Tissue‑specific effects of in ovo prebiotic stimulation on cytokine gene expression in broiler chickens

Tissue‑specific effects of in ovo prebiotic stimulation on cytokine gene expression in broiler chickens | 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 Research Article Tissue‑specific effects of in ovo prebiotic stimulation on cytokine gene expression in broiler chickens Maria Siwek, Elżbieta Pietrzak, Aleksandra Dunisławska This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8892868/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract The modulation of the immune system during the early life of broiler chickens has been shown to have a beneficial effect on disease resistance and overall health. The present study investigated the effects of in ovo administration of xylo- and mannooligosaccharides (XOS and MOS) on cytokine gene expression in two immune-relevant tissues: the spleen and cecal tonsils. The injection of fertilized eggs was conducted on embryonic day 12, with the substances xylotriose (XOS3), xylotetraose (XOS4), mannotriose (MOS3), mannotetraose (MOS4), or saline (control) utilised for this purpose. The gene expression of cytokines representing general immune regulation (IL1β, IL2, IL10), Th1 (IL8, IL12, IFNβ, IFNγ), Th2 (IL4, IL6), and Th17 (IL17) pathways was quantified in 42-day-old birds using RT-qPCR. A significant immunomodulatory effect was observed in the spleen, where XOS3 consistently increased the expression of cytokines associated with Th1-, Th2-, and Th17-mediated responses, indicating enhanced systemic immune activation. Conversely, no statistically significant differences were detected in the cecal tonsils, although numerical variation was present. This muted mucosal response is likely to reflect the inherent heterogeneity and tolerance-oriented function of gut-associated lymphoid tissue, consistent with the concept of acquired immunotolerance to commensal microbiota. Collectively, these findings demonstrate that in ovo prebiotic stimulation exerts tissue-specific effects, promoting systemic immune readiness while maintaining mucosal homeostasis, and highlight XOS3 as a particularly effective modulator of splenic cytokine expression in broiler chickens. cecal tonsils spleen xylooligosaccharides mannooligosaccharides Full Text Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 01 Apr, 2026 Reviewers invited by journal 05 Mar, 2026 Editor assigned by journal 23 Feb, 2026 First submitted to journal 16 Feb, 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. 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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-8892868","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601231953,"identity":"771937a8-b463-4c6c-a355-349f88b02fcf","order_by":0,"name":"Maria Siwek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYDACZgYGxoYKhgQQWwKIZYjUcgahhYcoixgb20jRIt/OY/hw5rw7efINzAdv8zDcIazF4DCPseHGbc+KDQ6wJVvzMDwjQgsz7zbJh9sOJ25g4DGT5mE4TITDmnm3/3w453Di/Ab+b8RpYTjMu41xY8PhxIYDPGzEaTE4zP9ZcsYxoF8OsxlbzjEgwi/y/ccSP/bUAEOsvfnhjTcVd+QIOwwCDoDjFGjpAWJ1MBzAYIyCUTAKRsEogAMAII48gBOciMoAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4148-5174","institution":"University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"","lastName":"Siwek","suffix":""},{"id":601231955,"identity":"cd78764a-71b5-4c26-a457-2b9d7049bf7f","order_by":1,"name":"Elżbieta Pietrzak","email":"","orcid":"","institution":"Bydgoszcz University of Science and Technology: Uniwersytet Technologiczno-Przyrodniczy im Jana i Jedrzeja Sniadeckich w Bydgoszczy","correspondingAuthor":false,"prefix":"","firstName":"Elżbieta","middleName":"","lastName":"Pietrzak","suffix":""},{"id":601231956,"identity":"9e737c39-0c73-4629-be8c-906f7e144215","order_by":2,"name":"Aleksandra Dunisławska","email":"","orcid":"","institution":"Bydgoszcz University of Science and Technology: Uniwersytet Technologiczno-Przyrodniczy im Jana i Jedrzeja Sniadeckich w Bydgoszczy","correspondingAuthor":false,"prefix":"","firstName":"Aleksandra","middleName":"","lastName":"Dunisławska","suffix":""}],"badges":[],"createdAt":"2026-02-16 11:45:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8892868/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8892868/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104325227,"identity":"b7624cf5-a4fd-418c-9bd2-8746e0d66d51","added_by":"auto","created_at":"2026-03-10 13:57:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":775793,"visible":true,"origin":"","legend":"","description":"","filename":"SiwekM.etalsubmission.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8892868/v1_covered_11a48588-41dd-44ec-be6e-46a1486a7087.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTissue‑specific effects of in ovo prebiotic stimulation on cytokine gene expression in broiler chickens\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joag","sideBox":"Learn more about [Journal of Applied Genetics](https://www.springer.com/journal/13353)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/joag/default.aspx","title":"Journal of Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cecal tonsils, spleen, xylooligosaccharides, mannooligosaccharides","lastPublishedDoi":"10.21203/rs.3.rs-8892868/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8892868/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The modulation of the immune system during the early life of broiler chickens has been shown to have a beneficial effect on disease resistance and overall health. The present study investigated the effects of in ovo administration of xylo- and mannooligosaccharides (XOS and MOS) on cytokine gene expression in two immune-relevant tissues: the spleen and cecal tonsils. The injection of fertilized eggs was conducted on embryonic day 12, with the substances xylotriose (XOS3), xylotetraose (XOS4), mannotriose (MOS3), mannotetraose (MOS4), or saline (control) utilised for this purpose. The gene expression of cytokines representing general immune regulation (IL1β, IL2, IL10), Th1 (IL8, IL12, IFNβ, IFNγ), Th2 (IL4, IL6), and Th17 (IL17) pathways was quantified in 42-day-old birds using RT-qPCR. A significant immunomodulatory effect was observed in the spleen, where XOS3 consistently increased the expression of cytokines associated with Th1-, Th2-, and Th17-mediated responses, indicating enhanced systemic immune activation. Conversely, no statistically significant differences were detected in the cecal tonsils, although numerical variation was present. This muted mucosal response is likely to reflect the inherent heterogeneity and tolerance-oriented function of gut-associated lymphoid tissue, consistent with the concept of acquired immunotolerance to commensal microbiota. 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