Host genetic variation in SPINK5 regulates rumen epithelial barrier function and shapes heritable microbial communities through SCFA-mediated mechanisms in dairy cattle

Host genetic variation in SPINK5 regulates rumen epithelial barrier function and shapes heritable microbial communities through SCFA-mediated mechanisms in dairy cattle | 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 Article Host genetic variation in SPINK5 regulates rumen epithelial barrier function and shapes heritable microbial communities through SCFA-mediated mechanisms in dairy cattle Zheng Lai, Zhang Chen, Junru Tian, Xinyi Zheng, Jichao Li, Jiyou Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8264600/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background : Despite standardized feeding and management protocols, substantial variations in rumen microbial composition and fermentation profiles among dairy cattle suggest significant host genetic influence. Although genome-wide association studies have identified genetic polymorphisms linked to microbial abundances, the physiological mechanisms through which host genetics regulate rumen microecology remain largely unexplored. This knowledge gap limits our understanding of host-microbiome interactions and hinders the development of precision breeding strategies for optimizing rumen function. Results: We integrated host genomic, rumen microbiomic, and metabolomic data from 603 lactating Holstein dairy cows across five commercial farms to elucidate genetic mechanisms governing rumen function. Population structure analysis revealed four distinct ancestral clusters with clear genetic differentiation. Genetic clustering significantly influenced rumen microbial β-diversity across bacteria, methanogens, anaerobic fungi, and protozoa, explaining 4-22% of variation while controlling for farm-specific environmental effects. Species-level heritability analysis identified 54 heritable microbial taxa (h² ≥ 0.2) concentrated among keystone species, collectively representing 23% of bacterial abundance and 44% of methanogenic abundance. Fermentation parameters showed high heritability, with propionate exhibiting the highest genetic control (h² = 0.670). Genome-wide association analysis identified a chromosomal hotspot containing 13 significant SNPs, with fine-mapping localizing strongest associations to SPINK5 (serine peptidase inhibitor Kazal type 5). Functional validation revealed that SPINK5 , expressed specifically in the spinous layer of rumen stratified squamous epithelium, controls epithelial keratinization and barrier permeability. SPINK5 knockdown accelerated keratinization by upregulating nine keratin genes and desmosomal markers, strengthening epithelial barriers and reducing SCFA absorption, while overexpression increased epithelial permeability. Co-occurrence network analysis demonstrated that moderate SCFA concentrations promoted maximum connectivity among heritable microbes, indicating optimal conditions for microbial syntrophic relationships. Conclusions: This study reveals an innovative physiological mechanism whereby host genetic variants regulate rumen microbiota through SPINK5 -mediated epithelial barrier control. SPINK5 functions as a molecular rheostat that modulates epithelial permeability and paracellular SCFA absorption, thereby controlling intraluminal fermentation environments that shape heritable microbial communities. These findings provide actionable genetic markers for incorporating microbiome-associated traits into genomic selection programs, offering new strategies for enhancing rumen fermentation efficiency, reducing methane emissions, and improving dairy productivity through precision breeding approaches. Biological sciences/Genetics Biological sciences/Microbiology Rumen microbiota Heritability SCFAs concentrations GWAS SPINK5 Keratinization Full Text Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx TableS2.xlsx TableS3.docx FigureS1.docx FigureS2.docx FigureS3.docx GraphicalAbstract.tif Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Mar, 2026 Reviews received at journal 15 Mar, 2026 Reviewers agreed at journal 09 Mar, 2026 Reviews received at journal 11 Feb, 2026 Reviewers agreed at journal 17 Jan, 2026 Reviewers agreed at journal 15 Jan, 2026 Reviewers invited by journal 14 Jan, 2026 Editor assigned by journal 23 Dec, 2025 Submission checks completed at journal 05 Dec, 2025 First submitted to journal 02 Dec, 2025 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8264600","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":576074146,"identity":"75c713a5-cca2-4db9-ae92-714e7c7fabbb","order_by":0,"name":"Zheng Lai","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Lai","suffix":""},{"id":576074149,"identity":"bdb0239f-89f7-442b-a09b-83e1ba68fdc5","order_by":1,"name":"Zhang Chen","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Chen","suffix":""},{"id":576074150,"identity":"22f7a5f7-be89-4b45-9b78-c50d166cd5c2","order_by":2,"name":"Junru