{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T06:07:53Z","timestamp":1788847673428,"version":"build-2803163510"},"reference-count":54,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    Pseudo-germ-free (PGF) animal models are typically established after 7\u202fdays of antibiotic treatment, mostly validated using 16S rRNA sequencing. However, this method cannot distinguish live from dead bacteria and may overestimate the required depletion time. We investigated whether PGF model establishment in Sprague\u2013Dawley (SD) rats could be completed in shorter than 7\u202fdays using an antibiotic cocktail. Gut microbiota was dynamically monitored with antibiotic cocktail treatment on days 0 to 7 by aerobic\/anaerobic bacterial culture, Gram staining, and 16S rRNA sequencing, alongside body weight, food intake, and water intake. Body weight continued to increase in the Test group, while feed and water intake decreased only transiently during the first 2\u202fdays and returned to normal from day 3. No visible aerobic or anaerobic bacterial growth after 4\u202fdays and persisting through day 7. 16S rRNA sequencing showed that shared amplicon sequence variants (ASVs) with the Control group dropped from 379 on day 0 to 27 on day 4, stabilizing at 19\u201331 ASVs thereafter. Alpha diversity indices showed no significant changes after day 3 (\n                    <jats:italic>p<\/jats:italic>\n                    \u202f&amp;gt;\u202f0.05). Beta diversity showed the peak period of microbiota restructuring on days 2\u20133 (\n                    <jats:italic>R<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    \u202f=\u202f0.558,\n                    <jats:italic>p<\/jats:italic>\n                    \u202f=\u202f0.001), while the effect size between adjacent time points dropped to an extremely low level after day 4 (\n                    <jats:italic>R<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    \u202f\u2264\u202f0.025). LEfSe identified\n                    <jats:italic>Escherichia-Shigella<\/jats:italic>\n                    as the dominant genus. The multi-dimensional data demonstrated that 4\u202fdays of antibiotic cocktail treatment were sufficient to establish a stable PGF state in SD rats, aligning with 3R principles.\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2026.1930662","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T05:36:04Z","timestamp":1788845764000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Multi-dimensional dynamic assessment revealed the antibiotic-induced pseudo-germ-free model in SD rats stabilized within 4 days"],"prefix":"10.3389","volume":"17","author":[{"given":"Yiduo","family":"Zhao","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Hebei Agricultural University","place":["Baoding, Hebei, China"]},{"name":"Technical Innovation Center of Traditional Chinese Veterinary Medicine, Key Laboratory of Veterinary Pharmaceutical Discovery, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences","place":["Lanzhou, Gansu, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Wang","sequence":"additional","affiliation":[{"name":"Technical Innovation Center of Traditional Chinese Veterinary Medicine, Key Laboratory of Veterinary Pharmaceutical Discovery, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences","place":["Lanzhou, Gansu, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Technical Innovation Center of Traditional Chinese Veterinary Medicine, Key Laboratory of Veterinary Pharmaceutical Discovery, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences","place":["Lanzhou, Gansu, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingyan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Technical Innovation Center of Traditional Chinese Veterinary Medicine, Key Laboratory of Veterinary Pharmaceutical Discovery, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences","place":["Lanzhou, Gansu, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiting","family":"Guo","sequence":"additional","affiliation":[{"name":"Technical Innovation Center of Traditional Chinese Veterinary Medicine, Key Laboratory of Veterinary Pharmaceutical Discovery, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences","place":["Lanzhou, Gansu, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaorong","family":"Lu","sequence":"additional","affiliation":[{"name":"Technical Innovation Center of Traditional Chinese Veterinary Medicine, Key Laboratory of Veterinary Pharmaceutical Discovery, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences","place":["Lanzhou, Gansu, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianxi","family":"Li","sequence":"additional","affiliation":[{"name":"Technical Innovation Center of Traditional Chinese Veterinary Medicine, Key Laboratory of Veterinary Pharmaceutical Discovery, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences","place":["Lanzhou, Gansu, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,8]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"473","DOI":"10.1007\/s00018-018-2943-4","article-title":"An insight into gut microbiota and its functionalities","volume":"76","author":"Adak","year":"2019","journal-title":"Cell. Mol. Life Sci."