Application of nanotechnology in animal reproduction

Introduction

The concept of "nanotechnology" was first introduced by Nobel Laureate Richard P. Feynman in his seminal 1959 lecture titled "There's Plenty of Room at the Bottom" (Ijaz et al., 2020). Nanotechnology involves the manipulation and study of materials at the nanoscale, typically ranging from 1 to 100 nanometers (10⁻⁹ to 10⁻⁷ meters) in at least one dimension (Jiang et al., 2008;Albanese et al., 2012). At this scale, materials exhibit distinct physicochemical properties compared to their bulk counterparts, primarily due to an increased surface area to volume ratio. This enlarged surface area leads to a higher proportion of atoms located on the surface of the particle, significantly influencing its chemical reactivity and physical behavior. For instance, while bulk copper is known for its ductility and is widely used in applications such as wiring and piping, its nanoscale counterpart exhibits reduced malleability. This is attributed to the predominance of surface atoms in nanoparticles, which resist deformation, unlike the interior atoms that facilitate bending in bulk copper (Sharma and Sharma, 2012). Nanoparticles can function either as independent entities or as carriers for active agents, which may be adsorbed onto their surfaces or encapsulated within. Their enhanced surface reactivity and small size make them highly versatile for a wide range of applications, including biomedical diagnostics, drug delivery, and gene therapy (Albanese et al., 2012;Swain et al., 2015). From a biological perspective, nanoparticles often demonstrate improved solubility, increased bioactivity, and structural stability. These attributes make them less susceptible to degradation through oxidative processes or other environmental stressors. In the field of animal sciences, particularly livestock production, nanotechnology has shown promising potential. Nano-formulated minerals have been reported to inhibit pathogenic microorganisms in animal feed, modulate rumen fermentation processes and improve reproductive performance in livestock species such as cattle and sheep (Laurent et al., 2008) [39, 40, 2, 71, 2, 75,47] .

The deployment of nanoscale drug delivery systems allows for more targeted and efficient therapeutic interventions, including the administration of antibiotics and hormones, thereby reducing both dosage requirements and treatment costs. This approach also contributes to minimizing the risk of antimicrobial resistance. Nanotechnology further offers novel opportunities in livestock reproductive management. For example, smart delivery platforms can enhance the bioavailability of reproductive hormones and therapeutic agents, thus improving fertility outcomes (Feugang, 2017;Feugang et al., 2019). Nanoparticles, whether naturally occurring or synthetically engineered, can be fabricated in various geometries (e.g., spherical, cubic) and may consist of a diverse range of materials including metals, salts, lipids, proteins, and polymers (Sun et al., 2014). These diverse material properties and morphologies expand the functional possibilities for nanotechnology in enhancing animal health, productivity, and overall farm sustainability [27, 29, 74] .

Applications of nanotechnology in the Animal Reproduction Biostimulation and Pheromones

Reproductive efficiency in farm animals is a cornerstone of livestock productivity. Several tools have been developed to enhance reproduction, including biostimulation methods (e.g., male effect), reproductive assisted techniques (such as estrous synchronization and artificial insemination), nutritional strategies, and disease prevention or treatment (Caraviello et al., 2006;Hashem et al., 2021). Among these, pheromones play a unique biological role. Pheromones are volatile chemical signals secreted by specific organs and scent glands in animals. In mammals, including livestock, pheromones are crucial for intra-species communication, mediating a wide range of behavioral and physiological responses. One of the most significant pheromone mediated reproductive phenomena in livestock is the "Male Effect" (Murata et al., 2014). The male effect refers to the stimulatory influence of a sexually active male on the reproductive activity of females. Pheromones present in the wool, wax, and urine of males can initiate puberty, resume ovarian activity in seasonally anestrous females, and reduce postpartum anestrus. This effect has been well-documented in sheep, goats, and swine (Ferreira-Silva et al., 2017;Meza-Herrera et al., 2017). Its practical utility in farm management includes advancing the breeding season by 4-6 weeks, achieving synchronization in estrus, and shortening the interval between parturitions, particularly when used 35-40 days postpartum (DeSantiago-Miramontes et al., 2008; Martin et al., 2004). In swine, the presence of boars significantly reduces the postpartum interval in lactating sows and advances puberty in gilts by approximately 30 days. Intriguingly, even the odor left by a boar in a pen can elicit these effects, underscoring the potency of olfactory cues (Signoret and Lindsay, 1982). Recent innovations such as BOARBETTER, a synthetic 3molecule boar pheromone spray, have been shown to improve sow reproductive performance (McGlone et al., 2019). Despite the demonstrated efficacy of the male effect, its practical implementation at scale faces logistical challenges, such as the seasonal sexual inactivity of males and the need for managing male-female contact. A promising solution is the use of aerosol nano-drug delivery systems. These systems can transport bioactive molecules, including pheromones, through olfactory pathways or systemic circulation, bypassing biological barriers such as the blood-brain barrier (Cerbu et al., 2021;Dugasa and Williams, 2016). By encapsulating pheromonal compounds-identified from wool wax, saliva, urine, or feces-into nanoparticles suitable for nasal spray formulations, it becomes feasible to mimic the male effect year-round. This approach could minimize reliance on live males and eliminate the need for priming sexually inactive males before use (Kekan et al., 2017;Archunan, 2020). The application of nanotechnology opens avenues for developing pheromone-based protocols not only for the male effect but also for broader reproductive events such as estrus detection, puberty induction, postpartum anestrus management, pregnancy diagnosis, and even modulating maternal behavior. Potential innovations include commercial bio-stimulating pheromones and pheromone based bioassay kits. For instance, urinary pheromones like 4-methyl phenol (p-Cresol), oleic acid (9-octadecenoic acid), and luteinizing hormone (LH) have been proposed as candidates for the development of nanoparticle-based estrus detection kits in buffaloes (Archunan, 2020) [9, 37, 57, 26, 55, 15, 51, 73, 52, 12, 20, 43, 6] .

