Evaluation of Indolicidin for Inactivation of Bovine Alphaherpesvirus 1 in Semen

Evaluation of Indolicidin for Inactivation of Bovine Alphaherpesvirus 1 in Semen | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Evaluation of Indolicidin for Inactivation of Bovine Alphaherpesvirus 1 in Semen João Paulo Yoshio Prado Cerqueira Kubota, Gabriel Henrique Santos, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8605122/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 4 You are reading this latest preprint version Abstract Bovine alphaherpesvirus 1 (BoAHV1) is a reproductively important pathogen whose presence in bovine semen restricts international trade. The increasing use of controlled breeding programmes highlights the need for effective viral inactivation strategies. This study evaluated the antiviral activity of the antimicrobial peptide indolicidin in bovine semen experimentally infected with BoAHV1. Indolicidin was synthesised by solid-phase peptide synthesis. Antiviral activity and minimum inhibitory concentration (MIC) were determined in MDBK cell cultures. The peptide was then tested in infected semen, and semen quality was assessed using computer-assisted sperm analysis (CASA). Indolicidin inhibited BoAHV1 in MDBK cells with an MIC of 8 µM. In semen, viral titre was significantly reduced at 10 µM with complete inactivation at 30 µM, confirmed by the absence of cytopathic effect in MDBK cells. CASA analysis demonstrated that semen quality parameters were preserved at concentrations up to 30 µM, while 100 µM caused a significant reduction in straight and progressive motility. Semen disinfection Indolicidin virus inactivation BoAHV1 Figures Figure 1 INTRODUCTION Antimicrobial peptides (AMPs) constitute a conserved molecule of the host’s first line of defense, exhibiting broad activity against viruses, bacteria, and fungi, including pathogens resistant to conventional antimicrobial agents 1 . Among these molecules, indolicidin, a cathelicidin peptide isolated from bovine neutrophils, has received particular attention. This 13-residue peptide (ILPWKWPWWPWRR-NH₂) is amphipathic, containing five tryptophan residues that facilitate its interaction with biological membranes. Such structural features support its ability to inhibit a range of viruses as well as Gram-positive and Gram-negative bacteria 2 . Although indolicidin has long been recognized for its antiviral activity, important gaps remain regarding its application in bovine semen and its potential effects on sperm motility parameters. Collected semen is routinely treated with broad-spectrum conventional antibiotics, prolonged use of these compounds can impair semen quality 3 and has become increasingly ineffective due to rising antimicrobial resistance. In addition, conventional antibiotics target only bacterial contaminants and provide no control over viruses, which represent a significant concern in reproductive biosecurity 4 . This study investigated the potential of indolicidin to inactivate viruses present in bovine semen. Bovine alphaherpesvirus 1 (BoAHV-1), classified by the World Organisation for Animal Health as a notifiable pathogen of relevance to international trade in animal genetic material 5 , was used as the experimental model to evaluate the feasibility of this antiviral approach. MATERIAL AND METHODS Indolicidin was synthesised by solid-phase peptide synthesis 6 and characterised by HPLC and mass spectrometry. Bovine alphaherpesvirus 1 (BoAHV-1; Los Angeles strain) was propagated in Madin–Darby bovine kidney (MDBK) cells cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Viral titres were determined using the trimmed Spearman–Karber method 7 . For in vitro antiviral assays, BoAHV-1 suspensions (10⁶·⁷⁵ TCID₅₀/mL) were incubated with indolicidin (4, 6, 8 or 10 µM) for 1 h at room temperature. Tenfold serial dilutions were inoculated into MDBK monolayers in 96-well plates, overlaid with MEM containing 10% FBS, and incubated for 72 h at 37°C in 5% CO₂. Cytopathic effect (CPE) was evaluated by inverted microscopy, and residual viral titres were calculated. Cell and virus controls were included. For semen assays, semen aliquots (500 µL) were experimentally inoculated with BoAHV-1 (10⁵·⁷⁵ TCID₅₀/mL) and incubated with indolicidin (10 or 30 µM) for 1 h at room temperature. Serial dilutions were inoculated into MDBK monolayers, overlaid with MEM containing 5% FBS, and incubated under the same conditions. CPE was assessed after 72 h; samples without CPE underwent three blind passages with freeze/thaw cycles 5 ˒ 8 . The same controls were used. The effect of indolicidin on spermatozoa was assessed using computer-assisted sperm analysis (CASA) 10 . Semen was collected from three Girolando bulls by electroejaculation and maintained at 37°C. Initial semen quality was assessed according to CBRA guidelines 9 . Baseline CASA evaluation (Hamilton-Thorne) was performed using a Makler® chamber (10 µL), assessing total motility, curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), straightness (STR), linearity (LIN), beat-cross frequency (BCF) and amplitude of lateral head displacement (ALH). Only samples with total motility ≥ 70% were included (two ejaculates). Semen aliquots (500 µL) were incubated with an overestimated concentration of the peptide (30 or 100 µM) for 15 min and reanalysed using the same CASA protocol. All assays were performed in triplicate. Data were analysed using analysis of variance followed by Tukey’s test in R 11 (p < 0.05) and are presented as mean ± SD. RESULTS In vitro antiviral activity of the peptide against BoAHV1 Indolicidin achieved complete inactivation of BoAHV-1 in MDBK cells at minimum inhibitory concentration (MIC) of 8 µM (Figue 1- graph 1) as also indicated by the absence of the characteristic CPE (panel MDBK - D). At 6 µM, the peptide did not fully prevent infection but produced a marked reduction in viral titre (> 4 log₁₀) compared with the untreated control. This reduction in infectivity is evident when contrasting the residual CPE in the 6 µM treatment (panel MDBK - C) compared with the widespread CPE observed in the positive control (panel MDBK - B), tipically characterized by the grape-like clusters of rounded cells gathered (arrows), characteristic of the cytopathic effect caused by BoAHV1 infection 5 . In vitro antiviral activity of the peptide in infected semen After confirming the antiviral effect of indolicidin in cell culture, the peptide was evaluated in bovine semen