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"397","DOI":"10.1016\/j.bbi.2010.10.023","article-title":"Exposure to a social stressor alters the structure of the intestinal microbiota: implications for stressor-induced immunomodulation","volume":"25","author":"Bailey","year":"2011","journal-title":"Brain Behav. Immun."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"458","DOI":"10.1016\/j.molmed.2016.04.003","article-title":"Antibiotic-induced changes in the intestinal microbiota and disease","volume":"22","author":"Becattini","year":"2016","journal-title":"Trends Mol. Med."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1128\/IAI.05496-11","article-title":"Profound alterations of intestinal microbiota following a single dose of clindamycin results in sustained susceptibility to Clostridium difficile-induced colitis","volume":"80","author":"Buffie","year":"2012","journal-title":"Infect. Immun."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"790","DOI":"10.1038\/nri3535","article-title":"Microbiota-mediated colonization resistance against intestinal pathogens","volume":"13","author":"Buffie","year":"2013","journal-title":"Nat. Rev. Immunol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"5884","DOI":"10.1128\/AEM.01763-14","article-title":"Dead or alive: molecular assessment of microbial viability","volume":"80","author":"Cangelosi","year":"2014","journal-title":"Appl. Environ. Microbiol."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"1765","DOI":"10.3389\/fmicb.2017.01765","article-title":"Next-generation beneficial microbes: the case of Akkermansia muciniphila","volume":"8","author":"Cani","year":"2017","journal-title":"Front. Microbiol."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"16242","DOI":"10.1038\/nmicrobiol.2016.242","article-title":"Relic DNA is abundant in soil and obscures estimates of soil microbial diversity","volume":"2","author":"Carini","year":"2017","journal-title":"Nat. Microbiol."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"621","DOI":"10.1038\/nature11400","article-title":"Antibiotics in early life alter the murine colonic microbiome and adiposity","volume":"488","author":"Cho","year":"2012","journal-title":"Nature"},{"key":"ref10","doi-asserted-by":"publisher","first-page":"2211","DOI":"10.1038\/s41467-022-29919-9","article-title":"Evolution of the murine gut resistome following broad-spectrum antibiotic treatment","volume":"13","author":"de Nies","year":"2022","journal-title":"Nat. Commun."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"1339","DOI":"10.1016\/j.cell.2016.10.043","article-title":"A dietary Fiber-deprived gut microbiota degrades the colonic mucus barrier and enhances pathogen susceptibility","volume":"167","author":"Desai","year":"2016","journal-title":"Cell"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"4554","DOI":"10.1073\/pnas.1000087107","article-title":"Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation","volume":"108","author":"Dethlefsen","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"f323","DOI":"10.1093\/jas\/skaf323","article-title":"The effects of rosmarinic acid and antibiotics on the intestinal morphology, function, and cecal microbiota of broiler chickens","volume":"103","author":"Ding","year":"2025","journal-title":"J. Anim. Sci."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"86","DOI":"10.1186\/s40168-017-0285-3","article-title":"Schr\u00f6dinger\u2019s microbes: tools for distinguishing the living from the dead in microbial ecosystems","volume":"5","author":"Emerson","year":"2017","journal-title":"Microbiome."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"2064","DOI":"10.1038\/s41564-019-0540-4","article-title":"Intestinal serotonin and fluoxetine exposure modulate bacterial colonization in the gut","volume":"4","author":"Fung","year":"2019","journal-title":"Nat. Microbiol."