Treatment and Diagnosis of Infectious Reproductive Disorders in Livestock

Bacterial and zoonotic infections of the reproductive tract pose significant challenges in livestock management due to their impact on fertility and productivity. The clinical manifestations of these infections are often driven either directly by microbial products such as endotoxins or indirectly through the host's immune response involving cytokines, eicosanoids, and oxidative mediators like nitric oxide (Sánchez-Sánchez et al., 2018). The emergence of antibiotic resistance, limited drug bioavailability, and tissuespecific delivery barriers further complicate therapeutic outcomes.

In this context, nanotechnology-based interventions are gaining traction as promising alternatives. A range of engineered nanomaterials-including liposomes, polymeric nanoparticles, solid lipid nanoparticles, nanogels, and inorganic nanoparticles-have been explored for their ability to overcome the limitations of conventional antibiotic therapies (Zhou et al., 2018;Wang et al., 2017;Paudel et al., 2019). These systems offer several advantages [69, 82, 78, 63]: • Enhanced targeting and retention at the site of infection A practical example of nanotechnology in action is the encapsulation of enrofloxacin, a widely used antibiotic in swine. Traditionally administered intramuscularly at 2.5-5 mg/kg body weight for 3-5 days, enrofloxacin faces limitations such as low oral bioavailability and potential side effects. Paudel et al. (2019) reported that oral administration of enrofloxacin loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles via drinking water reduced the minimum inhibitory concentration (MIC) against Escherichia coli by 23%, compared to the free drug. The nano-formulation also exhibited enhanced bioavailability, indicating a potential for lower dosage requirements and reduced risk of resistance development.

In addition to therapy, nanotechnology offers diagnostic benefits. Nanosensors incorporating highly sensitive biomolecular probes have been developed for the detection of reproductive tract infections, hormonal imbalances, and even for estrus detection. These nanosensors provide real-time, accurate, and non-invasive diagnostic capabilities, greatly enhancing reproductive monitoring and management in livestock (Saragusty and Arav, 2011). Nanotechnology has emerged as a transformative tool in combating reproductive tract infections in livestock. By facilitating targeted drug delivery, reducing antibiotic doses, and enhancing antimicrobial efficacy, nanomaterials offer a sustainable and effective solution to many challenges posed by conventional therapies. Additionally, advances in nanosensor technology promise earlier and more precise diagnostics, supporting better reproductive health management. Continued research and translational efforts are essential to fully harness the potential of these technologies in field conditions [78, 4, 26, 63, 70] .

Sustain Delivery of Hormone/Nutrition

Reproductive efficiency is a fundamental determinant of productivity in farm animals. Traditional hormonal treatments and nutritional interventions have improved animal fertility; however, they come with limitations such as low bioavailability, immunogenicity, and environmental risks. Recent advances in nanotechnology-based delivery systems offer promising solutions by enhancing hormone stability, reducing doses, and improving nutrient utilization. Some hormones commonly used in reproductive management, such as gonadotropin releasing hormone (GnRH) and prostaglandin F 2α (PGF 2α ), have low molecular weights and short half-lives, which compromise their sustained delivery and biological efficacy. In contrast, glycoprotein hormones like follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), and equine chorionic gonadotropin (eCG) are more stable but may elicit immune responses, leading to antibody formation and reduced responsiveness upon repeated administration (DeCastro et al., 2009;Forcada et al., 2011). Nanostructured drug delivery systems, particularly those utilizing biodegradable and biocompatible polymers, are being employed to overcome the pharmacokinetic limitations of conventional hormones (Lombardo et al., 2019). These systems protect hormones from enzymatic degradation, prevent rapid clearance, and extend their half-life. For example, Hashem and Sallam (2020) demonstrated that GnRH nanoparticles conjugated with chitosan-sodium tripolyphosphate (TPP) enabled a 75% dose reduction in goats without compromising fertility or prolificacy. Similarly, Casares-Crespo et al. (2018) incorporated nanoparticles into rabbit semen extenders, achieving a 50% reduction in hormone use while maintaining reproductive outcomes.