experimentally infected with BoAHV-1. Unlike the findings in MDBK monolayers, where complete viral inactivation occurred at 8 µM, treatment of infected semen required a MIC of 10 µM to achieve a reduction of nearly 4 log₁₀ in viral titre compared with the untreated semen control (Figue 1 - graph 2). At this concentration, however, residual infectivity was still detected following three blind passages in MDBK cells (panel SEMEN - C). For this reason, a higher, broadly effective concentration (30 µM) was tested. At 30 µM, indolicidin completely inactivated BoAHV-1 (Figue 1 - graph 2), also demonstrated by the absence of CPE in MDBK cells after serial passage (panel SEMEN - D) compared with the extensive CPE observed in the positive control (panel SEMEN - B). Notably, MDBK monolayers exposed to semen treated with this peptide concentration displayed normal cell growth patterns, indicating that indolicidin at 30 µM did not exert cytotoxic effects under the conditions evaluated. Effect of indolicidin on bovine semen To confirm the absence of adverse effects of indolicidin on bovine semen, the MIC (30 µM) identified in the in vitro antiviral activity in infected semen assay was assessed together with an intentionally high peptide concentration (100 µM) (Table 1 ). Treatment with 30 µM indolicidin showed no significant differences in any parameter when compared with the untreated control, indicating that this concentration did not impair sperm quality. Curiously, in very high concentrations (100 µM), indolicidin did not significantly affect VAP, VSL, VCL, ALH, STR or LIN values relative to the control group. Although BCF increased at 100 µM, this value remained within the range observed for the control. Despite maintaining most kinematic standards at this concentration, a significant reduction in straight and progressive motility (MT and MP) was detected, rendering semen treated at 100 µM unsuitable for insemination. Table 1 Safety test of indolicidin in bovine semen using the peptide at 30 µM and 100 µM incubated for 30 minutes and evaluated by CASA Semen quality parameters Control Indolicidin 30 µM Indolicidin 100 µM MT (%) 79.3 ± 2.9a 76.3 ± 2.1a 26.7 ± 10.6b MP (%) 37.7 ± 9.1a 34.7 ± 11.6a 12.7 ± 4b VAP (µm/s) 114.9 ± 34.8a 120.3 ± 41.4a 114.3 ± 37.6a VSL(µm/s) 76.6 ± 15.8a 76.4 ± 14.8a 74.1 ± 11a VCL(µm/s) 205.8 ± 73.1a 215.8 ± 85.8a 209.5 ± 80.2a ALH (µm) 8.3 ± 2.1a 8.5 ± 2.6a 7.3 ± 1.8a BCF (Hz) 33.1 ± 5.1ab 32 ± 4.9b 36 ± 5.2a STR (%) 71.3 ± 10.6a 67 ± 11.3a 70 ± 10.7a LIN (%) 40.3 ± 7.8a 40 ± 10.4a 43 ± 8.5a Means followed by the same lowercase letter between columns do not differ significantly from each other (p-value < 0.05). MT: Straight motility; MP: Progressive motility; VAP: Average path velocity; VSL: straight-line velocity; VCL: curvilinear velocity; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; STR: Straightness; LIN: Linearity. DISCUSSION Different mechanisms of action previously described for indolicidin may help explain the peptide activity against BoAHV-1 in this study. Falla et al. (1996) 12 reported that, in Gram-negative bacteria, the peptide is able to traverse the outer membrane and induce disruption of the plasma membrane. In contrast, Subbalakshmi et al. (2000) 13 did not observe membrane lysis in E. coli , but instead documented an increase in membrane permeability that facilitated peptide entry, followed by inhibition of DNA and protein synthesis within the cytoplasm. Given that BoAHV-1 consists of an icosahedral capsid covered by a host-derived lipid bilayer envelope 14 , it is plausible that these mechanisms could act in combination. Glycoproteins present in the viral envelope are directly responsible for the adsorption of the virus to specific cell receptors present in the host cell membrane during the initial stages of infection 15 . Therefore, direct disruption of the viral envelope would provide a virucidal effect, compromising the structural integrity of the virion, whereas interaction of indolicidin with viral DNA could exert an intracellular antiviral effect, preventing genome replication after entry into host cells. Together, these complementary actions offer a coherent explanation for the marked reduction in BoAHV-1 infectivity observed in our assays. When comparing the MIC of indolicidin in culture medium with that required in bovine semen, a marked increase in the concentration needed for complete viral inactivation was evident. This discrepancy is likely due to the complex biochemical composition of semen, which contains carbohydrate, proteins, lipids and other biomolecules capable of binding or sequestering antimicrobial peptides. Such interactions may reduce the amount of free, biologically active indolicidin available to interact with viral targets, thereby requiring higher concentrations to achieve effective BoAHV-1 inactivation. This matrix effect offers a plausible explanation for the MIC adjustment (30 µM) required in the semen applications. Although complete viral inactivation was achieved with 30 µM indolicidin, it is important to consider that the viral titre used for experimental contamination in this study (10⁵·⁷⁵ TCID₅₀/mL) was intentionally high and exceeds the levels typically detected in naturally infected semen. Considering that natural infections generally present titres around 10² TCID₅₀/mL 16 , it is reasonable to assume that 10 µM indolicidin could be sufficient for complete disinfection, as this concentration reduced the viral titre by approximately 4 logs. Sperm motility remains a key parameter for evaluating semen quality, as motile spermatozoa are essential for reaching the female reproductive tract and achieving fertilisation. Although all motilities parameters were maintained when 30 µM of indolicidin was used to semen disinfection, an overestimated peptide concentration (100 µM) was considered detrimental to sperm viability, as a minimum motility threshold of 50% is diserable for acceptable reproductive performance. Compared to the control, the significant reduction in straight and progressive motility observed at this concentration renders its use impractical for insemination. The antimicrobial activity of bioactive peptides is well recognised 17 – 18 , however, their use as disinfectants in semen remains incipient. A recent review by Kumar et al. (2024) 3 offers a comprehensive overview of the origin and potential application of AMPs in the semen of several species, including peptides in boar semen (hexapeptides