The applicabiliTy of nanobioTechnology-relaTed approaches To veTerinary medicine and assisTed animal reproducTion – a review*

The development and optimization of nanobiotechnology has recently contributed to the elaboration of a wide spectrum of nanoparticle-based strategies that are reliable and feasible for a broad panel of practical applications in different disciplines of biological, agricultural, nutritional, biopharmaceutical

The development of manufacturing methods and infrastructure for the physicochemical characterization of materials, observed for several decades, has contributed to the flourishing of science that deals with the functioning of materials at the nanoscale level.The phenomenon of nanotechnology results from obtaining new unique properties such as certain physical, chemical, optical, and electrical properties that differ at the nanoscale level when compared to the properties of materials at a macro scale.According to the definition developed and recommended by the European Commission, nanomaterials are materials that display a size ranging from 1 to 100 nm in at least one dimension or, in terms of a particle size distribution, at least 50% of the particles are in the nanometric scale (2011/696/EU OJ L 275, 20.10.2011, pp. 38-40).The division of nanoparticles, taking into account the material from which they are synthesized, includes the following types: magnetic, metallic, carbon, polymeric, and lipid nanoparticles.There are a variety of nanomaterial features that have contributed to the development of nanotechnology, ones which deal with the production and use of nanoparticles in specific applications.The size of nanoparticles is similar to the size of biomolecules, which allows them to be used in biomedical applications (Zhang et al., 2008).Moreover, relatively easy methods of nanoparticle synthesis and their specific features, such as a large specific surface area and the possibility of chemical surface modification, enable the design of nanoparticles with a strictly defined size and with certain biological properties (Ye and Loh., 2013;Loh et al., 2016;Dhand et al., 2015).
In human biomedicine, nanoparticles are applied for many different purposes.A great deal of work has been devoted to the application of nanoparticles as carriers of biologically active molecules, such as anticancer drugs, antibiotics, vitamins, antibodies, etc. (Mrówczyński et _________ *The present study was financially supported by the research grants Nos.04-19-11-21 and 04-19-05-00 from the National Research Institute of Animal Production in Balice near Kraków, Poland to Marcin Samiec and Monika Trzcińska. al., 2016;Ivashchenko et al., 2017 a;Woźniak et al., 2017 a;Pant et al., 2020).Due to the chemical possibility of attaching not only drugs to the surface, but also -for example -peptides, it is possible to give such a nanosystem the character of targeted therapy.This is extremely important because the molecules of the anticancer drug will be delivered only to the neoplastic cell, which, on its surface, shows an overexpression of its receptor, thus serving as the target site of the ligand from the nanoparticle surface.In addition, there is a possibility of the biofunctionalization of nanoparticles, e.g., a fluorescent marker or a contrast agent being used in nuclear magnetic resonance imaging, thus obtaining nanosystems with many biological characteristics (Babayevska et al., 2023).Such multimodal nanoparticles combine therapy and diagnostics, which has led to the creation of a new definition of theranostic nanoparticles (Funkhouser, 2002;Kelkar and Reineke, 2011).Therefore, the approach that uses modern nanocarriers in therapy exceeds the treatment possibilities of classic therapy due to greater specificity, improved quality of treatment, increased half-life in the blood, triggered release (pH, temperature, and magnetic field), and the possibility of reducing the therapeutic dose, the side effects of therapy, and its costs (Lale et al., 2014;Deptuła et al., 2015;Wu et al., 2021).
Nanoparticles are also successfully used as carriers of nucleic acids for genetic modification, gene therapy, and RNA interference (Grześkowiak et al., 2015;Chien et al., 2022;Chen et al., 2019).In turn, in regenerative medicine, biofunctionalized nanoparticles are used to create scaffolds and systems with a trabecular structure, facilitating -for example -the process of osseointegration or, as biocompatible implant coatings, giving them an antibacterial and/or anti-inflammatory character (Wang et al., 2019).An interesting group of nanoparticles is the group of metallic nanoparticles (e.g., gold, silver, copper), which themselves represent antibacterial properties (Sánchez-López et al., 2020).
In view of such a wide range of applications and implementations in human medicine, it is only a matter of time before the use of nanotechnology in veterinary medicine and reproductive biotechnology takes place.The specificity of the use of nanotechnology in the abovementioned research fields encompasses the following applications: therapy and diagnostics, biosensors, and animal health and nutrition.In recent years, issues regarding animal reproduction have developed, which is an extremely important topic due to the key role of livestock breeding.The breeding process of farm animals determines the efficiency of milk and meat production, and it directly affects management decisions and maintenance costs (El-Sayed and Kamel, 2020).
For all the above-indicated reasons, the current paper seeks to unravel not only the state of the art, but also future targets and directions related to the feasibility and reliability of a broad spectrum of nano-applications for veterinary medicine and assisted reproductive technologies, as well as their combinations with animal production.