Tian","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Junru","middleName":"","lastName":"Tian","suffix":""},{"id":576074151,"identity":"4bf54c22-4443-4074-8092-6249de38f536","order_by":3,"name":"Xinyi Zheng","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Zheng","suffix":""},{"id":576074152,"identity":"7ee4e8af-c85d-4c7d-9bbc-e835f524364a","order_by":4,"name":"Jichao Li","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jichao","middleName":"","lastName":"Li","suffix":""},{"id":576074153,"identity":"c58f923a-024b-445c-a8f2-7a096f37d32e","order_by":5,"name":"Jiyou Zhang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiyou","middleName":"","lastName":"Zhang","suffix":""},{"id":576074154,"identity":"42470c8b-83c9-435f-bd58-647ea3d426ba","order_by":6,"name":"Zan Huang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zan","middleName":"","lastName":"Huang","suffix":""},{"id":576074155,"identity":"c844cf53-0741-44ce-9975-e86e28869d80","order_by":7,"name":"Shengyong Mao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYJACZjjrAwMbmJYgWgvjDJK1MPNAGXi16LYfPvy5oOKO3YbjvYdf2+7gkzc4wHzwNg+DXR4uLWZn0tKkZ5x5lrzhzLk069wzbIYbDrAlW/MwJBfj1HIgx4yZt+1wssGNHDPj3DY2xg0HeMykeRgOJDbg0nL+jfFn3n9QLZZtbPYbDvB/w6/lRo6BNG/DYTugFuPHjG1siUBb2AhoeZYmzXPscILkmTNmjL1tbMkzD7MZW84xSMbjsOTDn3lqDtvzHe8x/vCz7Zht3/HmhzfeVNjh1AIDiQsOMLABo+MYNJoMCKgHAnv5BgbmDwwMNYSVjoJRMApGwYgDAEp1W9rIkphCAAAAAElFTkSuQmCC","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Shengyong","middleName":"","lastName":"Mao","suffix":""}],"badges":[],"createdAt":"2025-12-03 00:23:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8264600/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8264600/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100554904,"identity":"d4681799-3029-4713-9b48-82e033054293","added_by":"auto","created_at":"2026-01-19 08:39:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1265014,"visible":true,"origin":"","legend":"","description":"","filename":"MainManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8264600/v1_covered_d4b8a21c-154a-443a-bd42-34c84c5034af.pdf"},{"id":100549461,"identity":"541965fb-8141-4a86-a3d4-ad9ebf266508","added_by":"auto","created_at":"2026-01-19 08:23:23","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":34547,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8264600/v1/751982ee6ef927a3ad9fc325.xlsx"},{"id":100544583,"identity":"7f7b188a-2f67-4432-9c4d-5b177a331002","added_by":"auto","created_at":"2026-01-19 06:21:21","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":63949,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8264600/v1/2802719fbe9f313dd6ac1f12.xlsx"},{"id":100544580,"identity":"e244ec3a-9148-4db5-aaed-e83aba4eec95","added_by":"auto","created_at":"2026-01-19 06:21:20","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21480,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8264600/v1/36e8073a1ac92aab8ea42e90.docx"},{"id":100544585,"identity":"662bdc05-7914-41ee-b125-2cf22aac5af0","added_by":"auto","created_at":"2026-01-19 06:21:21","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":352095,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8264600/v1/ad4ba623cc32ace109f9f88e.docx"},{"id":100549273,"identity":"f33a1102-73a7-4067-880a-c6f18a8eeca9","added_by":"auto","created_at":"2026-01-19 08:22:59","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":51616,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8264600/v1/a5e180141dab00dc0c4bf78c.docx"},{"id":100544582,"identity":"32ebd5de-af95-4126-b9c3-097f2dfa0017","added_by":"auto","created_at":"2026-01-19 06:21:20","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":31539,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8264600/v1/062af285cc27a4ce435ec00a.docx"},{"id":100544614,"identity":"a0a5dc65-c9f0-42c1-b8f2-4ca9976bb8b0","added_by":"auto","created_at":"2026-01-19 06:21:37","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":465094568,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-8264600/v1/f82ed087b12bddef367ef28f.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Host genetic variation in SPINK5 regulates rumen epithelial barrier function and shapes heritable microbial communities through SCFA-mediated mechanisms in dairy cattle","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":"npj-biofilms-and-microbiomes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjbiofilms","sideBox":"Learn more about [npj Biofilms and Microbiomes](http://www.nature.com/npjbiofilms/)","snPcode":"41522","submissionUrl":"https://submission.springernature.com/new-submission/41522/3","title":"npj Biofilms and Microbiomes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rumen microbiota, Heritability, SCFAs concentrations, GWAS, SPINK5, Keratinization","lastPublishedDoi":"10.21203/rs.3.rs-8264600/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8264600/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Despite standardized feeding and management protocols, substantial variations in rumen microbial composition and fermentation profiles among dairy cattle suggest