Biomarker-Based Nanotechnology
for the Improvement
of Reproductive Performance in
Beef and Dairy Cattle
Nagina Gilani
10-arid-1768
Ph.D Zoology
Contents
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Reproductive performance
Factors affecting reproductive performance
Artificial insemination (AI)
Types of sperm defects
Gain in reproductive performance
Semen evaluation by light microscopy
Objectives
Nanotechnology
Biomarker-based Flow Cytometric Semen Analysis
Flow cytometry, advantages
Plant lectins and Ubiquitin in flow cytometry
Semen nanopurification
Conclusion and future aspects
2
Reproductive performance
• Productivity
of
the
animal
or
herd
in terms of offspring produced
• Measured in terms of,
– Pregnancy rate
(PR = CR x HDR)
– Calving Interval
– Days Open
3
Factors affecting reproductive
performance
• Reproductive performance is affected by,
– season of calving,
– maturity,
– reproductive disorders.
– timing of mating on the resultant sex ratio
– breeding Method
4
Artificial insemination (AI)
• Artificial insemination (AI) is a technique for the
deliberate introduction of sperm into a female's uterus
or cervix for the purpose of achieving a pregnancy .
• This technique has great potential to improve livestock
reproductive efficiency.
• It has been used since last six decades for breeding
cattle and buffaloes. However it may cause various
health and productivity problems
5
Types of sperm defects
• Sperm defects found in semen of bulls used for AI are:
– compensable defects such as sperm tail defects and
cytoplasmic droplets
– non-compensable
defects
including
nuclear
craters/diadems and knobbed acrosomes.
Acrosome can be damaged or prematurely activated
(premature ‘‘acrosome reaction’’) during freezethawing of the cryopreserved AI semen.
6
Continue…
• Compensable defects on AI outcome can be
decreased by increasing total number of spermatozoa
per AI dose.
• But adding more spermatozoa per dose does not
improve conception rates in bulls with high
percentage of spermatozoa with non compensable
defects.
7
Gain in reproductive performance
• Reproductive performance of bulls can be improved
at the level of
– individual sires (by eliminating bulls with inferior
fertility)
– individual semen collection (by discarding semen
collections/ejaculates
potential,
and/or
with
by
inferior
fertilizing
eliminating
defective
spermatozoa from collected semen).
8
Semen evaluation by light
microscopy
• Semen evaluation via traditional light microscopy
– determines sperm concentration per mL of semen,
– percentage of spermatozoa with visible structural
defects (sperm appearance/ morphology, presence of
abnormal and immature sperm forms),
– sperm motility (velocity of movement),
– presence of semen contaminants such as sperm
fragments, white blood cells, and bacteria.
9
Continue…
• This analysis provides useful information about the
semen sample, yet new methods for estimating future
fertility of a sire are still sought.
• Furthermore,
not
all
sperm
abnormalities
are
detectable with light microscopy.
• So, in depth analysis carried out quickly and with
repeatable precision on a large number of spermatozoa
is
of
paramount
biotechnology.
importance
to
farm
animal
10
Objectives
• Cost-efficient ways of improving semen analysis and
increasing the fertilizing potential of AI doses, with
particular
focus
on
nanotechnology-based
approaches
• Development of nanoparticle-based lateral flow
devices for fertility testing in male livestock animals
11
Nanotechnology
• Nanotechnology is conducted at nanoscale (up to 100
nanometers). Its applications in reproductive biology
are,
- Develop
new
methods
to
remove
defective
spermatozoa from semen collected from sires with
high genetic value.
12
Continue…
- Flow cytometric evaluation using biomarkers to
detect specific spermatozoan characteristics is
growing
in
popularity
in
both
andrology
laboratories and agricultural studs.
- Semen nano purification may be useful to
explore whether nano purification would increase
conception rates of bulls in the non-compensable
category.
13
Biomarker-based Flow Cytometric
Semen Analysis
• Fluorescently labeled biomarker probes are used to
image particular targets or pathways
• Candidate sperm quality/fertility biomarkers include
proteins acting as ‘‘negative’’ fertility biomarkers and
‘‘positive’’ biomarkers of sperm quality/fertility.
• Assess many properties of spermatozoa like:
– packaging of DNA in sperm head (chromatin
integrity);
14
Continue…
- presence and integrity of the sperm head cap for
sperm’s ability to penetrate the egg coat during
fertilization (sperm acrosomal status);
- ability of spermatozoa to produce and regenerate
energy for movement (mitochondrial membrane
potential);
- sperm cell viability (percentage of live/dead
spermatozoa in collected semen).
15
Continue…
• In addition to semen analysis, biomarkers present on
the sperm surface, such as sperm protein ubiquitin
and binding partners (ligands) of several plant
lectins, are potential targets for nanoparticle-based
semen purification, which will be discussed later
16
Flow cytometry
• Flow cytometry is a method in which fluorescently
labeled cells (spermatozoa) travel individually at
high speed (hundreds or thousands of spermatozoa
per second) through the flow cell of a flow
cytometer, where they are illuminated by one or
more lasers.