The importance of nanobiotechnology for diagnostics and preventive/therapeutic treatments against microbes responsible for the etiopathogenesis of reproductive-related disorders
Reproductive-related diseases are mainly caused by different bacterial species (i.e., Escherichia coli, Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Prevotella melaninogenica, Arcanobacterium pyogenes, and Toxoplasma gondii).The consequences of mastitis and pregnancy-associated, or postpartum, diseases can lead to a significant decrease in milk quality, decrease in sperm quality, decrease in ovarian and uterine function, disturbances in embryonic development, and a drastic increase in treatment costs.If herds are diagnosed with reproductive disorders (abortions, stillbirth, reduced conception rate, the need for multiple mating, extended calving intervals), diagnostics and effective treatment should be implemented (Gurunathan et al., 2018;Vallejo-Timaran et al., 2020;Szweda et al., 2013).
Analytical techniques usually include the measurement of somatic cell counts (SCC), the detection of pathogens, and the determination of inflammatory statuses.On the other hand, diagnostics tests detecting pathogens are dedicated to the detection of antibodies (serological), antigens, or live viruses (viral), as well as tests detecting the genetic material of the pathogen from the biological material derived from animals.These tests have many advantages (e.g., universality), but they also have many limitations (e.g., they are time and cost consuming, and they require the use of advanced equipment).
The modern diagnostic approach focuses on the use of devices that are characterized by high detection sensitivity, repeatability of results, and low cost, as well as rapid and universal availability.Nanomaterials with unique electronic, optical, mechanical, and thermal properties have been recognized as some of the most promising materials for next-generation biosensors.Reactions take place on the surface of nanoparticles by attaching a label-free target antigen, peptide, or aptamer change in the physical properties of nanoparticles (e.g., optical).This phenomenon is used in biosensors based on nanoparticles (mainly metallic NPs: Au, Ag, and Fe 3 O 4 ), which allows the reading of changes after the analyte is attached by using the measurements of localized surface plasmon resonance spectra, scattered light, absorption, spectrophotometry, color change that is visible to the eye, magnetic separation, magnetoresistance, or chemiluminescence (Howes et al., 2014;Duarte et al., 2017;Viveiros et al., 2020;Nirala et al., 2020).Biosensors based on nanoparticles can generate qualitative and quantitative results, and the spectrum of their use is wide.These include microarray or strip tests that are soaked with nanoparticles, which are biofunctionalized with an antibody or aptamers, and the presence of which analyte (e.g., antigens, exotoxins, amplicons, DNA, and RNA) in the test sample determines its specific attachment and color change (Piriya et al., 2017;Park et al., 2003;Le et al., 2014;Mujawar et al., 2013).The aforementioned use of nano-and micro-array techniques, as well as miniaturization, allows for the creation of systems in which it is possible to prepare a sample for analysis, to detect it, and to analyze the result (Freitas, 2002).This is the idea of a "Lab on a chip", which can be performed on a farm for the purpose of rapid diagnostics (less than 3 h, 1 pg/ mL of analyte) (Mujawar et al., 2013).
Taking advantage of the functionality, accuracy, realtime reading, and miniaturization of biosensors, portable concepts of nanoelectromechanical systems (NEMS) detecting DNA, proteins, viruses, and bacteria have thus been developed.These systems connect analyte-binding with the mechanical motions of devices in nanometer scales, and are then converted into detectable electrical or optical signals (Yeri and Gao, 2011).To summarize, modern biosensors based on nanotechnology enable the detection of analytes at an incredibly low concentration, with high accuracy and with a repeatability of results in a short period of time.
Examples of the use of nanotechnology in the diagnostics of pathogens, specifically those involved in reproductive-related infection diseases in farm animals, are presented in Table 1.
The treatments with classic antibiotic therapy against the microbes responsible for the etiopathogenesis of reproductive-related disorders often does not provide effective results, which forces increased doses of the drug.Unfortunately, this approach may lead to drug resistance, as well as the negative effects that arise from the potential risk of antibiotic residues transferring onto meat and milk, which releases antibiotics into the environment.Recently, a great deal of attention has been paid to multidrug resistance (MDR).A common cause of this phenomenon is the incorrect use of antibiotics, which can be as per the following: administering too high or too low a dose, irregular drug intake, the unreasonable prescription of antibiotics, the overuse of broad-spectrum antibiotics, too short or too long of a duration of antibiotic therapy, and antibiotic therapy that is conducted inconsistently with the antibiogram.The many effects of drug resistance to antibiotics include longer disease durations, increased risk in the transmission of resistant microorganisms to other animals in the herd, increased risk of death, potential for more adverse events, and increased treatment costs.To counteract these effects, it is therefore necessary to monitor infections and to introduce targeted therapy.Due to the unique and excellent traits of nanoparticles as drug carriers, many different material types have been reported as being effectively biofunctionalized with antibiotics (Ivashchenko et al., 2017 b;Hasan et al., 2019;Kalhapure et al., 2014).Moreover, certain types of metallic nanoparticles (e.g., silver, gold, copper) can be strongly bound by the cell membrane of both Grampositive and Gram-negative bacteria.The antibacterial effect of these nanoparticles consists, among others, in preventing the migration of bacteria, mitigating the for-mation of new cell wall layers, intensifying/ameliorating cell wall degradation, changing the permeability of the cell membrane, inducing oxidative stress, deactivating proteolytic enzymes, and inducing apoptosis (Banach et al., 2007;Kędziora et al., 2016;Szymczak, 2018).What is more, this action does not only apply to single microbial cells, but also the entire bacterial biofilm system.Due to urgent multidrug resistance problems, the non-antibiotic approach to infectious disease treatment represents a great challenge (Li et al., 2022).To date, many examples of the use of these modern therapies in farm animals have been described, examples of which are included in Table 2.