Beyond dose reduction, NHDS enable alternative administration routes (e.g., intranasal delivery), enhancing animal welfare and minimizing hormone exposure risks for workers and technicians (Hashem et al., 2015;Tomoda et al., 2012). Conventional hormonal delivery systems, such as progesterone-impregnated intravaginal devices, rely on nonbiodegradable silicon polymers and require high hormone concentrations to be effective. These devices pose environmental hazards due to hormone residues and risk of human exposure (Rathbone and Burke, 2013; DeGraaff and Grimard, 2018). In contrast, biodegradable nanofiber-based systems, such as poly (lactic acid) progesterone nanofibers, provide controlled hormone release while eliminating ecological and health concerns (Oliveira et al., 2013). Nutrition plays a critical role in modulating reproductive function across key physiological stages-fetal development, puberty, and reproductive maturity (CordovaIzquierdo, 2016). Specific short and long-term nutritional strategies have been employed to optimize breeding efficiency. For example, short-term supplementation (7-10 days pre-mating) or longterm feeding (about 60 days before mating) of energy-dense diets (e.g., protected fats, glycerol) has shown to enhance reproductive parameters in small ruminants (Hashem and El-Zarkouny, 2014; Meza-Herrera et al., 2017; Cannas et al., 2019). Moreover, certain nutrients exhibit 'functional effects' on reproductive physiology. Amino acids, for example, are essential for the development of the oviductal and uterine histotroph, and for the composition of amniotic and allantoic fluids, which support embryo development (Abdelnour et al., 2021). In dairy cows, linseed oil supplementation, rich in linolenic acid (C18:3), has been associated with increased plasma progesterone levels and improved first-AI conception rates (Castro et al., 2019). On the other hand, nutritional imbalances can lead to poor body condition, delayed puberty, longer open days, and infertility due to hormonal dysfunction and ovarian inactivity. The integration of nanotechnology into livestock nutrition holds promise for enhancing the effectiveness of feed additives and supplements. Nano-formulated nutrients exhibit improved solubility, bioavailability, and cellular uptake due to their reduced particle size and increased surface area (Gawad and Fellner, 2019). This allows for sustained and site-specific nutrient release, optimizing their biological function and reducing wastage [30, 48, 35, 10, 36, 77, 67, 16, 62, 13, 33, 55, 8, 1, 11, 32] . Nanotechnology represents a transformative approach in reproductive and nutritional management of livestock. From enhancing the efficacy and safety of hormonal treatments to improving nutrient absorption and utilization, nano-delivery systems promise to elevate animal productivity while ensuring environmental sustainability and occupational safety. As research progresses, further integration of nanotechnology into livestock management protocols could redefine current reproductive and nutritional paradigms.

Semen Purification

Semen purification plays a critical role in enhancing reproductive efficiency by isolating high-quality sperm subpopulations-those with superior motility and morphology while simultaneously eliminating defective cells, cellular debris, and potential contaminants from the ejaculate. Conventional semen purification methods employed in farm animals include density gradient centrifugation (Sieme et al., 2003), the swim-up technique (Arias et al., 2017), column filtration (Galarza et al., 2018), and single-layer centrifugation (Nongbua et al., 2017). While these approaches are widely used, they often involve significant manual handling, are time-intensive, and require moderate technical infrastructure. Recent advancements have introduced nanotechnology-based purification strategies as promising alternatives. Specifically, magnetic nanoparticles (Fe₃O₄) functionalized with biological ligands-such as peanut agglutinin (PNA), Pisum sativum agglutinin (PSA), annexin V, and anti-ubiquitin antibodieshave shown efficacy in selectively binding and removing defective spermatozoa in bovine semen samples (Faezah et al., 2014). These biofunctionalized nanoparticles facilitate the enrichment of viable sperm by targeting apoptotic or ubiquitinated cells, thus enhancing semen quality. Such Nano-magnetic purification methods have demonstrated several advantages over traditional techniques. They minimize excessive semen manipulation, reduce labour intensity, and shorten processing times (typically under one hour), while also eliminating the need for costly laboratory equipment (Feugang, 2017;Meles et al., 2022). Consequently, the integration of nanoparticle-based strategies offers a streamlined, efficient, and cost-effective solution for obtaining high-quality spermatozoa, particularly in resourcelimited settings.

Semen Preservation (cryopreservation)