c-WFW (10 µM) and c-WWW (20 µM), PMAP-37 peptide (3µM) and bacteriocin (1%), nisin peptide (50 µg/mL) in rat, LL-37 (10 µM) peptide in goat and a human β-defensin (10 µg/mL). Even less explored is the application of such peptides in bovine semen. Sánchez-Acosta et al. (2020) 19 demonstrated that dermaseptin S4 derivatives (15 µM) exhibited in vitro antibacterial activity without impairing sperm motility, however, no previous study has assessed the use of AMPs for viral inactivation in semen. Therefore, indolicidin emerges as a promising candidate to address this gap, offering a feasible alternative for broadening the range of strategies applicable to semen preservation. Previous studies have reported bactericidal activity of indolicidin at concentrations up to 32 µM 20 . Thus, the complete viral inactivation observed neste trabalho in semen with 30 µM indolicidin is particularly relevant, as this concentration may provide a broad-spectrum disinfectant effect in a single step. Such an approach could help reduce or even eliminate the use of conventional antibiotics in semen extenders, thereby contributing to antimicrobial management. Moreover, the methodology employed here is readily adaptable to routine procedures in semen collection and processing centres, offering a favourable cost-benefit profile. Indolicidin also possesses physicochemical features of interest for industrial application. Its short length allows high yield synthesis, while the feasibility of recombinant production offers a cost-effective route for large scale manufacturing, supporting its potential commercial use. Despite BoAHV-1 being used exclusively in this study as a model virus to evaluate the disinfectant potential of indolicidin, the peptide’s mode of action indicates that its applicability may extend beyond this pathogen. Also considering the bacterias, several other viruses of reproductive importance are enveloped, and indolicidin’s known membranolytic and intracellular activities suggest that it may exert enhanced antiviral effects against this broader group of agents. This raises the prospect that indolicidin could serve as a versatile tool for controlling a range of semen-borne pathogens in reproductive technologies. CONCLUSION Indolicidin demonstrated marked in vitro antiviral activity against BoAHV-1, achieving complete viral inactivation in cell culture and experimentally infected bovine semen. A concentration of 30 µM was required for full inactivation in semen, likely due to matrix effects, and did not impair sperm kinematic parameters, whereas higher concentrations negatively affected sperm motility. These findings indicate that indolicidin can effectively inactivate BoAHV-1 in semen under experimental conditions while preserving sperm quality, supporting its potential use as a semen disinfectant. This peptide represents a promising alternative to conventional antimicrobial strategies in semen processing and warrants further investigation under field and fertility conditions. Declarations STATEMENT OF ANIMAL ETHICS All procedures were approved by the Ethics Committee on Animal Experimentation of the Federal University of Goiás (protocol 012/22). CONFLICTS OF INTEREST The authors declare that there are no conflicts of interest. Author Contribution João Paulo Yoshio Prado Cerqueira Kubota: Writing – original draft, Methodology, Investigation. Gabriel Henrique Santos: Writing – original draft, Methodology, InvestigationTaise Maria dos Anjos Oliveira: Methodology, Investigation. Maria Lúcia Gambarini: Methodology, Investigation, Formal analysis. Keyla de Oliveira Ribeiro: Methodology, Investigation, Data analysis. Klayto José Gonçalves dos Santos: Methodology (CASA).Guilherme Sastre de Souza: Methodology (peptide synthesis, purification, and characterization). Maria Carolina Oliveira de Arruda Brasil: Methodology (peptide synthesis, purification, and characterization).Eduardo Maffud Cilli: Resources, Methodology, Supervision.Esteban Nicolás Lorenzón: Resources, Writing – review & editing, Conceptualization, Methodology, Investigation, Supervision, Formal analysis. Guilherme Rocha Lino de Souza: Resources, Writing – review & editing, Conceptualization, Methodology, Investigation, Supervision, Formal analysis. Acknowledgement The authors gratefully acknowledge the financial support by National Council for Scientific and Technological Development (CNPq), grant no. 427961/2018-1, 302390/2023-5, 141840/2023-4 and Goiás Research Foundation (FAPEG), grant no. 202310267000475. References Le CF, Fang CM, Sekaran SD (2017) Intracellular Targeting Mechanisms by Antimicrobial Peptides. Antimicrob Agents Chemother 61(4):e02340–e02316 Selsted ME, Novotny MJ, Morris WL et al (1992) Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J Biol Chem 267(7):4292–4295 Kumar M, Ranjan R, Bhardwaj A (2024) Antimicrobial peptide and their role in sperm cryopreservation. J Appl Biol Biotechnol 12(3):8–16 Morrell JM, Malaluang P, Cojkic A et al (2022) Alternatives to Antibiotics in Semen Extenders Used in Artificial Insemination. In: The Global Antimicrobial Resistance Epidemic - Innovative Approaches and Cutting-Edge Solutions [Internet]. IntechOpen ; Available from: https://www.intechopen.com/chapters/81891 . 10.5772/intechopen.104226 (accessed 25 Nov 2025) Animal Diseases. WOAH - World Organisation for Animal Health (2025) Available from: https://www.woah.org/en/what-we-do/animal-health-and-welfare/animal-diseases/ (accessed 25 Nov 2025) Merrifield RB (1963) Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J Am Chem Soc 85(14):2149–2154 Hamilton MA, Russo RC, Thurston RV (1977) Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environ Sci Technol 11(7):714–719 Brunner D, Engels M, Schwyzer M et al (1988) A Comparison of Three Techniques for Detecting Bovine Herpesvirus Type 1 (BHV-1) in Naturally and Experimentally Contaminated Bovine Semen. Reprod Domest Anim 23(1):1–9 de Colégio B (2025) Reprodução Animal (CBRA). CBRA – Colégio Brasileiro de Reprodução Animal. Available from: http://www.cbra.org.br/portal/ (accessed 25 Nov 2025) Amann RP, Waberski D (2014) Computer-assisted sperm analysis (CASA): capabilities and potential developments. Theriogenology 81(1):5–17. e1-3 R Core Team. R: The R Project for Statistical Computing [Internet] (2025) Available from: https://www.r-project.org/ (accessed 25 Nov 2025) Falla T, Karunaratne N, Hancock REW (1996) Mode of Action of the Antimicrobial Peptide Indolicidin. J Biol Chem 271(32):19298–19303 