The importance of nanobiotechnology for hormonally assisted reproduction in livestock species
Reproductive management in livestock improves herd fertility.Standard assisted reproductive technologies (ARTs) include, among others, hormonal synchronization, estrus synchronization/induction, superovulation, and artificial insemination.The most commonly used hormones in animals for the improvement of reproductive performance, cycle management, and the treatment of certain reproductive diseases are progesterone, estradiol, gonadotropin, prostaglandin, testosterone, and melatonin (Hashem et al., 2015).The action of these substances in the body depends on many kinetic parameters; hence, their formulation into delivery nanocarriers (organic -chitosan and inorganic -metal oxides) can contribute to increasing stability and biological activity, as well as their bioavailability.This generates more efficient absorption and protects them from rapid degradation, thus reducing the number of used doses (Abdelnour et al., 2021;Hassanein et al., 2021).The added value of such an approach is also decreasing the risk of the presence of hormonal residues in milk and meat, as well as the release of hormonal wastes to the environment (Hashem and Gonzalez-Bulnes, 2020).
To achieve success in the implementation of ARTs such as in the case of artificial insemination, attention should be paid to the condition of the semen.The process of purification, storing, freezing, and then thawing the sperm of animals is crucial to maintain their viability; hence, the methods of their preservation must be effective.Numerous studies conducted in this area show that the use of metallic nanoparticles as additives to cryoprotectants can improve the fertility rate.This type of nanoparticles exhibits the activity of antioxidant enzyme cofactors, which directly affects the reduction in apoptosis, as well as the peroxidation of proteins, fatty acids, and nucleic acids, and thus improves the integrity of the plasma membrane and the survival rate of spermatozoa (Khalil et al., 2019;Abdelnour et al., 2021;Heidari et al., 2019).The sperm purification process can be enhanced using biocompatible magnetic nanoparticles and the magnetic separation process (Farini et al., 2016).MNPs in a magnetic field biosensor generated a fringe field when exposed to an external magnetic field, promoting a proportional change in the sensor's electrical resistance, which was detected by the portable electronic reader.