• Sperm acrosomal status can be probed by flow
cytometry using fluorescently labeled lectins
17
Advantages
• Flow cytometry is fast, accurate, highly repeatable, and
can analyze more spermatozoa per sample (e.g., 10,000
cells in few seconds in single sample). It measures,
– sperm viability,
– mitochondrial function,
– chromatin structure,
– and acrosomal status
18
Process
Flow of fluorescently labelled spermatozoa causes
– Light scattering and fluorescence excitation of
biomarker-recognizing fluorescent probes
– Biomarkers are bound to a specific site on the
spermatozoa;
– the signals are detected and quantitated by photodetectors,
– the data are routed to a computer program.
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Continue…
• The program presents the information in form of
relative fluorescent intensity units, which are typically
displayed as either scatter plots or histograms.
• Analysis of scatter plots/histograms allows specific
sperm populations to be gated off, giving information
regarding
– fluorescence intensity in spermatozoa,
– percentage
of
sperm
population
with
certain
fluorescence characteristics within a total sample.
20
Plant lectins in flow cytometry
• Proteins that recognize and bind glucosidic residues in
different parts of acrosomal membrane of sperm
• Plant lectins coupled to fluorescent dyes are ued for
flow cytometric analysis and to metallic nanoparticles
for semen purification.
– Pisum sativum agglutinin (PSA) derived from the
pea plant,
– Arachis hypogaea agglutinin (PNA, or peanut
agglutinin) derived from the peanut plant
21
Continue…
• In a compromised acrosome, lectin ligands become
exposed on sperm head surface and are available for
lectin
binding
making
lectins
suitable
for
nanodepletion-based semen purification.
• Metallic nanoparticles coated with a lectin will
readily bind to the surface of spermatozoa with
damaged acrosomes upon nanoparticle mixing with
semen.
22
Continue…
• A magnet is used to concentrate defective, nanoparticlecoated sperms on bottom of test tube used for
nanopurification.
• Normal spermatozoa are skimmed off, while the
defective spermatozoa are held on the bottom
• Spermatozoa with reacted, damaged, or abnormally
formed acrosomes acquire green fluorescence after
labeling with PNA lectin and spermatozoa with intact,
normal acrosomes have no fluorescence.
23
Fig. 1. Patterns of biomarker labeling in normal and
defective bull spermatozoa. Lectin PNA (green) binds
to the caps of the sperm heads in spermatozoa with
compromised acrosomes (arrow), but shows no
labeling of intact spermatozoa (A).
24
Lectin LCA (green) binds to the acrosomal caps of
normal spermatozoa and intensely labels the whole
sperm head and tail of the defective spermatozoa
(arrow; split tail) (B).
25
Ubiquitin in flow cytometry
• Ubiquitin is present on the surface of defective
spermatozoa
and
can
be
easily
targeted
by
nanoparticles with specific affinity to it (coated with
ubiquitin-binding
antibodies
or
with
genetically
engineered proteins that interact with ubiquitin).
• It recognizes not only spermatozoa with damaged
acrosomes, but also with other types of head and tail
defects, including cells with compromised DNA.
26
Continue…
• During sperm maturation in the epididymis (a sperm
storage gland) attached to the testicular surface
abnormal spermatozoa are tagged on their surfaces by
ubiquitin through process of protein ubiquitination.
• Increased binding of fluorescently labeled antiubiquitin antibodies to the sperm surface causes
increase in fluorescence reflecting the occurrence of
sperm abnormalities .
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Continue…
• It has also been used for validation of other biomarkers
of sperm quality, including platelet-activating factor
receptor (PAFR), and more recently, post-acrosomal,
WW domain binding protein (PAWP)
• Defective spermatozoa displays various anomalies of
PAWP labeling and often show the presence of
ubiquitin on their surface .
28
Dual immuno fluorescence labeling of sperm proteins
PAWP (red) and ubiquitin (green) in bull spermatozoa
(C). A spermatozoon displaying green ubiquitin labeling
has an abnormal sperm tail and lacks PAWP on its
head. Normal spermatozoa display a red band of PAWP
(a biomarker reflective of normal sperm head structure)
on their heads and lack green labeling of ubiquitin (a
biomarker associated with sperm head and tail defects). 29
Nanoparticle-based
Fertility Test
• Heparin-binding proteins (HBP) in bull spermatozoa
and seminal plasma (HBP-30) is known as fertilityassociated antigen (FAA) in the AI industry
• Groups of FAA-positive bulls were consistently more
fertile (by 9–40%) than groups of FAA negative bulls.
• AI trials on beef cows inseminated using semen from
FAA positive or FAA negative bulls established FAA as
a biomarker of sperm quality
30
Continue…
• The ReproTest is a lateral flow device based on the
colloidal gold nanoparticle design, has been developed
and marketed.