Subbalakshmi C, Bikshapathy E, Sitaram N et al (2000) Antibacterial and hemolytic activities of single tryptophan analogs of indolicidin. Biochem Biophys Res Commun 274(3):714–716 van Oirschot JT (1995) Bovine herpesvirus 1 in semen of bulls and the risk of transmission: A brief review. Vet Q 17(1):29–33 Muylkens B, Thiry J, Kirten P et al (2007) Bovine herpesvirus 1 infection and infectious bovine rhinotracheitis. Vet Res 38(2):181–209 Flores EF, Weiblen R, Vogel FSF et al (2005) A infecção pelo vírus da diarréia viral bovina (BVDV) no Brasil: histórico, situação atual e perspectivas. Pesq Vet Bras 25:125–134 Vasilchenko AS, Vasilchenko AV, Pashkova TM et al (2017) Antimicrobial activity of the indolicidin-derived novel synthetic peptide In-58. J Pept Sci 23(12):855–863 Shagaghi N, Palombo EA, Clayton AHA et al (2016) Archetypal tryptophan-rich antimicrobial peptides: properties and applications. World J Microbiol Biotechnol 32(2):31 Sánchez-Acosta YA, Castillo Vargas JA, Ramírez Quintero KJ et al (2020) Peptide derivatives of dermaseptin S4 in fresh bovine semen for bacterial contamination control: Physicochemical and structural characterization, antibacterial potency, and effects on red blood and sperm cells. Reprod Domest Anim 55(8):905–914 Vergis J, Malik SS, Pathak R et al (2019) Antimicrobial Efficacy of Indolicidin Against Multi-Drug Resistant Enteroaggregative Escherichia coli in a Galleria mellonella Model. Front Microbiol 10:2723 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 22 Jan, 2026 Editor assigned by journal 21 Jan, 2026 Submission checks completed at journal 21 Jan, 2026 First submitted to journal 14 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8605122","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":578575112,"identity":"11d52b04-eb6e-4a29-8ee4-21e491019b4b","order_by":0,"name":"João Paulo Yoshio Prado Cerqueira Kubota","email":"","orcid":"","institution":"Universidade Federal de Goiás","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"Paulo Yoshio Prado Cerqueira","lastName":"Kubota","suffix":""},{"id":578575113,"identity":"31bc0ab1-5154-468e-8d65-621f38c0d5f9","order_by":1,"name":"Gabriel Henrique 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21:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8605122/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8605122/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101992076,"identity":"9c200aed-bc3f-45e1-bca9-a1e3e4cf5b70","added_by":"auto","created_at":"2026-02-05 20:26:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":194955,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphs\u003c/strong\u003e\u003cem\u003e:\u003c/em\u003e viral titres as a function of indolicidin concentration in MDBK cell culture and in bovine semen experimentally infected with BoAHV-1. \u003cstrong\u003ePanel MDBK\u003c/strong\u003e: Optical microscopy (20×) showing the effect of indolicidin on the viability of the BoAHV1 in MDBK cell culture. A - control cell culture - no CPE; B - positive control (BoAHV1 wthout peptide treatment – with CPE characteristic of the MDBK cells virus infection); C - BoAHV1 previously treated with 6 µM of peptide infecting MDBK cell culture, exhibiting lower CPE; D - BoAHV1 previously treated with 8 µM of peptide infecting MDBK cell culture, with no CPE, indicating viral inactivation. \u003cstrong\u003ePanel SEMEN\u003c/strong\u003e: MDBK culture showing the effect of indolicidin on the viability of the BoAHV1 in the semen artificially infected. A - control cell culture - no CPE. B - positive control (BoAHV1 wthout peptide treatment – with CPE characteristic of the MDBK cells virus infection). C - sample treated with 10 µM of peptide, with low CPE. D - MDBK cells treated with 30 µM of peptide - no CPE. \u003cstrong\u003eArrows\u003c/strong\u003e: typical grape-like clusters of rounded cells gathered, characteristic of the cytopathic effect caused by BoAHV1 infection. \u003cstrong\u003e*\u003c/strong\u003e p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8605122/v1/a53679c5a58ad1222d7a3542.png"},{"id":102295281,"identity":"2c8c13d3-e461-4787-8d5a-32cba167361e","added_by":"auto","created_at":"2026-02-10 10:10:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":749633,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8605122/v1/1467c462-19e6-43b4-b70c-9a666af65998.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEvaluation of Indolicidin for Inactivation of Bovine Alphaherpesvirus 1 in Semen\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAntimicrobial peptides (AMPs) constitute a conserved molecule of the host\u0026rsquo;s first line of defense, exhibiting broad activity against viruses, bacteria, and fungi, including pathogens resistant to conventional antimicrobial agents\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among these molecules, indolicidin, a cathelicidin peptide isolated from bovine neutrophils, has received particular attention. This 13-residue peptide (ILPWKWPWWPWRR-NH₂) is amphipathic, containing five tryptophan residues that facilitate its interaction with biological membranes. Such structural features support its ability to inhibit a range of viruses as well as Gram-positive and Gram-negative bacteria\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although indolicidin has long been recognized for its antiviral activity, important gaps remain regarding its application in bovine semen and its potential effects on sperm motility parameters.\u003c/p\u003e \u003cp\u003eCollected semen is routinely treated with broad-spectrum conventional antibiotics, prolonged use of these compounds can impair semen quality\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and has become increasingly ineffective due to rising antimicrobial resistance. In addition, conventional antibiotics target only bacterial contaminants and provide no control over viruses, which represent a significant concern in reproductive biosecurity\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study investigated the potential of indolicidin to inactivate viruses present in bovine semen. Bovine alphaherpesvirus 1 (BoAHV-1), classified by the World Organisation for Animal Health as a notifiable pathogen of relevance to international trade in animal genetic material\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, was used as the experimental model to evaluate the feasibility of this antiviral approach.