Animal Reproduction (AR)
Animal Reproduction (AR)

Silver nanoparticle as an alternate to antibiotics in cattle semen during cryopreservation

Arushi Kanwar; Meenakshi Virmani; Sant Lal; Kartik Chaudhary; Sandeep Kumar; Ankit Magotra; Anand Kumar Pandey

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The proposed study was to determine if the silver nanoparticles can be used as potential antimicrobial agents and can replace the use of conventional antibiotics in semen without affecting the motility and fertility of semen. The silver nanoparticles prepared by chemical reduction method were confirmed by determination of the wavelength of surface plasmon resonance peak and further characterized using Zetasizer by determining their size, polydispersity index, and zeta potential. The nanoparticles were assessed for antibacterial activity and their concentration was optimized for use in semen extender for cryopreservation. Cryopreserved semen was further evaluated for seminal parameters, antioxidant parameter, and microbial load. Prepared silver NPs showed a plasmon resonance peak at 417 nm wavelength. NPs were found to possess antibacterial activity and were supplemented in semen extender @ 125 and 250 µg/ml for semen cryopreservation. There was a significant increase in pre and post-freezing motility and other seminal parameters. The microbial load of frozen-thawed semen of control and supplemented groups were well within the permissible limits. Lipid peroxidation levels were reduced in NPs supplemented groups, and reactive oxygen species (ROS) levels were significantly reduced in semen supplemented with 125 µg/ml NPs. Thus it can be conclude that silver NPs can be successfully used as a substitute for antibiotics in cattle bull semen cryopreservation with good antimicrobial activity and no adverse effects on sperm characteristics.


CASA, freezing, nanoparticle, silver, sperm


Abdelhamid HN, Talib A, Wu HF. Facile synthesis of water soluble silver ferrite (AgFeO2) nanoparticles and their biological application as antibacterial agents. RSC Advances. 2015;5(44):34594-602.

Abdelhamid HN, Wu HF. Facile synthesis of nano silver ferrite (AgFeO2) modified with chitosan applied for biothiol separation. Mater Sci Eng C Mater Biol Appl. 2014;45:438-45. PMid:25491849.

Akçan R, Aydogan HC, Yildirim MŞ, Taştekin B, Sağlam N. Nanotoxicity: a challenge for future medicine. Turk J Med Sci. 2020;50(4):1180-96. PMid:32283898.

Alghoraibi I, Zein R. Silver nanoparticles: advances in research and applications is approaching. In: Edwards B, editor. Silver nanoparticles: advances in research and applications. New York: Nova Science Publishers; 2017. p. 106-45.

Baracaldo MI, Barth AD, Bertrand W. Steps for freezing bovine semen: from semen collection to the liquid nitrogen tank. In: Revah I, editor. Reviews in veterinary medicine. La Jolla: IVIS; 2006. p. 1-29.

Biel MA, Sievert C, Usacheva M, Teichert M, Balcom J. Antimicrobial photodynamic therapy treatment of chronic recurrent sinusitis biofilms. Int Forum Allergy Rhinol. 2011;1(5):329-34. PMid:22287461.

Birla SS, Tiwari VV, Gade AK, Ingle AP, Yadav AP, Rai MK. Fabrication of silver nanoparticles by Phomaglomerata and its combined effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Lett Appl Microbiol. 2009;48(2):173-9. PMid:19141039.

Bisla A, Rautela R, Yadav V, Saini G, Singh P, Ngou AA, Kumar A, Ghosh S, Kumar A, Bag S, Mahajan S, Srivastava N. Synthesis of iron oxide nanoparticles–antiubiquitin antibodies conjugates for depletion of dead/damaged spermatozoa from buffalo (Bubalus bubalis) semen. Biotechnol Appl Biochem. 2021;68(6):1453-68. PMid:33135803.

Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, Giwercman A. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod. 2007;22(1):174-9. PMid:16921163.

Campbell RC, Hancock JL, Shaw IG. Cytological characteristics and fertilizing capacity of bull spermatozoa. J Agric Sci. 1960;55(1):91-9.

DHADF. Compendium of minimum standards of protocol and standard operating procedures for bovine breeding. New Delhi: Department of Animal Husbandry, Dairying & Fisheries/Ministry of Agriculture; 2014.

Domínguez E, Moreno-Irusta A, Castex HR, Bragulat AF, Ugaz C, Clemente H, Giojalas L, Losinno L. Sperm sexing mediated by magnetic nanoparticles in donkeys, a preliminary in vitro study. J Equine Vet Sci. 2018;65:123-7.

Dong ZY, Rao MPN, Xiao M, Wang H-F, Hozzein WN, Chen W, Li W-J. Antibacterial activity of silver nanoparticles against Staphylococcus warneri synthesized using endophytic bacteria by photo-irradiation. Front Microbiol. 2017;8:1090. PMid:28659903.