Viveiros et al., 2020
Surface active maghemite nanoparticles (SAMNs) Proteome modifications, biomarkers for cow mastitis SAMNs showed a high selectivity toward protein corona content.Thus it can be quantified by gel electrophoresis and mass spectrometry.

Bovine haptoglobin
The hemoglobin-modified plate complexed with haptoglobin from the sample.The resulting complex inhibited the catalytic activity of hemoglobin within the luminol system and reduced the chemiluminescence signal.The addition of cross-linked GNPs onto the luminol mixture intensified the radiation read with a microplate reader.

Nirala and Shtenberg, 2019
Antibodies and magnetic nanoparticles, and a lab-on-a-chip magnetoresistive cytometer with microfluidic handling

Staphylococcus aureus
The antigens in the sample bound to S. aureus antibodies decorated magnetic nanoparticles; thus, they can be analyzed using a lab-on-a-chip magnetoresistive cytometer.

Duarte et al., 2017
Table 2.The implementation of nanotechnology to a broad range of treatments aimed at the reproductive-related infection diseases in mammals

Type of nanoparticle biofunctionalization
Antimicrobial activity

References
Silver nanoparticles, quercetin nanoparticles (QA NPs) The multidrug-resistant Escherichia coli strain, which was isolated from a dairy cow with mastitis QA NPs inhibited the growth of E. coli; it can disintegrate the structure of biofilm via its regulatory role in biofilm-associated gene expressions (no detailed path of action was given).

Ranjani et al., 2022
Gold nanoparticles biosynthesized with plant extracts (Artemisia herba-alba and Morus alba) Escherichia coli and Salmonella spp.samples were isolated from cattle, buffaloes, sheep, and goats suffering from respiratory signs, diarrhea, and mastitis AuNPs showed an inhibitory effect due to entering the cytoplasm of the thin wall Gram-negative bacteria; this effect may be also improved by the presence of leaf extracts, which contain flavonoids, tannins, and polyphenolic compounds.

Pseudomonas aeruginosa virulence in mice
The tested MET, MET-NEs, Ag-MET-NEs, and AgNPs significantly decreased the expression of the quorumsensing regulatory genes of P. aeruginosa, which thus inhibited the virulence factors of these bacteria.

Gomaa et al., 2022
Silver nanoparticles Multiple drug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa from mastitis-infected goats The activity of AgNPs was due to the generation of reactive oxygen species, malondialdehyde, and leakage of proteins and sugars in bacterial cells.Yuan et al., 2017 Nanovaccines: TgP2-pVAX1/PLGA nanospheres TgP2-pVAX1/CS nanospheres

Immunization in mice
The DNA vaccine encoding Toxoplasma gondii ribosomal P2 protein was delivered by nanomaterials (poly-lactic-co-glycolic acid (PLGA) and chitosan) that stimulated an immune response in vivo (analyzed parameters: antibody, cytokine, dendritic cell maturation, splenocyte proliferation, and T lymphocyte proportion).