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eIndolicidin was synthesised by solid-phase peptide synthesis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and characterised by HPLC and mass spectrometry. Bovine alphaherpesvirus 1 (BoAHV-1; Los Angeles strain) was propagated in Madin\u0026ndash;Darby bovine kidney (MDBK) cells cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Viral titres were determined using the trimmed Spearman\u0026ndash;Karber method\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. For in vitro antiviral assays, BoAHV-1 suspensions (10⁶\u0026middot;⁷⁵ TCID₅₀/mL) were incubated with indolicidin (4, 6, 8 or 10 \u0026micro;M) for 1 h at room temperature. Tenfold serial dilutions were inoculated into MDBK monolayers in 96-well plates, overlaid with MEM containing 10% FBS, and incubated for 72 h at 37\u0026deg;C in 5% CO₂. Cytopathic effect (CPE) was evaluated by inverted microscopy, and residual viral titres were calculated. Cell and virus controls were included.\u003c/p\u003e \u003cp\u003eFor semen assays, semen aliquots (500 \u0026micro;L) were experimentally inoculated with BoAHV-1 (10⁵\u0026middot;⁷⁵ TCID₅₀/mL) and incubated with indolicidin (10 or 30 \u0026micro;M) for 1 h at room temperature. Serial dilutions were inoculated into MDBK monolayers, overlaid with MEM containing 5% FBS, and incubated under the same conditions. CPE was assessed after 72 h; samples without CPE underwent three blind passages with freeze/thaw cycles\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e˒\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The same controls were used.\u003c/p\u003e \u003cp\u003eThe effect of indolicidin on spermatozoa was assessed using computer-assisted sperm analysis (CASA)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Semen was collected from three Girolando bulls by electroejaculation and maintained at 37\u0026deg;C. Initial semen quality was assessed according to CBRA guidelines\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Baseline CASA evaluation (Hamilton-Thorne) was performed using a Makler\u0026reg; chamber (10 \u0026micro;L), assessing total motility, curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), straightness (STR), linearity (LIN), beat-cross frequency (BCF) and amplitude of lateral head displacement (ALH). Only samples with total motility\u0026thinsp;\u0026ge;\u0026thinsp;70% were included (two ejaculates). Semen aliquots (500 \u0026micro;L) were incubated with an overestimated concentration of the peptide (30 or 100 \u0026micro;M) for 15 min and reanalysed using the same CASA protocol. All assays were performed in triplicate. Data were analysed using analysis of variance followed by Tukey\u0026rsquo;s test in R\u003csup\u003e11\u003c/sup\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro antiviral activity of the peptide against BoAHV1\u003c/h2\u003e \u003cp\u003eIndolicidin achieved complete inactivation of BoAHV-1 in MDBK cells at minimum inhibitory concentration (MIC) of 8 \u0026micro;M (Figue 1-\u003cem\u003egraph\u003c/em\u003e 1) as also indicated by the absence of the characteristic CPE (panel \u003cem\u003eMDBK\u003c/em\u003e - D). At 6 \u0026micro;M, the peptide did not fully prevent infection but produced a marked reduction in viral titre (\u0026gt;\u0026thinsp;4 log₁₀) compared with the untreated control. This reduction in infectivity is evident when contrasting the residual CPE in the 6 \u0026micro;M treatment (panel \u003cem\u003eMDBK -\u003c/em\u003e C) compared with the widespread CPE observed in the positive control (panel \u003cem\u003eMDBK -\u003c/em\u003e B), tipically characterized by the grape-like clusters of rounded cells gathered (arrows), characteristic of the cytopathic effect caused by BoAHV1 infection\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro antiviral activity of the peptide in infected semen\u003c/h3\u003e\n\u003cp\u003eAfter confirming the antiviral effect of indolicidin in cell culture, the peptide was evaluated in bovine semen experimentally infected with BoAHV-1. Unlike the findings in MDBK monolayers, where complete viral inactivation occurred at 8 \u0026micro;M, treatment of infected semen required a MIC of 10 \u0026micro;M to achieve a reduction of nearly 4 log₁₀ in viral titre compared with the untreated semen control (Figue 1 - \u003cem\u003egraph\u003c/em\u003e 2). At this concentration, however, residual infectivity was still detected following three blind passages in MDBK cells (panel \u003cem\u003eSEMEN -\u003c/em\u003e C). For this reason, a higher, broadly effective concentration (30 \u0026micro;M) was tested. At 30 \u0026micro;M, indolicidin completely inactivated BoAHV-1 (Figue 1 - \u003cem\u003egraph\u003c/em\u003e 2), also demonstrated by the absence of CPE in MDBK cells after serial passage (panel \u003cem\u003eSEMEN -\u003c/em\u003e D) compared with the extensive CPE observed in the positive control (panel \u003cem\u003eSEMEN -\u003c/em\u003e B). Notably, MDBK monolayers exposed to semen treated with this peptide concentration displayed normal cell growth patterns, indicating that indolicidin at 30 \u0026micro;M did not exert cytotoxic effects under the conditions evaluated.\u003c/p\u003e\n\u003ch3\u003eEffect of indolicidin on bovine semen\u003c/h3\u003e\n\u003cp\u003eTo confirm the absence of adverse effects of indolicidin on bovine semen, the MIC (30 \u0026micro;M) identified in the in vitro antiviral activity in infected semen assay was assessed together with an intentionally high peptide concentration (100 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Treatment with 30 \u0026micro;M indolicidin showed no significant differences in any parameter when compared with the untreated control, indicating that this concentration did not impair sperm quality. Curiously, in very high concentrations (100 \u0026micro;M), indolicidin did not significantly affect VAP, VSL, VCL, ALH, STR or LIN values relative to the control group. Although BCF increased at 100 \u0026micro;M, this value remained within the range observed for the control. Despite maintaining most kinematic standards at this concentration, a significant reduction in straight and progressive motility (MT and MP) was detected, rendering semen treated at 100 \u0026micro;M unsuitable for insemination.