The integration of nanotechnology into semen extender formulations has emerged as a promising strategy to enhance the preservation of sperm quality during storage and cryopreservation. Among various nanomaterials, cerium oxide nanoparticles (CeO₂ NPs), recognized for their oxygen buffering capacity, have demonstrated significant potential in protecting spermatozoa. Supplementation of semen extenders with CeO₂ NPs during storage at 4°C for up to 96 hours has been shown to enhance sperm motility, particularly after 48 hours, and maintain plasma membrane and DNA integrity (Falchi et al., 2018a). Selenium nanoparticles (Se NPs), known for their reactive oxygen species (ROS) scavenging ability, have also been employed to mitigate oxidative damage in sperm cells. Their inclusion in semen preservation protocols has resulted in improved post-thaw sperm motility, morphology, and decreased apoptotic and necrotic cell populations, ultimately contributing to higher in vivo fertility rates (Khalil et al., 2019). Similarly, nano-lecithin-based extenders, specifically those containing 2% lecithin, have been found superior to conventional egg yolk-or lecithin-based media in preserving goat spermatozoa, evidenced by increased motility, viability, and functional membrane integrity, along with reduced apoptosis (Nadri et al., 2019). Advancements in magnetic nanoparticle technology have enabled the targeted purification of sperm populations. For instance, Fe₃O₄ nanoparticles functionalized with lectins or antibodies have been effectively used to isolate viable spermatozoa from both fresh and extended porcine semen (Durfey et al., 2019), as well as from cryopreserved bull semen (Odhiambo et al., 2014). Other studies have utilized avidin-conjugated Fe₃O₄ nanoparticles along with DNA aptamers to sort both unsorted and sex-sorted bull sperm (Farini et al., 2016). In addition to metal oxide nanoparticles, "nanowater" has emerged as a novel extender medium. When used for ram semen, nanowater improved fertilizing capacity, with significant increases in conception and lambing rates (Murawski et al., 2015). Moreover, the antioxidant properties of various nanomaterials-such as cerium oxide, selenium, vitamin E nanoemulsions, and noble metal nanoparticles (gold, platinum)-have been associated with improved sperm motility, membrane integrity, and overall semen quality during chilled or frozen storage (Falchi et al., 2018b; Jurado-Campos et al., 2023; Hashem and Gonzales-Bulnes, 2020; Khalique et al., 2024). Cryopreservation of semen often results in functional impairments, including DNA fragmentation, premature capacitation, and disruption of acrosomal and plasma membrane integrity (Yoon et al., 2015;Yeste, 2016). To mitigate these damages, liposome vesicles have been investigated for their ability to fuse with sperm membranes and deliver protective agents intracellularly. Liposomes incorporated into egg yolk-free extenders (e.g., OptiXcell®) have proven effective in minimizing cryodamage across several species, including bulls, buffaloes (Ansari et Organic nanoparticles, particularly liposomes and exosomes, offer further potential due to their biocompatibility and membrane-binding ability. These nanocarriers have been shown to reduce the population of non-viable spermatozoa and may facilitate the repair of damaged sperm membranes. While synthetic liposomes can be engineered to deliver specific molecules (Feugang, et al., 2022), naturally derived exosomes from biofluids-including seminal plasma-contain diverse bioactive cargos such as proteins, miRNAs, lipids, and mRNAs (Piehl et al., 2013;Yang et al., 2017;Dilsiz, 2022;Dlamini et al., 2023). These attributes make both liposomes and exosomes promising vehicles for enhancing sperm viability and fertility potential during and after cryopreservation [7, 31, 60, 22, 27, 54, 23, 44, 59, 21, 61, 25, 58, 24, 42, 34, 45, 81, 80, 5, 3, 76, 68, 46, 38, 49, 50, 56, 65, 53, 28, 64, 79, 17, 18] .

Nanotechnology-Based Strategies for Sperm Sexing and Fetal Sex Determination

Recent proteomic investigations have identified distinct surface protein profiles in bull spermatozoa, revealing that 12 proteins are upregulated in X-chromosome-bearing sperm, while 3 are more abundant in Y-bearing sperm (Quelhas et al., 2021). These molecular markers offer promising targets for selective sperm separation through immune affinity approaches. The application of magnetic nanoparticles (MNPs), functionalized with specific ligands or antibodies that recognize these differential protein expressions, presents a novel and efficient technique for immune selection of sexspecific sperm cells. Furthermore, surface-engineered magnetic nanoparticles have been successfully employed to exploit the differential surface charge between X-and Y-chromosome-bearing spermatozoa. By modifying nanoparticle coatings to selectively bind Ybearing sperm based on their electrostatic properties, researchers have achieved effective enrichment of X-bearing sperm within semen samples (Domínguez et al., 2018). For practical application, scalable production of such functionalized nanoparticles is crucial to support the broader adoption of nanotechnology-assisted sex-sorting methods in livestock reproduction. In addition to sperm sexing, advancements in nanoscale biosensing have contributed to non-invasive fetal sex determination. Technologies such as biochips integrated with nanomaterials have shown potential for accurately identifying fetal sex at early stages of pregnancy by detecting sex-specific biomarkers in maternal biological fluids (Joanitti and Silva, 2014). These platforms combine high sensitivity and specificity with rapid analysis, offering a valuable tool for reproductive management in both veterinary and human medicine [66, 19, 41] .

Conclusion

In conclusion, the integration of nanotechnology into gynecology and reproductive management in livestock presents a transformative shift toward precision, efficiency, and sustainability. From enhancing biostimulation via pheromone delivery and improving diagnosis and treatment of reproductive infections to enabling sustained hormone and nutrient release, nanotechnology offers novel solutions to long-standing challenges. Moreover, innovations in semen purification, preservation, and sex selection-along with noninvasive fetal sex determination-underscore its broad applicability across reproductive stages. As research continues to evolve, these nanotechnological advancements promise to revolutionize animal reproduction, optimize productivity, and contribute to more effective and environmentally responsible livestock management systems.