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"1941711","DOI":"10.1080\/19490976.2021.1941711","article-title":"Investigating causality with fecal microbiota transplantation in rodents: applications, recommendations and pitfalls","volume":"13","author":"Gheorghe","year":"2021","journal-title":"Gut Microbes"},{"key":"ref17","doi-asserted-by":"publisher","first-page":"144","DOI":"10.1016\/j.aninu.2025.12.002","article-title":"Total flavonoids from Abrus cantoniensis alleviate fatty liver hemorrhagic syndrome in laying hens by regulating inflammation, oxidative stress, and cecal metabolites and microbiota","volume":"25","author":"Guo","year":"2026","journal-title":"Anim. Nutr."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"2","DOI":"10.1186\/s13568-025-01982-7","article-title":"Strain-level dynamics of Akkermansia muciniphila in the human gut microbiota","volume":"16","author":"Han","year":"2026","journal-title":"AMB Express"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"967","DOI":"10.1126\/science.1240527","article-title":"Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment","volume":"342","author":"Iida","year":"2013","journal-title":"Science"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"192ra85","DOI":"10.1126\/scitranslmed.3006055","article-title":"Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells","volume":"5","author":"Kalghatgi","year":"2013","journal-title":"Sci. Transl. Med."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"1534","DOI":"10.3389\/fphys.2018.01534","article-title":"Mouse microbiota models: comparing germ-free mice and antibiotics treatment as tools for modifying gut bacteria","volume":"9","author":"Kennedy","year":"2018","journal-title":"Front. Physiol."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"90","DOI":"10.1111\/imr.12563","article-title":"The intestinal microbiota: antibiotics, colonization resistance, and enteric pathogens","volume":"279","author":"Kim","year":"2017","journal-title":"Immunol. Rev."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"208","DOI":"10.1128\/CMR.00110-14","article-title":"Current and past strategies for bacterial culture in clinical microbiology","volume":"28","author":"Lagier","year":"2015","journal-title":"Clin. Microbiol. Rev."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1186\/s40168-019-0637-2","article-title":"Sequence and cultivation study of Muribaculaceae reveals novel species, host preference, and functional potential of this yet undescribed family","volume":"7","author":"Lagkouvardos","year":"2019","journal-title":"Microbiome."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1186\/s13073-016-0294-z","article-title":"The effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation","volume":"8","author":"Langdon","year":"2016","journal-title":"Genome Med."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"3289","DOI":"10.3389\/fmicb.2018.03289","article-title":"Comparative evaluation of microbiota engraftment following fecal microbiota transfer in mice models: age, kinetic and microbial status matter","volume":"9","author":"Le Roy","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"155831","DOI":"10.1016\/j.phymed.2024.155831","article-title":"Lung-intestinal axis, Shuangshen granules attenuate lung metastasis by regulating the intestinal microbiota and related metabolites","volume":"132","author":"Li","year":"2024","journal-title":"Phytomedicine"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"e270","DOI":"10.1002\/imt2.270","article-title":"Gut\u2013X axis","volume":"4","author":"Lin","year":"2025","journal-title":"iMeta"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"3669","DOI":"10.1038\/s41467-023-39403-7","article-title":"Gut microbiota Turicibacter strains differentially modify bile acids and host lipids","volume":"14","author":"Lynch","year":"2023","journal-title":"Nat. Commun."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"1481","DOI":"10.1136\/gut.2005.064261","article-title":"Psychological stress in IBD: new insights into pathogenic and therapeutic implications","volume":"54","author":"Mawdsley","year":"2005","journal-title":"Gut"},{"key":"ref32","doi-asserted-by":"publisher","first-page":"1681","DOI":"10.1016\/j.celrep.2015.02.034","article-title":"Tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research","volume":"10","author":"Moullan","year":"2015","journal-title":"Cell Rep."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"96","DOI":"10.1038\/nature12503","article-title":"Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens","volume":"502","author":"Ng","year":"2013","journal-title":"Nature"},{"key":"ref34","doi-asserted-by":"publisher","first-page":"101982","DOI":"10.1016\/j.xcrm.2025.101982","article-title":"The gut microbiota-immune-brain axis: therapeutic implications","volume":"6","author":"O\u2019Riordan","year":"2025","journal-title":"Cell Rep. Med."