shape parameters
to include area, length, width, perimeter, aspect ratio, roundness
and solidity, and nuclear texture measures of angular second
moment, contrast, correlation, inverse difference moment, and
entropy following deconvolution analysis. A second aliquot of semen
will be stained with the same procedure but evaluated for
fluorescence intensity with flow cytometry. Remaining samples will
be stored refrigerated in a locked room. Suitable procedures will
be followed throughout to maintain patient confidentiality.
[0105] It is expected that the correlation between increased DNA
staining intensity and decreased fertility that has been seen in
other species (e.g. bull and boar) will also be observed in human
samples. It is also expected that results of DNA staining intensity
will correlate with the results of FHA analysis and that both
analysis methods will be predictive of fertility. It is further
expected that the deconvolution of sperm DNA will produce measures
of DNA distribution within sperm nuclei that are predictive of
fertility. The approaches are also expected to produce methods to
evaluate if sperm are suitable for IUI or ICSI.
Example 4
Sperm Intensity Measurements Using Flow Cytometry
[0106] While other embodiments disclosed herein utilize fluorescent
microscopy of a sperm attached to a substrate such as a glass
slide, in some embodiments sperm DNA intensity staining will be
evaluated using flow cytometry. Most sperm are flat and paddle
shaped, which is the case for several of the species discussed
herein. By attaching paddle-shaped sperm to a slide, sperm heads
tend to be imaged perpendicular to that flat surface. Given this
orientation this is likely the lowest fluorescent intensity
obtainable, while changing sperm orientation relative to the
fluorescent light beam would mean the light would pass a greater
amount of the DNA and would produce increased fluorescence
intensity.
[0107] An alternative technology to measuring fluorescence with a
microscopy system is flow cytometry. However, as sperm pass through
the sheath fluid of a flow cytometry system, the sperm heads are
randomly oriented but with the head pointing forward. Due to this
random orientation of sperm heads there tend to be varying amounts
of fluorescence emitted from the heads, depending on what
proportion of the sperm head DNA is exposed to the light beam.
Nonetheless, using a flow cytometer one can take measurements on
20,000 sperm or more as compared to 100-200 sperm with fluorescence
microscopy and manual image analysis. In some embodiments,
orientation-dependent DNA staining intensity measurements from flow
cytometry may average out if a large enough population of sperm are
measured. To determine the extent to which this is true, one set of
experiments will be directed to measuring and comparing both
microscope-derived and flow cytometer-derived fluorescence
intensity for the same semen samples. In some embodiments, a
sorting flow cytometer will be used which includes an orienting
nozzle for sperm heads so that the intensity measurements are
obtained when each sperm head is in approximately the same
orientation in order to even out variation due to the angle at
which the excitation beam strikes the sperm head. In various
embodiments, sperm samples from a number of different species will
be evaluated using flow cytometry, including bull, boar, human,
dog, stallion, and other species including those listed herein.
[0108] Given the complexity of flow cytometry output (e.g. which
can include graphs of 1- and 2-dimensional data with peaks in
various locations on the graphs), additional studies will be
performed to determine how to interpret the flow cytometry results.
For example, studies will be performed to identify which peaks are
best at predicting whether the sperm is fertile or infertile. In
some embodiments (particularly when sperm heads travel through the
flow cytometer in random orientations), there may be several peaks
of intensity seen on the flow cytometry data graphs; therefore,
additional studies will be performed to determine which peak(s) are
predictive of fertility/infertility (or ratios of peaks, or
threshold peak levels, etc.).
[0109] Flow cytometer experiments were conducted using the same
bull sperm samples as were used to obtain the data listed in Table
2. Straws containing sperm samples (0.25 ml or 0.5 ml) were thawed
at 37.degree. C. for 60 sec and the contents were expelled into 1.5
ml microcentrifuge tubes. The sperm were diluted 1:1 in 2.9% Sodium
Citrate dihydrate solution to a final volume of 500 or 1000 .mu.l.