significant host genetic influence. Although genome-wide association studies have identified genetic polymorphisms linked to microbial abundances, the physiological mechanisms through which host genetics regulate rumen microecology remain largely unexplored. This knowledge gap limits our understanding of host-microbiome interactions and hinders the development of precision breeding strategies for optimizing rumen function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe integrated host genomic, rumen microbiomic, and metabolomic data from 603 lactating Holstein dairy cows across five commercial farms to elucidate genetic mechanisms governing rumen function. Population structure analysis revealed four distinct ancestral clusters with clear genetic differentiation. Genetic clustering significantly influenced rumen microbial β-diversity across bacteria, methanogens, anaerobic fungi, and protozoa, explaining 4-22% of variation while controlling for farm-specific environmental effects. Species-level heritability analysis identified 54 heritable microbial taxa (h² ≥ 0.2) concentrated among keystone species, collectively representing 23% of bacterial abundance and 44% of methanogenic abundance. Fermentation parameters showed high heritability, with propionate exhibiting the highest genetic control (h² = 0.670). Genome-wide association analysis identified a chromosomal hotspot containing 13 significant SNPs, with fine-mapping localizing strongest associations to \u003cem\u003eSPINK5\u003c/em\u003e(serine peptidase inhibitor Kazal type 5). Functional validation revealed that \u003cem\u003eSPINK5\u003c/em\u003e, expressed specifically in the spinous layer of rumen stratified squamous epithelium, controls epithelial keratinization and barrier permeability. \u003cem\u003eSPINK5\u003c/em\u003eknockdown accelerated keratinization by upregulating nine keratin genes and desmosomal markers, strengthening epithelial barriers and reducing SCFA absorption, while overexpression increased epithelial permeability. Co-occurrence network analysis demonstrated that moderate SCFA concentrations promoted maximum connectivity among heritable microbes, indicating optimal conditions for microbial syntrophic relationships.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis study reveals an innovative physiological mechanism whereby host genetic variants regulate rumen microbiota through \u003cem\u003eSPINK5\u003c/em\u003e-mediated epithelial barrier control. \u003cem\u003eSPINK5\u003c/em\u003e functions as a molecular rheostat that modulates epithelial permeability and paracellular SCFA absorption, thereby controlling intraluminal fermentation environments that shape heritable microbial communities. These findings provide actionable genetic markers for incorporating microbiome-associated traits into genomic selection programs, offering new strategies for enhancing rumen fermentation efficiency, reducing methane emissions, and improving dairy productivity through precision breeding approaches.\u003c/p\u003e","manuscriptTitle":"Host genetic variation in SPINK5 regulates rumen epithelial barrier function and shapes heritable microbial communities through SCFA-mediated mechanisms in dairy cattle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 06:21:14","doi":"10.21203/rs.3.rs-8264600/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-18T05:04:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-15T12:37:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12984213764126616695125345079058873917","date":"2026-03-09T23:34:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-12T04:23:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235895054512431468950104912504746472413","date":"2026-01-17T14:12:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36074533501737168279745142136775432867","date":"2026-01-15T05:56:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-15T04:28:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T11:51:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-06T04:19:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Biofilms and Microbiomes","date":"2025-12-03T00:14:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-biofilms-and-microbiomes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjbiofilms","sideBox":"Learn more about [npj Biofilms and Microbiomes](http://www.nature.com/npjbiofilms/)","snPcode":"41522","submissionUrl":"https://submission.springernature.com/new-submission/41522/3","title":"npj Biofilms and Microbiomes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"550134db-5953-47a1-8fe7-2465dc1c0163","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61298167,"name":"Biological sciences/Genetics"},{"id":61298168,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-25T15:38:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 06:21:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8264600","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8264600","identity":"rs-8264600","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}