Durfey CL, Burnett DD, Liao SF, Steadman CS, Crenshaw MA, Clemente HJ, Willard ST, Ryan PL, Feugang JM. Nanotechnology-based selection of boar spermatozoa: growth development and health assessments of produced offspring. Livest Sci. 2017;205:137-42.

Falchi L, Galleri G, Dore GM, Zedda MT, Pau S, Bogliolo L, Ariu F, Pinna A, Nieddu S, Innocenzi P, Ledda S. Effect of exposure to CeO2 nanoparticles on ram spermatozoa during storage at 4 C for 96 hours. Reprod Biol Endocrinol. 2018;16(1):19. PMid:29510737.

Farini VL, Camaño CV, Ybarra G, Viale DL, Vichera G, Yakisich JS, Radrizzani M. Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles. J Biotechnol. 2016;229:33-41. PMid:27164256.

Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R. Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria. Nanomedicine. 2010;6(1):103-9. PMid:19447203.

Gade A, Gaikwad S, Tiwari V, Yadav A, Ingle A, Rai M. Biofabrication of silver nanoparticles by Opuntiaficus-indica: in vitro antibacterial activity and study of the mechanism involved in the synthesis. Curr Nanosci. 2010;6(4):370-5.

Gopal J, Abdelhamid HN, Huang JH, Wu HF. Nondestructive detection of the freshness of fruits and vegetables using gold and silver nanoparticle mediated graphene enhanced Raman spectroscopy. Sens Actuators B Chem. 2016;224:413-24.

Ingle A, Gade A, Pierrat S, Sonnichsen C, Rai M. Mycosynthesis of silver nanoparticles using the fungus Fusarium acuminatum and its activity against some human pathogenic bacteria. Curr Nanosci. 2008;4(2):141-4.

Isaac AV, Kumari S, Nair R, Urs DR, Salian SR, Kalthur G, Adiga SK, Manikkath J, Mutalik S, Sachdev D, Pasricha R. Supplementing zinc oxide nanoparticles to cryopreservation medium minimizes the freeze-thaw-induced damage to spermatozoa. Biochem Biophys Res Commun. 2017;494(3-4):656-62. PMid:29074362.

Ismail AA, Abdel-Khalek AE, Khalil W, El-Harairy MA. Influence of adding green synthesized gold nanoparticles to tris-extender on sperm characteristics of cryopreserved goat semen. J Anim Poult Prod. 2020;11(2):39-45.

Jahanbin R, Yazdanshenas P, Rahimi M, Hajarizadeh A, Tvrda E, Nazari SA, Mohammadi-Sangcheshmeh A, Ghanem N. In vivo and in vitro evaluation of bull semen processed with zinc (Zn) nanoparticles. Biol Trace Elem Res. 2021;199(1):126-35. PMid:32495179.

Khalil WA, El-Harairy MA, Zeidan AE, Hassan MA. Impact of selenium nano-particles in semen extender on bull sperm quality after cryopreservation. Theriogenology. 2019;126:121-7. PMid:30551018.

Lazar V. Quorum sensing in biofilms–how to destroy the bacterial citadels or their cohesion/power? Anaerobe. 2011;17(6):280-5. PMid:21497662.

Maheshwari M, Ahmad I, Althubiani AS. Multidrug resistance and transferability of blaCTX-M among extended-spectrum β-lactamase-producing enteric bacteria in biofilm. J Glob Antimicrob Resist. 2016;6:142-9. PMid:27530857.

Mohanty TK, Lone SA, Kumaresan A, Bhakat M, Kumar R, Baithalu RK, Sinha R, Paray AR, Yadav HP, Sahu SK, Mohanty AK. Sperm dosage and site of insemination in relation to fertility in bovines. Asian Pac J Reprod. 2018;7(1):1-5.

Ohkawa H, Ohishi W, Yagi K. Colorimetric method for determination of MDA activity. Biochemistry. 1979;95:351-8.

Ong I [homepage on the Internet]. Huntersville: MICROBAN; 2022. Zone of inhibition test: why bigger isn't always better; 2022 [cited 2022 Aug 14]. Available from:

Oves M, Aslam M, Rauf MA, Qayyum S, Qari HA, Khan MS, Alam MZ, Tabrez S, Pugazhendhi A, Ismail IM. Antimicrobial and anticancer activities of silver nanoparticles synthesized from the root hair extract of Phoenix dactylifera. Mater Sci Eng C Mater Biol Appl. 2018;89:429-43. PMid:29752116.

Pérez-Duran F, Acosta-Torres LS, Serrano-Díaz PN, Toscano-Torres IA, Olivo-Zepeda IB, García-Caxin E, Nuñez-Anita RE. Toxicity and antimicrobial effect of silver nanoparticles in swine sperms. Syst Biol Reprod Med. 2020;66(4):281-9. PMid:32456478.

Periasamy S, Joo HS, Duong AC, Bach TH, Tan VY, Chatterjee SS, Cheung GY, Otto M. How Staphylococcus aureus biofilms develop their characteristic structure. Proc Natl Acad Sci USA. 2012;109(4):1281-6. PMid:22232686.