Yu et al., 2022
Table 3.The implementation of nanotechnology to a wide array of treatments focused on animal reproductive-related issues

References
Nano-bromocriptine Egg production and prolactin expression in brown laying hens Nanoformulated bromocriptine (dopamine agonist) significantly reduced prolactin gene expression in treated birds when compared to the low bioavailability of bromocriptine.

Bovine semen purification
The synthetic biotin-labeled aptamers that recognized the membrane-damaged cells were used to complex with the avidin-coated (SPION) materials for enhanced separation and semen purification.

Farini et al., 2016
Cesium oxide nanoparticles (CeO 2 NPs) Ram semen storage processes The exposure of ram spermatozoa to increasing doses of CeO 2 NPs had a beneficial effect on the kinematic and morphologic parameters of sperm cells.
The authors showed that these effects were not connected with the antioxidant activity of CeO 2 NPs, and thus need further analyses.

Supplementation of bull semen extender
The semen samples supplemented with ZnNPs were frozen and thawed, and then the sperm motility and motion parameters were assessed.In the analyzed groups, Zn NPs showed cell-protective actions due to improving the plasma membrane functionality.

Jahanbin et al., 2015
Copper nanoparticles (CuO-NPs), zinc nanoparticles (ZnO-NPs) The addition of nanominerals to the culture media of bovine oocytes and embryos (in vitro maturation, in vitro fertilization, and embryo culture media) CuO-NPs or ZnO-NPs-treated bovine oocytes during IVM showed a low level of DNA fragmentation and the increased intracellular glutathione content of cumulus cells due to antioxidant action of these mineral supplements.

2018
Magnetic nanoparticles (MNP) The nanoselection of apoptotic boar spermatozoa Annexin V-MNP or Lectin-MNP were incubated with boar semen samples.
Positive complexes were then selected from healthy sperm cells using magnetic fields.

Durfey et al., 2019
Chitosan nanoparticle with human chorionic gonadotrophin (CS-NPh) Increasing the induction of dairy cattle ovulation The nasal spray administration of CS-NPh resulted in enhancing the induction of ovulation in dairy cattle, which indicated the use of nanodelivery systems to overcome the rapid degradation of hormones.

Pamungkas et al., 2016
Gonadotropin-releasing hormone-loaded chitosan nanoparticles (GnRH-ChNPs) The induction of ovulation in rabbits GnRH-ChNP administration resulted in a reduction in the conventional intramuscular GnRH dose to half, without compromising the fertility.It was added to semen extenders, and did not show a negative effect on fertility.

Hassanein et al., 2021
Gonadorelin chitosan-sodium tripolyphosphate nanoparticles A reduction in the conventional dose, increasing the ovulation rate and the number of goat embryos The nanoformulation of GnRH allowed the researches to reduce (75%) the conventional dose of gonadorelin without affecting fertility due to the increased bioavailability of GnRH in the nanodelivery system.
Hashem and Sallam, 2020 Nanofabricated hormones gonadorelin cloprostenol An improvement on the estrous synchronization outcomes of goats, thus enabling lower hormone dose administration Nanofabricated hormones improved the estrus synchronization outcomes of goats while enabling a lower (half) hormone dose administration.Hashem et al., 2022 In the reproduction process, attention should be paid to the correct course of pregnancy and efficient lactation.One of the most important problems connected with these issues is the quality of nutrition and dietary supplementation.The nanoformulation of nutrients and supplements favors nourishment due to the increased ability of the nanoformulation materials in the protection, absorption, and targeted delivery of bioactive compounds.The main groups of nanocarriers for the feeds described in the literature include polymeric nanoparticles (i.e., chitosan), nanoemulsions, and nanofibers, as well as nanohydrogels (Siddiqui et al., 2022;Konvicna et al., 2015).The implementation of nanotechnology to the treatment modalities targeted at reproductive-related issues in a variety of livestock species is shown in Table 3.