References

1. Abdelnour SA, Al-Gabri NA, Hashem NM, GonzalezBulnes A. Supplementation with proline improves haematobiochemical and reproductive indicators in male rabbits affected by environmental heat-stress. Animals. 2021;11(2):373. DOI:10.3390/ani11020373.
2. Albanese A, Tang PS, Chan WCW. The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu Rev Biomed Eng. 2012;14:116. DOI:10.1146/annurev-bioeng-071811-150124.
3. Al-Bulushi S, Manjunatha BM, Bathgate R, Rickard JP, De Graaf SP. Liquid storage of dromedary camel semen in different extenders. Anim Reprod Sci. 2019;207:95106. DOI:10.1016/j.anireprosci.2019.06.008.
4. Algharib SA, Dawood A, Xie S. Nanoparticles for treatment of bovine Staphylococcus aureus mastitis. Drug Deliv. 2020;27(1):292-308. DOI:10.1080/10717544.2020.1724200.
5. Ansari MS, Rakha BA, Akhter S, Ashiq M. OPTIXcell improves the post-thaw quality and fertility of buffalo bull sperm. Theriogenology. 2016;85(3):528-532. DOI:10.1016/j.theriogenology.2015.09.035.
6. Archunan G. Reproductive enhancement in buffalo: Looking at urinary pheromones and hormones. Iran J Vet Res. 2020;21(3):163-170.
7. Arias ME, Andara K, Briones E, Felmer R. Bovine sperm separation by swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression. Reprod Biol. 2017;17(2):126-132. DOI:10.1016/j.repbio.2017.04.006.
8. Cannas A, Tedeschi LO, Atzori AS, Lunesu MF. How can nutrition models increase the production efficiency of sheep and goat operations? Anim Front. 2019;9(4):33-44. DOI:10.1093/af/vfz040.
9. Caraviello D, Weigel K, Fricke P, Wiltbank M, Florent M, Cook N, et al. Survey of management practices on reproductive performance of dairy cattle on large US commercial farms. J Dairy Sci. 2006;89(12):4723-4735. DOI:10.3168/jds.S0022-0302(06)72522-X.
10. Casares-Crespo L, Fernández-Serrano P, Viudes-DeCastro MP. Protection of GnRH analogue by chitosandextran sulfate nanoparticles for intravaginal application in rabbit artificial insemination. Theriogenology. 2018;116:49-52. DOI:10.1016/j.theriogenology.2018.05.008.
11. Castro T, Martinez D, Isabel B, Cabezas A, Jimeno V. Vegetable oils rich in polyunsaturated fatty acids supplementation of dairy cows' diets: Effects on productive and reproductive performance. Animals. 2019;9(5):205. DOI:10.3390/ani9050205.
12. Cerbu C, Kah M, White JC, Astete CE, Sabliov CM. Fate of biodegradable engineered nanoparticles used in veterinary medicine as delivery systems from a one health perspective. Molecules. 2021;26(2):523. DOI:10.3390/molecules26020523.
13. Cordova-Izquierdo A. Best practices in animal reproduction: Impact of nutrition on reproductive performance livestock. J Adv Dairy Res. 2016;4(3):152. DOI:10.4172/2329-888X.1000152.
14. De Castro MV, Cortell C, Mocé E, Marco-Jiménez F, Joly T, Vicente J. Effect of recombinant gonadotropins on embryo quality in superovulated rabbit does and immune response after repeated treatments. Theriogenology. 2009;72(5):655-662. DOI:10.1016/j.theriogenology.2009.04.018.
15. De Santiago-Miramontes M, Rivas-Muñoz R, MuñozGutiérrez M, Malpaux B, Scaramuzzi R, Delgadillo J. The ovulation rate in anoestrous female goats managed under grazing conditions and exposed to the male effect is increased by nutritional supplementation. Anim Reprod Sci. 2008;105(3-4):409-416. DOI:10.1016/j.anireprosci.2007.04.007.
16. DeGraaff W, Grimard B. Progesterone-releasing devices for cattle estrus induction and synchronization: Device optimization to anticipate shorter treatment durations and new device developments. Theriogenology. 2018;112:3443. DOI:10.1016/j.theriogenology.2018.01.026.
17. Dilsiz N. Hallmarks of exosomes. Future Sci OA. 2022;8(1):FSO764. DOI:10.2144/fsoa-2021-0102.
18. Dlamini NH, Nguyen T, Gad A, Tesfaye D, Liao SF, Willard ST, et al. Characterization of extracellular vesicle-coupled miRNA profiles in seminal plasma of boars with divergent semen quality status. Int J Mol Sci. 2023;24(4):3194. DOI:10.3390/ijms24043194.
19. Domínguez E, Moreno-Irusta A, Castex HR, Bragulat AF, Ugaz C, Clemente H, et al. Sperm sexing mediated by magnetic nanoparticles in donkeys: A preliminary in vitro study. J Equine Vet Sci. 2018;65:123-127. DOI:10.1016/j.jevs.2017.11.011.
20. Dugasa HL, Williams RO III. Nanotechnology for pulmonary and nasal drug delivery. In: Nanotechnologybased drug delivery. Volume 2: Nanotechnology-based strategies for nanomedicine against severe diseases. Elsevier; 2016. p. 102-120. DOI:10.1016/B978-0-323-42978-8.00005-0.