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSafety test of indolicidin in bovine semen using the peptide at 30 \u0026micro;M and 100 \u0026micro;M incubated for 30 minutes and evaluated by CASA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemen quality parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIndolicidin\u003c/p\u003e \u003cp\u003e30 \u0026micro;M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIndolicidin\u003c/p\u003e \u003cp\u003e100 \u0026micro;M\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMT (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMP (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVAP (\u0026micro;m/s)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e114.9\u0026thinsp;\u0026plusmn;\u0026thinsp;34.8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e120.3\u0026thinsp;\u0026plusmn;\u0026thinsp;41.4a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e114.3\u0026thinsp;\u0026plusmn;\u0026thinsp;37.6a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVSL(\u0026micro;m/s)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.6\u0026thinsp;\u0026plusmn;\u0026thinsp;15.8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.4\u0026thinsp;\u0026plusmn;\u0026thinsp;14.8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVCL(\u0026micro;m/s)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e205.8\u0026thinsp;\u0026plusmn;\u0026thinsp;73.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e215.8\u0026thinsp;\u0026plusmn;\u0026thinsp;85.8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e209.5\u0026thinsp;\u0026plusmn;\u0026thinsp;80.2a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eALH (\u0026micro;m)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBCF (Hz)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSTR (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67\u0026thinsp;\u0026plusmn;\u0026thinsp;11.3a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLIN (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43 \u0026plusmn;\u0026thinsp;8.5a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMeans followed by the same lowercase letter between columns do not differ significantly from each other (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). MT: Straight motility; MP: Progressive motility; VAP: Average path velocity; VSL: straight-line velocity; VCL: curvilinear velocity; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; STR: Straightness; LIN: Linearity.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDifferent mechanisms of action previously described for indolicidin may help explain the peptide activity against BoAHV-1 in this study. Falla et al. (1996)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e reported that, in Gram-negative bacteria, the peptide is able to traverse the outer membrane and induce disruption of the plasma membrane. In contrast, Subbalakshmi et al. (2000)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e did not observe membrane lysis in \u003cem\u003eE. coli\u003c/em\u003e, but instead documented an increase in membrane permeability that facilitated peptide entry, followed by inhibition of DNA and protein synthesis within the cytoplasm.\u003c/p\u003e \u003cp\u003eGiven that BoAHV-1 consists of an icosahedral capsid covered by a host-derived lipid bilayer envelope\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, it is plausible that these mechanisms could act in combination. Glycoproteins present in the viral envelope are directly responsible for the adsorption of the virus to specific cell receptors present in the host cell membrane during the initial stages of infection\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Therefore, direct disruption of the viral envelope would provide a virucidal effect, compromising the structural integrity of the virion, whereas interaction of indolicidin with viral DNA could exert an intracellular antiviral effect, preventing genome replication after entry into host cells. Together, these complementary actions offer a coherent explanation for the marked reduction in BoAHV-1 infectivity observed in our assays.\u003c/p\u003e \u003cp\u003eWhen comparing the MIC of indolicidin in culture medium with that required in bovine semen, a marked increase in the concentration needed for complete viral inactivation was evident. This discrepancy is likely due to the complex biochemical composition of semen, which contains carbohydrate, proteins, lipids and other biomolecules capable of binding or sequestering antimicrobial peptides. Such interactions may reduce the amount of free, biologically active indolicidin available to interact with viral targets, thereby requiring higher concentrations to achieve effective BoAHV-1 inactivation. This matrix effect offers a plausible explanation for the MIC adjustment (30 \u0026micro;M) required in the semen applications.\u003c/p\u003e \u003cp\u003eAlthough complete viral inactivation was achieved with 30 \u0026micro;M indolicidin, it is important to consider that the viral titre used for experimental contamination in this study (10⁵\u0026middot;⁷⁵ TCID₅₀/mL) was intentionally high and exceeds the levels typically detected in naturally infected semen. Considering that natural infections generally present titres around 10\u0026sup2; TCID₅₀/mL\u003csup\u003e16\u003c/sup\u003e, it is reasonable to assume that 10 \u0026micro;M indolicidin could be sufficient for complete disinfection, as this concentration reduced the viral titre by approximately 4 logs.\u003c/p\u003e \u003cp\u003eSperm motility remains a key parameter for evaluating semen quality, as motile spermatozoa are essential for reaching the female reproductive tract and achieving fertilisation. Although all motilities parameters were maintained when 30 \u0026micro;M of indolicidin was used to semen disinfection, an overestimated peptide concentration (100 \u0026micro;M) was considered detrimental to sperm viability, as a minimum motility threshold of 50% is diserable for acceptable reproductive performance. Compared to the control, the significant reduction in straight and progressive motility observed at this concentration renders its use impractical for insemination.