The approaches applied to nanoparticle-mediated nucleic acid delivery provide the powerful and hopeful tools for the transgenization of nuclear donor cells and their subsequent use for somatic cell cloningbased arTs
Genetic engineering-mediated strategies serve as an effective tool for modern animal biotechnology.By taking into account thoroughly planned reproductive procedures, measurable effects can be produced in terms of improving the performance characteristics of animals or in implementing gene therapy.
Nanotechnology-based assisted animal reproduction mainly concerns genetic modification and nucleic acid delivery.The literature data show that non-viral, nanodelivery systems are more efficient when compared to the classical methods (i.e., lipoplexes) (Mykhaylyk et al., 2007).Nanoparticle complexation with nucleic acids refers to a plethora of molecular and physicochemical conditions, i.e., nanoparticle size and shape, surface charge, type of nanoparticles, concentration of the nanocomplex, nanoparticle nucleic acid ratio, time of incubation, and type of cell lines (Mitchell et al., 2021;Behzadi et al., 2017;Faria et al., 2018;Conte et al., 2021;Grześkowiak et al., 2015Grześkowiak et al., , 2016)).Most of these factors determine the efficacy of cellular uptake and internalization (nanofection process), as well as the molecular activity in the cell and nucleus.Most of the literature findings suggest that the size of nanoparticles for biomedical application should not extend more than 100 nm, but this generally depends on the cellular internalization process, which is frequently based on endocytosis.The same reason determines the shape of the NPs (Hoshyar et al., 2016;Woźniak et al., 2017 b).The properties of surface chemistry -in terms of surface charge -electrochemical zeta potential, as well as hydrophilicity determine nanoparticle behavior in biological fluids and should be synthetized in the formulation, thus preventing the NPs' aggregation process (Jin et al., 2011).As such, designing the nanoparticles for nucleic acids delivery should address cationic nanomaterials (due to the electrostatic interaction with cellular membranes, as well as the possibility to bind negative charged nucleic acids and endosomal releases) ranged up to 100 nm.The complexation with nucleic acids can be achieved via electrostatic interactions, rather than via strong covalent binding that releases nucleic acid cargo in the cytoplasm or in the nucleus.To effectively transfect particular cell populations, the DNA:nanoparticles ratio should also be experimentally assessed to avoid cytotoxic effects (Grześkowiak et al., 2015(Grześkowiak et al., , 2016)).
The literature data describe many different types of nanoparticles dedicated to the delivery of nucleic acids: stable biological fluids with spherical lipid NPs (Hou et al., 2021); particularly biocompatible nanospheres or dendrimer-shaped polymeric NPs (Mendes et al., 2017); and well known non-toxic inorganic NPs, i.e., AuNPs (Ding et al., 2014).The selection of cell types for nanotransfection is often determined by the purpose of application, e.g., gene editing, gene addiction, or gene inhibition.As nanotechnology-based approaches to assisted animal reproduction mainly concern gene editing, two different strategies are usually taken into account: the nanofection of somatic cell primary cultures or clonal lines (for the purpose of somatic cell nuclear transfer; SCNT) or the nanoparticle-mediated transgenization of zygotes (via intrapronuclear microinjection) (Grześkowiak et al., 2015;Dzięgiel et al., 2022).
A particularly special procedure for the non-viral, nanoparticle-mediated delivery of exogenous DNA molecules (gene constructs) is magnetofection.This process is associated with magnetic nanoparticles that are used as a carrier and are enhanced by external magnetic field delivery (Plank et al., 2003).The huge advantage of this technique is connected with its elimination of slow diffusion, rather low efficacy, and cytotoxic properties of well-known vehicles, such as viral vectors and polyplexes (Mykhaylyk et al., 2007).
Considering all the above-indicated facts, the major advantages for the nanodelivery of genes are the improvement in the kinetics of the delivery process, the significant reduction in the applied vector doses for effective gene expression, as well as the low cytotoxicity of the process.
Thus far, these advanced nanodelivery approaches have been used for the transgenization of ex vivo expanded porcine somatic cells (i.e., neonatal kidneyderived fibroblast-like cells) as an alternative strategy to the standard methods of electroporation, lipofection, and nucleofection (Richter et al., 2012).Magnetofection was successfully used to preferentially incorporate pCD59-GFPBsd gene constructs into porcine somatic cells (i.e., fetal fibroblasts) as a result of the action of an external magnetic field (Grześkowiak et al., 2016).The above-indicated strategies for the nanoparticle-assisted transfection of in vitro cultured somatic cells, in a variety of mammalian species, may be reliable and feasible for propagating and multiplying the abundant sources of genetically engineered or genome-edited nuclear donor cells for the purposes of cloning via SCNT technique (Perisse et al., 2021;Skrzyszowska and Samiec, 2021;Samiec, 2022;Samiec et al., 2022).Finally, nanoparticle-mediated gene delivery seems to be also a promising tool for the transgenization of in vivo fertilized rabbit zygotes by their intrapronuclear microinjection with pmaxFP-Red-N (eRFP-based) gene constructs; this might also open up new possibilities for the nanotransfection of cloned embryos at the early 1-blastomere stages that are generated in the post artificial activation of SCNTderived oocytes (Dzięgiel et al., 2022).