21. Durfey CL, Swistek SE, Liao SF, Crenshaw MA, Clemente HJ, Thirumalai RV, et al. Nanotechnologybased approach for safer enrichment of semen with best spermatozoa. J Anim Sci Biotechnol. 2019;10(1):14. DOI:10.1186/s40104-019-0315-4.
22. Faezah SS, Zuraina FM, Farah JH, Khairul O, Hilwani NI, Iswadi MI, et al. The effects of magnetic separation on cryopreserved bovine spermatozoa motility, viability and cryo-capacitation status. Zygote. 2014;22(3):378386. DOI:10.1017/S0967199413000071.
23. Falchi L, Galleri G, Dore GM, Zedda MT, Pau S, Bogliolo L, et al. Effect of exposure to CeO₂ nanoparticles on ram spermatozoa during storage at 4 °C for 96 hours. Reprod Biol Endocrinol. 2018;16(1):19. DOI:10.1186/s12958-018-0339-9.
24. Falchi L, Galleri G, Zedda MT, Pau S, Bogliolo L, Ariu F, et al. Liquid storage of ram semen for 96 hours: Effects on kinematic parameters, membranes and DNA integrity, and ROS production. Livest Sci. 2018;207:1-6. DOI:10.1016/j.livsci.2017.11.001.
25. Farini VL, Camaño CV, Ybarra G, Viale DL, Vichera G, Yakisich JS, et al. Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles. J Biotechnol. 2016;229:33-41. DOI:10.1016/j.jbiotec.2016.05.004.
26. Ferreira-Silva JC, Burnett TA, Souto PFMP, Filho PCBG, Pereira LC, Araujo MV, et al. Progesterone (P4), luteinizing hormone (LH) levels and ovarian activity in postpartum Santa Inês ewes subject to a male effect. Arch Anim Breed. 2017;60(1):95-100. DOI:10.5194/aab-60-95-2017.
27. Feugang JM. Novel agents for sperm purification, sorting, and imaging. Mol Reprod Dev. 2017;84(9):832841. DOI:10.1002/mrd.22820.
28. Feugang JM, Ishak GM, Eggert MW, Arnold RD, Rivers OS, Willard ST, et al. Intrafollicular injection of nanomolecules for advancing knowledge on folliculogenesis in livestock. Theriogenology. 2022;192:132-140. DOI:10.1016/j.theriogenology.2022.08.032.
29. Feugang JM, Rhoads CE, Mustapha PO, Tardif S, Parrish JJ, Willard ST, et al. Treatment of boar sperm with nanoparticles for improved fertility. Theriogenology. 2019;137:75-81. DOI:10.1016/j.theriogenology.2019.05.020.
30. Forcada F, Ait Amer-Meziane M, Abecia JA, Maurel MC, Cebrián-Pérez JA, Muiño-Blanco T, et al. Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep. Theriogenology. 2011;75(4):769-776. DOI:10.1016/j.theriogenology.2010.10.029.
31. Galarza DA, Lopez-Sebastian A, Woelders H, Blesbois E, Santiago-Moreno J. Sephadex filtration as successful alternative to density-gradient centrifugation procedures for ram sperm selection with improved kinetics. Anim Reprod Sci. 2018;192:261-270. DOI:10.1016/j.anireprosci.2018.03.022.
32. Gawad R, Fellner V. Evaluation of glycerol encapsulated with alginate and alginate-chitosan polymers in gut environment and its resistance to rumen microbial degradation. Asian-Australas J Anim Sci. 2019;32(1):7280. DOI:10.5713/ajas.18.0089.
33. Hashem N, El-Zarkouny S. Effect of short-term supplementation with rumen-protected fat during the late luteal phase on reproduction and metabolism of ewes. J Anim Physiol Anim Nutr. 2014;98(1):65-71. DOI:10.1111/jpn.12042.
34. Hashem NM, Gonzales-Bulnes A. State-of-the-art and prospective of nanotechnologies. Animals. 2020;10(5):840. DOI:10.3390/ani10050840.
35. Hashem NM, Sallam SM. Reproductive performance of goats treated with free gonadorelin or nanoconjugated gonadorelin at estrus. Domest Anim Endocrinol. 2020;71:106390. DOI:10.1016/j.domaniend.2019.106390.
36. Hashem NM, El-Zarkouny SZ, Taha TA, Abo-Elezz ZR. Oestrous response and characterization of the ovulatory wave following oestrous synchronization using PGF₂α alone or combined with GnRH in ewes. Small Rumin Res. 2015;129:84-87. DOI:10.1016/j.smallrumres.2015.06.002.
37. Hashem NM, Hassanein EM, Simal-Gandara J. Improving reproductive performance and health of mammals using honeybee products. Antioxidants. 2021;10(3):336. DOI:10.3390/antiox10030336.
38. He L, Bailey JL, Buhr MM. Incorporating lipids into boar sperm decreases chilling sensitivity but not capacitation potential. Biol Reprod. 2001;64(1):69-79. DOI:10.1095/biolreprod64.1.69.
39. Ijaz I, Gilani E, Nazir A, Bukhari A. Detail review on chemical, physical and green synthesis, classification, characterizations and applications of nanoparticles. Green Chem Lett Rev. 2020;13(3):223-245. DOI:10.1080/17518253.2020.1802517.
40. Jiang W, Kim BYS, Rutka JT, Chan WCW. Nanoparticle-mediated cellular response is sizedependent. Nat Nanotechnol. 2008;3(3):145-150. DOI:10.1038/nnano.2008.30.