• In this device, a semen sample is loaded into a sample
well and flows through a series of pads soaked with
specific antibodies.
• The first pad releases primary antibodies conjugated to
nanogold particles that bind specifically to the target
protein, in this case FAA.
31
Continue…
• Nanogold-tagged sample solution flows toward a test
line region where secondary antibody (recognizes a
different region of the target protein) is immobilized on
several pads in a concentration gradient-like fashion.
• Nanogold tagged target protein molecules are captured
on these lines, turn red indicating a FAA positive test.
• Extra primary antibodies are captured by antiimmunoglobulin G (IgG) antibodies at far end of strip
(positive control).
32
33
Semen Nanopurification
• Swim-up
technique
is
mostly
used
for
semen
purification, but is too lengthy and too low volume can
be used for bulk processing of bull semen in an AI stud.
• A gradient separation method involves centrifugation at
high speed that retards defective spermatozoa but
allows normal, motile spermatozoa but this method of
sperm separation is time, reagent, and equipment
intensive, and therefore not suitable for bulk processing
of semen for AI.
34
Improving reproductive performance in
cattle
• Consequently, magnetic separation methods have been
explored by andrologists.
• Based
on
spermatozoa
surface
in
bull
ubiquitination
epididymis,
of
defective
method
for
nanodepletion of defective spermatozoa during semen
preparation for AI doses is developed.
• Nanoparticles composed of magnetite and mixed iron
oxides were prepared using a method for conjugation of
antibodies and lectins.
35
Fig. 2. Nanopurification of bull semen for
artificial insemination. A magnet is used to
attract
the
nanoparticle-coated
defective
spermatozoa to the bottom of the collection tube
(A).
36
Discarded pellet of defective spermatozoa,
sperm fragments, and nanoparticles (B).
37
Morphologically normal spermatozoa in
supernatant collected after nanopurification (C).
38
Continue…
• Different batches of particles were coated with
commercially available anti-ubiquitin antibodies or
lectin PNA.
– The ubiquitin-binding particles were designed to
bind to ubiquitin protein, found exclusively on the
surface of defective bull spermatozoa.
– Particles coated with lectin PNA bind to glycans
exposed by the damage to or premature remodeling
of the sperm head acrosome.
39
Continue…
• Preliminary laboratory tests were conducted to assess
the effect of nanopurification on sperm viability and
semen content of defective spermatozoa and to
optimize the nanopurification protocol (Fig. 2).
• Based on these tests, IVF trails were conducted,
followed by two field insemination trials.
• Data from both trials showed improvement in
conception rates after insemination for some treatments
and animal groups.
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Continue…
• In the first trial, a total of 499 cows and heifers were
inseminated by semen from three bulls.
• In second trial, 422 cows inseminated by same bulls.
• In both cases, a full dose of 20 million non-purified
spermatozoa, a half dose (10 million) of non-purified
spermatozoa, a half dose of spermatozoa nanodepleted
with ubiquitin-binding nanoparticles, or a half dose of
spermatozoa
nanopurified
nanoparticles was used
with
PNA
coated
41
Continue…
• In both trials, conception rates achieved with a half
dose of PNA-particle purified spermatozoa were
significantly higher than that of a non-purified halfdose.
• Differences in conception rates with nanopurified
semen suggests that heifers and some sires may benefit
from nanopurification more than others.
• Importantly, no adverse effects on inseminated animals
were observed
42
Conclusions
• Field trials with flow cytometry by PNA and ubiquitin
nanoparticles were preceded by extensive laboratory
research
• Depending on the observed pregnancy rates it is
possible that nanopurification treatments could be
tailored specifically to boost fertility in replacement
females entering the breeding programs.
• No side effects related to residual nano-particles in
nano-purified semen have been observed thus far.
43
Future aspects
• The main focus of the ongoing and future AI trials will be
on optimization of nanoparticle doses and nano
purification protocols, with the goal of maximizing the
number of AI doses per semen collection from sires with
high genetic value.
• Research will be pursued to identify additional nano
purification targets, the negative fertility biomarkers
expressed on surface of defective sperm cells.
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Continue…
• These results will encourage further exploration of
nanodepletion protocols for use by the AI industry.
• Further testing will also be needed to assure the safety
of nanodepleted semen; residual nanoparticles that
could remain in semen after nanodepletion could enter
female body through the reproductive system and have
adverse effects on reproduction and other bodily
functions (nanoreprotoxicity).
45
Reference
• Biomarker-Based Nanotechnology for the
Improvement of Reproductive Performance in
Beef and Dairy Cattle
Peter Sutovsky1,2 and Chelsey E. Kennedy1
1Division of Animal Sciences and 2Department of
Obstetrics, Gynecology and Women’s Health,
University of Missouri-Columbia, Columbia, MO,
VOL. 9 NO. 1 FEBRUARY 2013 INDUSTRIAL
BIOTECHNOLOGY.
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