conclusions and future goals
The usefulness of various applications of nanobiotechnology-assisted animal breeding and veterinary medicine encompasses a wide spectrum of practical utilization, including pathogen diagnostics and treatment modalities aimed at reproductive-related disorders, hormonal stimulation procedures, antibiotic therapies in dairy research, and advanced ARTs, such as SCNT-based cloning combined with either gene targeting or genome editing.The benefits that can be achieved by using the strategies of nanoparticle-mediated biotechnology appear to be a tremendously attractive option.The advantages that are of paramount importance for agricultural and biomedical research are associated with the following: 1) reducing the concentration of the effective bioactive compounds that are sufficient for improving either the quality parameters of milk, meat, or environmental protection; 2) increasing animal welfare; and 3) decreasing herd maintenance costs.However, great care must be taken when implementing novel approaches to nanobiotechnology and every effort should be made to assess the potential toxicity of nanoparticles, their bioaccumulation and their environmental impact.

Table 1 .
The implementation of nanotechnology to the diagnostics of reproductive-related infection diseases in animals Type of nanoparticle biofunctionalization Diagnostics activity Methods for the detection of the analyte References NK-lysin peptide-functionalized nanoporous anodized aluminum oxide (NAAO) The detection of endotoxins of Gram-negative Mannheimia haemolytica and a Gram-positive Staphylococcus aureus NK-lysin peptides showed membranolysis activity and can be used to detect bacterial LPS and LTA molecules.NK-lysin peptide-functionalized NAAO, equipped with electrochemical electrodes, monitored the changes in the transmembrane impedance of functionalized NAAO membranes after analyte detection.Jiang et al., 2022 Biotinylated amplicons magnetically labeled with streptavidin-coated superparamagnetic nanoparticles (MNPs) Major bovine mastitis-causing pathogens: Escherichia coli, Klebsiella sp., Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae The targeted analytes (biotinylated amplicons) labeled with streptavidin-coated Mycoplasma bovisThe antibody modified the nitrocellulose-membrane-created complex with double tagged amplicons (microarray immunoassay); then, this complex was bound to black carbon NPs, which were read via flatbed scanning or the naked eye.
Arcanobacterum pyogenes in dairy cattleAgNPs inhibited cell viability and biofilm formation via oxidative stress; this effect may be also improved by the presence of apigenin.The authors concluded that the specific mechanism of the antibacterial effect of AgNPs against P. melaninogenica and A. pyogenes remains unknown.