Pineda Y, Santander J. Evaluación de la flora bacteriana delsemen de verracosen granjas porcinas de Venezuela. Zootec Trop. 2007;25(3):173-7.

Prabhu S, Poulose EK. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. Int Nano Lett. 2012;2(1):32.

Qais FA, Shafiq A, Khan HM, Husain FM, Khan RA, Alenazi B, Alsalme A, Ahmad I. Antibacterial effect of silver nanoparticles synthesized using Murraya koenigii (L.) against multidrug-resistant pathogens. Bioinorg Chem Appl. 2019;2019:4649506. PMid:31354799.

Radzig MA, Nadtochenko VA, Koksharova OA, Kiwi J, Lipasova VA, Khmel IA. Antibacterial effects of silver nanoparticles on gram-negative bacteria: influence on the growth and biofilms formation, mechanisms of action. Colloids Surf B Biointerfaces. 2013;102:300-6. PMid:23006569.

Raheman F, Deshmukh S, Ingle A, Gade A, Rai M. Silver nanoparticles: novel antimicrobial agent synthesized from an endophytic fungus Pestalotia sp. isolated from leaves of Syzygiumcumini (L). Nano Biomed Eng. 2011;3(3):174-8.

Rahman A, Kumar S, Bafana A, Dahoumane SA, Jeffryes C. Biosynthetic conversion of Ag+ to highly stable Ag0 nanoparticles by wild type and cell wall deficient strains of Chlamydomonas reinhardtii. Molecules. 2018;24(1):98. PMid:30597856.

Rai M, Kon K, Ingle A, Duran N, Galdiero S, Galdiero M. Broad-spectrum bioactivities of silver nanoparticles: the emerging trends and future prospects. Appl Microbiol Biotechnol. 2014;98(5):1951-61. PMid:24407450.

Rai MK, Deshmukh SD, Ingle AP, Gade AK. Silver nanoparticles: the powerful nanoweapon against multidrug‐resistant bacteria. J Appl Microbiol. 2012;112(5):841-52. PMid:22324439.

Rinna A, Magdolenova Z, Hudecova A, Kruszewski M, Refsnes M, Dusinska M. Effect of silver nanoparticles on mitogen-activated protein kinases activation: role of reactive oxygen species and implication in DNA damage. Mutagenesis. 2015;30(1):59-66. PMid:25527729.

Samuel MS, Jose S, Selvarajan E, Mathimani T, Pugazhendhi A. Biosynthesized silver nanoparticles using Bacillus amyloliquefaciens; Application for cytotoxicity effect on A549 cell line and photocatalytic degradation of p-nitrophenol. J Photochem Photobiol B. 2020;202:111642. PMid:31734434.

Schuppe HC, Meinhardt A, Allam JP, Bergmann M, Weidner W, Haidl G. Chronic orchitis: a neglected cause of male infertility? Andrologia. 2008;40(2):84-91. PMid:18336456.

Shukla MK. Applied veterinary andrology and frozen semen technology. New Delhi: New India Publishing Agency; 2011.

Tang S, Zheng J. Antibacterial activity of silver nanoparticles: structural effects. Adv Healthc Mater. 2018;7(13):e1701503. PMid:29808627.

Taraszkiewicz A, Fila G, Grinholc M, Nakonieczna J. Innovative strategies to overcome biofilm resistance. BioMed Res Int. 2013;2013:150653. PMid:23509680.

Ugur MR, Abdelrahman AS, Evans HC, Gilmore AA, Hitit M, Arifiantini RI, Purwantara B, Kaya A, Memili E. Advances in cryopreservation of bull sperm. Front Vet Sci. 2019;6:268. PMid:31552277.

Valgas C, Souza SM, Smânia EF, Smânia A Jr. Screening methods to determine antibacterial activity of natural products. Braz J Microbiol. 2007;38(2):369-80.

Watson PF, Martin IC. A comparison of changes in the acrosomes of deep-frozen ram and bull spermatozoa. J Reprod Fertil. 1972;28(1):99-101. PMid:5008009.

Wu D, Fan W, Kishen A, Gutmann JL, Fan B. Evaluation of the antibacterial efficacy of silver nanoparticles against Enterococcus faecalis biofilm. J Endod. 2014;40(2):285-90. PMid:24461420.

Yousef MS, Abdelhamid HN, Hidalgo M, Fathy R, Gómez-Gascón L, Dorado J. Antimicrobial activity of silver-carbon nanoparticles on the bacterial flora of bull semen. Theriogenology. 2021;161:219-27. PMid:33340755.

Yu DG. Formation of colloidal silver nanoparticles stabilized by Na+ –poly(γ-glutamic acid)–silver nitrate complex via chemical reduction process. Colloids Surf B Biointerfaces. 2007;59(2):171-8. PMid:17583483.

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