41. Joanitti G, Silva LP. The emerging potential of byproducts as platforms for drug delivery systems. Curr Drug Targets. 2014;15(5):478-485. DOI:10.2174/13894501113149990171.
42. Jurado-Campos A, Soria-Meneses PJ, Arenas-Moreira M, Alonso-Moreno C, Rodríguez-Robledo V, Soler AJ, et al. Minimizing sperm oxidative stress using nanotechnology for breeding programs in rams. J Anim Sci Biotechnol. 2023;14(1):106. DOI:10.1186/s40104-023-00907-3.
43. Kekan PM, Ingole SD, Sirsat SD, Bharucha SV, Kharde SD, Nagvekar AS. The role of pheromones in farm animals: A review. Agric Rev. 2017;38(1):65-72. DOI:10.18805/ag.R-1740.
44. Khalil WA, El-Harairy MA, Zeidan AE, Hassan MA. Impact of selenium nanoparticles in semen extender on bull sperm quality after cryopreservation. Theriogenology. 2019;126:121-127. DOI:10.1016/j.theriogenology.2018.12.020.
45. Khalique MA, Andrabi SMH, Majeed KA, Yousaf MS, Ahmad N, Tahir SK, et al. Cerium oxide nanoparticles improve the post-thaw quality and in vivo fertility of Beetal buck spermatozoa. Theriogenology. 2024;214:166-172. DOI:10.1016/j.theriogenology.2023.10.022.
46. Kumar P, Saini M, Kumar D, Balhara AK, Yadav SP, Singh P, et al. Liposome-based semen extender is suitable alternative to egg yolk-based extender for cryopreservation of buffalo (Bubalus bubalis) semen. Anim Reprod Sci. 2015;159:38-45. DOI:10.1016/j.anireprosci.2015.05.010.
47. Laurent S, Forge D, Port M, Roch A, Robic C, Vander Elst L, et al. Magnetic iron oxide nanoparticles: Synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chem Rev. 2008;108(6):2064-2110. DOI:10.1021/cr068445e.
48. Lombardo D, Kiselev MA, Caccamo MT. Smart nanoparticles for drug delivery application: Development of versatile nanocarrier platforms in biotechnology and nanomedicine. J Nanomater. 2019;2019:3702518. DOI:10.1155/2019/3702518.
49. Luna-Orozco JR, González-Ramos MA, Calderón-Leyva G, Gaytán-Alemán LR, Arellano-Rodríguez MA, ÁngelGarcía O, et al. Comparison of different diluents based on liposomes and egg yolk for ram semen cooling and cryopreservation. Iran J Vet Res. 2019;20(2):126-130.
50. Mafolo KS, Pilane CM, Chitura T, Nedambale TL. Use of phosphatidylcholine in Tris-based extender with or without egg yolk to freeze Bapedi ram semen. S Afr J Anim Sci. 2020;50(3):389-396. DOI:10.4314/sajas.v50i3.5.
51. McGlone JJ, Devaraj S, Garcia A. A novel boar pheromone mixture induces sow estrus behaviors and reproductive success. Appl Anim Behav Sci. 2019;219:104832. DOI:10.1016/j.applanim.2019.104832.
52. Medina-León AZ, Domínguez-Mancera B, CazalezPeñino N, Cervantes-Acosta P, Jácome-Sosa E, RomeroSalas D, et al. Cryopreservation of horse semen with a liposome and trehalose added extender. Aust J Vet Sci. 2019;51(3):119-123. DOI:10.4067/S0719-81322019000300119.
53. Meles DK, Mustofa I, Hariadi M, Wurlina W, Susilowati S, Amaliya A, et al. The enriched Y-bearing sperm combined with delayed fixed-time artificial insemination for obtaining male Simmental crossbred offspring. Vet World. 2022;15(1):102-109. DOI:10.14202/vetworld.2022.102-109.
54. Meza-Herrera CA, Cano-Villegas O, Flores-Hernandez A, Veliz-Deras FG, Calderon-Leyva G, Guillen-Munoz JM, et al. Reproductive outcomes of anestrous goats supplemented with spineless Opuntia megacantha SalmDyck protein-enriched cladodes and exposed to the male effect. Trop Anim Health Prod. 2017;49:1511-1516. DOI:10.1007/s11250-017-1366-7.
55. Mortazavi S, Eslami M, Farrokhi-Ardabili F. Comparison of different carrier-compounds and varying concentrations of oleic acid on freezing tolerance of ram spermatozoa in tris-citric acid-egg yolk plasma semen diluent. Anim Reprod Sci. 2020;219:106533. DOI:10.1016/j.anireprosci.2020.106533.
56. Murata K, Tamogami S, Itou M, Ohkubo Y, Wakabayashi Y, Watanabe H, et al. Identification of an olfactory signal molecule that activates the central regulator of reproduction in goats. Curr Biol. 2014;24:681-686. DOI:10.1016/j.cub.2014.01.073.
57. Murawski M, Schwarz T, Grygier J, Patkowski K, Oszczęda Z, Jelkin I, et al. The utility of nanowater for ram semen cryopreservation. Exp Biol Med (Maywood). 2015;240:611-617. DOI:10.1177/1535370214558020.
58. Nadri T, Towhidi A, Zeinoaldini S, Martínez-Pastor F, Mousavi M, Noei R, et al. Lecithin nanoparticles enhance the cryosurvival of caprine sperm. Theriogenology. 2019;133:38-44.
59. Nongbua T, Johannisson A, Edman A, Morrell JM. Effects of single layer centrifugation (SLC) on bull spermatozoa prior to freezing on post-thaw semen characteristics. Reprod Domest Anim. 2017;52:596-602.
