Biomarker-Based Nanotechnology for the Improvement of Reproductive Performance in Beef and Dairy Cattle Nagina Gilani 10-arid-1768 Ph.D Zoology 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 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. 19 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 . 27 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. 40 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. 44 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. 46 47 48