Biomarker-Based Nanotechnology for the Improvement

of Reproductive Performance in Beef and Dairy Cattle

Nagina Gilani

10-arid-1768

Ph.D Zoology

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Contents• 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

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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  

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

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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

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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 freeze-

thawing of the cryopreserved AI semen.

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• 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.

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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 with inferior fertilizing

potential, and/or by eliminating defective

spermatozoa from collected semen).

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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.

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• 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 importance to farm animal

biotechnology.

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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

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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.

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- 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.

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Biomarker-based Flow CytometricSemen 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);

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- 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).

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• 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

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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

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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

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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 photo-

detectors,

– the data are routed to a computer program.

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• 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.

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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

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• 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.

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Continue…• A magnet is used to concentrate defective, nanoparticle-

coated 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.

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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).

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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).

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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.

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• 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 anti-

ubiquitin antibodies to the sperm surface causes

increase in fluorescence reflecting the occurrence of

sperm abnormalities .

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• 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 .

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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).

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Nanoparticle-basedFertility Test

• Heparin-binding proteins (HBP) in bull spermatozoa

and seminal plasma (HBP-30) is known as fertility-

associated 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

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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.

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• 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 anti-

immunoglobulin G (IgG) antibodies at far end of strip

(positive control).

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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.

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Improving reproductive performance in cattle

• Consequently, magnetic separation methods have been

explored by andrologists.

• Based on surface ubiquitination of defective

spermatozoa in bull epididymis, 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.

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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).

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Discarded pellet of defective spermatozoa, sperm fragments, and nanoparticles (B).

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Morphologically normal spermatozoa in supernatant collected after nanopurification (C).

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• 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.

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• 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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• 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 with PNA coated

nanoparticles was used

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• In both trials, conception rates achieved with a half

dose of PNA-particle purified spermatozoa were

significantly higher than that of a non-purified half-

dose.

• 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

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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.

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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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• 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).

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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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