60. Odhiambo JF, DeJarnette JM, Geary TW, Kennedy CE, Suarez SS, Sutovsky M, et al. Increased conception rates in beef cattle inseminated with nanopurified bull semen. Biol Reprod. 2014;91(4):97:1-10.
61. Oliveira JE, Medeiros ES, Cardozo L, Voll F, Madureira EH, Mattoso LHC, et al. Development of poly(lactic acid) nanostructured membranes for the controlled delivery of progesterone to livestock animals. Mater Sci Eng C. 2013;33(2):844-849.
62. Paudel S, Cerbu C, Astete CE, Louie SM, Sabliov C, Rodrigues DF. Enrofloxacin-impregnated PLGA nanocarriers for efficient therapeutics and diminished generation of reactive oxygen species. ACS Appl Nano Mater. 2019;2(8):5035-5043.
63. Piehl LL, Fischman ML, Hellman U, Cisale H, Miranda PV. Boar seminal plasma exosomes: Effect on sperm function and protein identification by sequencing. Theriogenology. 2013;79(7):1071-1082. DOI:10.1016/j.theriogenology.2013.01.028.
64. Pillet E, Labbé C, Batellier F, Duchamp G, Beaumal V, Anton M, et al. Liposomes as an alternative to egg yolk in stallion freezing extender. Theriogenology. 2012;77(2):268-279. DOI:10.1016/j.theriogenology.2011.08.001.
65. Quelhas J, Santiago J, Matos B, Rocha A, Lopes G, Fardilha M. Bovine semen sexing: Sperm membrane proteomics as candidates for immunological selection of X-and Y-chromosome-bearing sperm. Vet Med Sci. 2021;7(5):1633-1641. DOI:10.1002/vms3.540.
66. Rathbone MJ, Burke CR. Controlled release intravaginal veterinary drug delivery. In: Rathbone MJ, editor. Long acting animal health drug products. New York: Springer; 2013. p. 247-270.
67. Röpke T, Oldenhof H, Leiding C, Sieme H, Bollwein H, Wolkers WF. Liposomes for cryopreservation of bovine sperm. Theriogenology. 2011;76(8):1465-1472.
68. Sánchez-Sánchez R, Vázquez P, Ferre I, Ortega-Mora LM. Treatment of toxoplasmosis and neosporosis in farm ruminants: State of knowledge and future trends. Curr Top Med Chem. 2018;18(15):1304-1323.
69. Saragusty J, Arav A. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction. 2011;141(1):1-19. DOI:10.1530/REP-10-0236.
70. Sharma HS, Sharma A. Neurotoxicity of engineered nanoparticles from metals. CNS Neurol Disord Drug Targets. 2012;11(1):65-80.
71. Sieme H, Martinsson G, Rauterberg H, Walter K, Aurich C, Petzoldt R, et al. Application of techniques for sperm selection in fresh and frozen-thawed stallion semen. Reprod Domest Anim. 2003;38(2):134-140.
72. Signoret JP, Lindsay DR. The male effect in domestic mammals: Effect on LH secretion and ovulation importance of olfactory cues. In: Olfaction and endocrine regulation. London: Academic Press; 1982. p. 63-72.
73. Sun T, Zhang YS, Pang B, Hyun DC, Yang M, Xia Y. Engineered nanoparticles for drug delivery in cancer therapy. Angew Chem Int Ed Engl. 2014;53(46):1232012364. DOI:10.1002/anie.201403036.
74. Swain PS, Rajendran D, Rao SBN, Dominic G. Preparation and effects of nano mineral particle feeding in livestock: A review. Vet World. 2015;8(7):888-891. DOI:10.14202/vetworld.2015.888-891.
75. Swelum AA, Saadeldin IM, Ba-Awadh H, Al-Mutary MG, Moumen AF, Alowaimer AN, et al. Efficiency of commercial egg yolk-free and egg yolk-supplemented Tris-based extenders for dromedary camel semen cryopreservation. Animals. 2019;9(11):999. DOI:10.3390/ani9110999.
76. Tomoda K, Watanabe A, Suzuki K, Inagi T, Terada H, Makino K. Enhanced transdermal permeability of estradiol using combination of PLGA nanoparticles system and iontophoresis. Colloids Surf B Biointerfaces. 2012;97:84-89.
77. Wang L, Hu C, Shao L. The antimicrobial activity of nanoparticles: Present situation and prospects for the future. Int J Nanomedicine. 2017;12:1227-1249. DOI:10.2147/IJN.S121956.
78. Yang C, Guo WB, Zhang WS, Bian J, Yang JK, Zhou QZ, et al. Comprehensive proteomics analysis of exosomes derived from human seminal plasma. Andrology. 2017;5(5):1007-1015. DOI:10.1111/andr.12412.
79. Yeste M. Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenology. 2016;85(1):47-64. DOI:10.1016/j.theriogenology.2015.09.047.
80. Yoon SJ, Kwon WS, Rahman MS, Lee JS, Pang MG. A novel approach to identifying physical markers of cryodamage in bull spermatozoa. PLoS One. 2015;10(5):e0126232. DOI:10.1371/journal.pone.0126232.
81. Zhou K, Li C, Chen D, Pan Y, Tao Y, Qu W, et al. A review on nanosystems as an effective approach against infections of Staphylococcus aureus. Int J Nanomedicine. 2018;13:7333-7347. DOI:10.2147/IJN.S178285.