If the volume was 1000 .mu.l, then 500 .mu.l was placed in a new
tube for staining of sperm. The sperm cells were then stained by
adding 2.5 .mu.l of Hoechst-33342 stain solution and incubated at
37.degree. C. for 30 min. The sample was next split, with half (250
.mu.l) remaining in the stain solution termed unwashed and the
other half (250 .mu.l) processed to remove non-bound stain. To
remove excess non-bound stain, the 250 .mu.l sperm sample was mixed
with 500 .mu.l of the Parrish Fixative solution and centrifuged at
6,000.times.g for 15 sec. The sperm pellet was then mixed with 750
.mu.l of Parrish Fixative solution, centrifuged at 6,000.times.g
for 15 sec, sperm pellet resuspended with 750 .mu.l of Dulbecco's
PBS (No Ca.sup.2+or Mg.sup.2+), centrifuged at 6,000.times.g for 15
sec, and the sperm pellet resuspended with 150 .mu.l of Dulbecco's
PBS. The sample was then transported to a 5 laser BD LSRII flow
cytometer for analysis.
[0110] An aliquot of the sample (75 .mu.l) was mixed with 1425
.mu.l of Dulbecco's PBS and run on the flow cytometer until at
least 20,000 sperm cells were evaluated. The unwashed sample was
simply diluted with Dulbecco's PBS after staining. Removing excess
stain with the washing procedure similar to that done for
microscopy analysis was found to provide better separation of high
and low fertility bull semen samples and so the procedure that was
used to obtain the results that are further discussed. A typical
output is shown in FIG. 7 and demonstrates that 2 peaks are
present. We will define the lower intensity peak as peak 1 and the
higher intensity peak as peak 2. The 2 peaks represent different
orientation of sperm as they are detected by the system. The
distribution of sperm in each peak is also a function of the speed
at which the sperm are traveling through the flow cytometer;
therefore, a standard speed must be used.
[0111] Quantifying the peak intensity of each peak proved difficult
as peaks are not symmetrical and substantial variation in sperm
exist. Thus the mean and median were considered less useful for
analysis. However, the mode of the lower intensity peak, peak 1,
was 96,400.+-.1005 for 51 high fertility bulls and 98,283.+-.873
for 54 lower fertility bulls with a trend for the lower fertility
bulls having a higher mode value, p=0.08. For the higher intensity
peak, peak 2, the mode was similar between the 2 fertility groups.
It is expected that better modeling and processing of the peak
data, including smoothing of the curve for example, will produce
more accurate values for the modes and will result in a better
relationship to fertility. As anticipated, the orientation of sperm
passing through the flow cytometer was a problem. In some
embodiments, this may be resolved by using a flow orienting nozzle
such as that used for sorting x or y bearing sperm on the flow
cytometer.
Example 5
Additional Species
[0112] In various embodiments, a correlation between sperm DNA
staining intensity and male fertility will be evaluated for other
species including dogs and stallions. Preliminary trials have
already found that staining can be evaluated in the same manner in
dogs and stallion semen as has been used for bull and boar semen.
For obtaining final data for dogs and stallions, samples of semen
will be sought from males of each species having at least 25
breedings via artificial insemination for which conception rate
data is available. These are expected to come from commercial semen
banks for dogs and from breeders selling semen from stallions. In
these and other species, breedings will likely be based on lifetime
records as it is unlikely to get any male with sufficient numbers
of breedings in a single year. In the United States, dogs and
stallions do not suffer seasonal infertility as has been described
herein for boars due to restricted breeding seasons (horses) or
housing conditions (dogs).
[0113] It is expected that dogs, stallions, and other species will
show the same correlation between increased DNA staining intensity
and decreased male fertility as seen in bulls and boars.
REFERENCES
[0114] Each of the following references is incorporated herein by
reference in its entirety: [0115] Parrish J J, Ostermeier. Fourier
harmonic analysis of sperm morphology. 1998. 17.sup.th Meeting of
the National Association of Animal Breeders, Columbia Mo. Pp.
25-313. [0116] Parrish J J, Enwall L, Kaya A, Pawshe C, Siddiqui M
A, Shamusuddin M. Sperm shape research: an update. 2006. 21.sup.st
Meeting of the National Association of Animal Breeders, Columbia
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[0127] Various features and advantages of the invention are set
forth in the following claims.
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