\u003c/p\u003e \u003cp\u003eThe antimicrobial activity of bioactive peptides is well recognised\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, however, their use as disinfectants in semen remains incipient. A recent review by Kumar et al. (2024)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e offers a comprehensive overview of the origin and potential application of AMPs in the semen of several species, including peptides in boar semen (hexapeptides c-WFW (10 \u0026micro;M) and c-WWW (20 \u0026micro;M), PMAP-37 peptide (3\u0026micro;M) and bacteriocin (1%), nisin peptide (50 \u0026micro;g/mL) in rat, LL-37 (10 \u0026micro;M) peptide in goat and a human β-defensin (10 \u0026micro;g/mL). Even less explored is the application of such peptides in bovine semen. S\u0026aacute;nchez-Acosta et al. (2020)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e demonstrated that dermaseptin S4 derivatives (15 \u0026micro;M) exhibited in vitro antibacterial activity without impairing sperm motility, however, no previous study has assessed the use of AMPs for viral inactivation in semen. Therefore, indolicidin emerges as a promising candidate to address this gap, offering a feasible alternative for broadening the range of strategies applicable to semen preservation.\u003c/p\u003e \u003cp\u003ePrevious studies have reported bactericidal activity of indolicidin at concentrations up to 32 \u0026micro;M\u003csup\u003e20\u003c/sup\u003e. Thus, the complete viral inactivation observed neste trabalho in semen with 30 \u0026micro;M indolicidin is particularly relevant, as this concentration may provide a broad-spectrum disinfectant effect in a single step. Such an approach could help reduce or even eliminate the use of conventional antibiotics in semen extenders, thereby contributing to antimicrobial management. Moreover, the methodology employed here is readily adaptable to routine procedures in semen collection and processing centres, offering a favourable cost-benefit profile.\u003c/p\u003e \u003cp\u003eIndolicidin also possesses physicochemical features of interest for industrial application. Its short length allows high yield synthesis, while the feasibility of recombinant production offers a cost-effective route for large scale manufacturing, supporting its potential commercial use.\u003c/p\u003e \u003cp\u003eDespite BoAHV-1 being used exclusively in this study as a model virus to evaluate the disinfectant potential of indolicidin, the peptide\u0026rsquo;s mode of action indicates that its applicability may extend beyond this pathogen. Also considering the bacterias, several other viruses of reproductive importance are enveloped, and indolicidin\u0026rsquo;s known membranolytic and intracellular activities suggest that it may exert enhanced antiviral effects against this broader group of agents. This raises the prospect that indolicidin could serve as a versatile tool for controlling a range of semen-borne pathogens in reproductive technologies.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIndolicidin demonstrated marked in vitro antiviral activity against BoAHV-1, achieving complete viral inactivation in cell culture and experimentally infected bovine semen. A concentration of 30 \u0026micro;M was required for full inactivation in semen, likely due to matrix effects, and did not impair sperm kinematic parameters, whereas higher concentrations negatively affected sperm motility. These findings indicate that indolicidin can effectively inactivate BoAHV-1 in semen under experimental conditions while preserving sperm quality, supporting its potential use as a semen disinfectant. This peptide represents a promising alternative to conventional antimicrobial strategies in semen processing and warrants further investigation under field and fertility conditions.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eSTATEMENT OF ANIMAL ETHICS\u003c/h2\u003e \u003cp\u003eAll procedures were approved by the Ethics Committee on Animal Experimentation of the Federal University of Goi\u0026aacute;s (protocol 012/22).\u003c/p\u003e \u003ch2\u003eCONFLICTS OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJo\u0026atilde;o Paulo Yoshio Prado Cerqueira Kubota: Writing \u0026ndash; original draft, Methodology, Investigation. Gabriel Henrique Santos: Writing \u0026ndash; original draft, Methodology, InvestigationTaise Maria dos Anjos Oliveira: Methodology, Investigation. Maria L\u0026uacute;cia Gambarini: Methodology, Investigation, Formal analysis. Keyla de Oliveira Ribeiro: Methodology, Investigation, Data analysis. Klayto Jos\u0026eacute; Gon\u0026ccedil;alves dos Santos: Methodology (CASA).Guilherme Sastre de Souza: Methodology (peptide synthesis, purification, and characterization). Maria Carolina Oliveira de Arruda Brasil: Methodology (peptide synthesis, purification, and characterization).Eduardo Maffud Cilli: Resources, Methodology, Supervision.Esteban Nicol\u0026aacute;s Lorenz\u0026oacute;n: Resources, Writing \u0026ndash; review \u0026amp; editing, Conceptualization, Methodology, Investigation, Supervision, Formal analysis. Guilherme Rocha Lino de Souza: Resources, Writing \u0026ndash; review \u0026amp; editing, Conceptualization, Methodology, Investigation, Supervision, Formal analysis.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge the financial support by National Council for Scientific and Technological Development (CNPq), grant no. 427961/2018-1, 302390/2023-5, 141840/2023-4 and Goi\u0026aacute;s Research Foundation (FAPEG), grant no. 202310267000475.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLe CF, Fang CM, Sekaran SD (2017) Intracellular Targeting Mechanisms by Antimicrobial Peptides. Antimicrob Agents Chemother 61(4):e02340\u0026ndash;e02316\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelsted ME, Novotny MJ, Morris WL et al (1992) Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J Biol Chem 267(7):4292\u0026ndash;4295\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar M, Ranjan R, Bhardwaj A (2024) Antimicrobial peptide and their role in sperm cryopreservation. J Appl Biol Biotechnol 12(3):8\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorrell JM, Malaluang P, Cojkic A et al (2022) Alternatives to Antibiotics in Semen Extenders Used in Artificial Insemination. In: The Global Antimicrobial Resistance Epidemic - Innovative Approaches and Cutting-Edge Solutions [Internet]. \u003cem\u003eIntechOpen\u003c/em\u003e; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.intechopen.com/chapters/81891\u003c/span\u003e\u003cspan address=\"https://www.intechopen.com/chapters/81891\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5772/intechopen.104226\u003c/span\u003e\u003cspan address=\"10.5772/intechopen.104226\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 25 Nov 2025)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnimal Diseases. WOAH - World Organisation for Animal Health (2025) Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.woah.org/en/what-we-do/animal-health-and-welfare/animal-diseases/\u003c/span\u003e\u003cspan address=\"https://www.woah.org/en/what-we-do/animal-health-and-welfare/animal-diseases/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 25 Nov 2025)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerrifield RB (1963) Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J Am Chem Soc 85(14):2149\u0026ndash;2154\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamilton MA, Russo RC, Thurston RV (1977) Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environ Sci Technol 11(7):714\u0026ndash;719\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrunner D, Engels M, Schwyzer M et al (1988) A Comparison of Three Techniques for Detecting Bovine Herpesvirus Type 1 (BHV-1) in Naturally and Experimentally Contaminated Bovine Semen. Reprod Domest Anim 23(1):1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Col\u0026eacute;gio B (2025) Reprodu\u0026ccedil;\u0026atilde;o Animal (CBRA). CBRA \u0026ndash; Col\u0026eacute;gio Brasileiro de Reprodu\u0026ccedil;\u0026atilde;o Animal. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbra.org.br/portal/\u003c/span\u003e\u003cspan address=\"http://www.cbra.org.br/portal/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 25 Nov 2025)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmann RP, Waberski D (2014) Computer-assisted sperm analysis (CASA): capabilities and potential developments. Theriogenology 81(1):5\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ee1-3\u003c/span\u003e\u003cspan address=\"http://e1-3\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Core Team. R: The R Project for Statistical Computing [Internet] (2025) Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.r-project.org/\u003c/span\u003e\u003cspan address=\"https://www.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 25 Nov 2025)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalla T, Karunaratne N, Hancock REW (1996) Mode of Action of the Antimicrobial Peptide Indolicidin. J Biol Chem 271(32):19298\u0026ndash;19303\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubbalakshmi C, Bikshapathy E, Sitaram N et al (2000) Antibacterial and hemolytic activities of single tryptophan analogs of indolicidin. Biochem Biophys Res Commun 274(3):714\u0026ndash;716\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Oirschot JT (1995) Bovine herpesvirus 1 in semen of bulls and the risk of transmission: A brief review. Vet Q 17(1):29\u0026ndash;33\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuylkens B, Thiry J, Kirten P et al (2007) Bovine herpesvirus 1 infection and infectious bovine rhinotracheitis. Vet Res 38(2):181\u0026ndash;209\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlores EF, Weiblen R, Vogel FSF et al (2005) A infec\u0026ccedil;\u0026atilde;o pelo v\u0026iacute;rus da diarr\u0026eacute;ia viral bovina (BVDV) no Brasil: hist\u0026oacute;rico, situa\u0026ccedil;\u0026atilde;o atual e perspectivas. Pesq Vet Bras 25:125\u0026ndash;134\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVasilchenko AS, Vasilchenko AV, Pashkova TM et al (2017) Antimicrobial activity of the indolicidin-derived novel synthetic peptide In-58. J Pept Sci 23(12):855\u0026ndash;863\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShagaghi N, Palombo EA, Clayton AHA et al (2016) Archetypal tryptophan-rich antimicrobial peptides: properties and applications. World J Microbiol Biotechnol 32(2):31\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026aacute;nchez-Acosta YA, Castillo Vargas JA, Ram\u0026iacute;rez Quintero KJ et al (2020) Peptide derivatives of dermaseptin S4 in fresh bovine semen for bacterial contamination control: Physicochemical and structural characterization, antibacterial potency, and effects on red blood and sperm cells. Reprod Domest Anim 55(8):905\u0026ndash;914\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVergis J, Malik SS, Pathak R et al (2019) Antimicrobial Efficacy of Indolicidin Against Multi-Drug Resistant Enteroaggregative Escherichia coli in a Galleria mellonella Model. Front Microbiol 10:2723\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Semen disinfection, Indolicidin, virus inactivation, BoAHV1","lastPublishedDoi":"10.21203/rs.3.rs-8605122/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8605122/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBovine alphaherpesvirus 1 (BoAHV1) is a reproductively important pathogen whose presence in bovine semen restricts international trade. The increasing use of controlled breeding programmes highlights the need for effective viral inactivation strategies. This study evaluated the antiviral activity of the antimicrobial peptide indolicidin in bovine semen experimentally infected with BoAHV1. Indolicidin was synthesised by solid-phase peptide synthesis. Antiviral activity and minimum inhibitory concentration (MIC) were determined in MDBK cell cultures. The peptide was then tested in infected semen, and semen quality was assessed using computer-assisted sperm analysis (CASA). Indolicidin inhibited BoAHV1 in MDBK cells with an MIC of 8 \u0026micro;M. In semen, viral titre was significantly reduced at 10 \u0026micro;M with complete inactivation at 30 \u0026micro;M, confirmed by the absence of cytopathic effect in MDBK cells. CASA analysis demonstrated that semen quality parameters were preserved at concentrations up to 30 \u0026micro;M, while 100 \u0026micro;M caused a significant reduction in straight and progressive motility.\u003c/p\u003e","manuscriptTitle":"Evaluation of Indolicidin for Inactivation of Bovine Alphaherpesvirus 1 in Semen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-05 20:26:30","doi":"10.21203/rs.3.rs-8605122/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-22T07:46:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-22T01:05:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-22T01:05:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2026-01-14T21:13:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7c3d0c63-3d8b-43ff-8d58-43df81ad268b","owner":[],"postedDate":"February 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-25T07:40:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-05 20:26:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8605122","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8605122","identity":"rs-8605122","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}