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"36","DOI":"10.1186\/s40168-016-0181-2","article-title":"Genomic characterization of the uncultured Bacteroidales family S24-7 inhabiting the guts of homeothermic animals","volume":"4","author":"Ormerod","year":"2016","journal-title":"Microbiome."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"1123","DOI":"10.13381\/j.cnki.cjm.202310002","article-title":"Comparison between two methods of administration in establishing pseudaseptic rat model with combined antibiotics","volume":"35","author":"Qi","year":"2023","journal-title":"Chin. J. Microecol."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"1929","DOI":"10.1073\/pnas.1424886112","article-title":"Analysis of gene\u2013environment interactions in postnatal development of the mammalian intestine","volume":"112","author":"Rakoff-Nahoum","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"e17996","DOI":"10.1371\/journal.pone.0017996","article-title":"Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression","volume":"6","author":"Reikvam","year":"2011","journal-title":"PLoS One"},{"key":"ref40","doi-asserted-by":"publisher","first-page":"1682","DOI":"10.1038\/s42255-024-01107-7","article-title":"Broad-spectrum antibiotics disrupt homeostatic efferocytosis","volume":"6","author":"Saavedra","year":"2024","journal-title":"Nat. Metab."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1126\/science.aaf2477","article-title":"Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases","volume":"352","author":"Safarian","year":"2016","journal-title":"Science"},{"key":"ref42","doi-asserted-by":"publisher","first-page":"e00893","DOI":"10.1128\/mBio.00893-14","article-title":"Recovery of the gut microbiome following Fecal microbiota transplantation","volume":"5","author":"Seekatz","year":"2014","journal-title":"MBio"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"2431643","DOI":"10.1080\/19490976.2024.2431643","article-title":"Probiotic significance of Lactobacillus strains: a comprehensive review on health impacts, research gaps, and future prospects","volume":"16","author":"Shah","year":"2024","journal-title":"Gut Microbes"},{"key":"ref44","doi-asserted-by":"publisher","first-page":"5004","DOI":"10.7150\/ijbs.86717","article-title":"High-fat diet promotes colitis-associated tumorigenesis by altering gut microbial butyrate metabolism","volume":"19","author":"Shao","year":"2023","journal-title":"Int. J. Biol. Sci."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"229","DOI":"10.1016\/j.chom.2018.01.003","article-title":"Strain tracking reveals the determinants of bacterial engraftment in the human gut following fecal microbiota transplantation","volume":"23","author":"Smillie","year":"2018","journal-title":"Cell Host Microbe"},{"key":"ref46","first-page":"120","article-title":"Russell and Burch's 3Rs then and now: the need for clarity in definition and purpose","volume":"54","author":"Tannenbaum","year":"2015","journal-title":"J. Am. Assoc. Lab. Anim. Sci."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"3114","DOI":"10.1038\/ncomms4114","article-title":"Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection","volume":"5","author":"Theriot","year":"2014","journal-title":"Nat. Commun."},{"key":"ref48","first-page":"600","article-title":"Administration of substances to laboratory animals: routes of administration and factors to consider","volume":"50","author":"Turner","year":"2011","journal-title":"J. Am. Assoc. Lab. Anim. Sci."},{"key":"ref49","doi-asserted-by":"publisher","first-page":"971","DOI":"10.1126\/science.1240537","article-title":"The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide","volume":"342","author":"Viaud","year":"2013","journal-title":"Science"},{"key":"ref50","doi-asserted-by":"publisher","first-page":"4985","DOI":"10.1128\/AEM.00753-08","article-title":"Ecological role of lactobacilli in the gastrointestinal tract: implications for fundamental and biomedical research","volume":"74","author":"Walter","year":"2008","journal-title":"Appl. Environ. Microbiol."},{"key":"ref51","doi-asserted-by":"publisher","first-page":"1536","DOI":"10.1128\/IAI.01104-10","article-title":"Antibiotic treatment alters the colonic mucus layer and predisposes the host to ExacerbatedCitrobacter rodentium -induced colitis","volume":"79","author":"Wlodarska","year":"2011","journal-title":"Infect. Immun."},{"key":"ref52","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1186\/s40168-022-01255-6","article-title":"The microbiota-gut-brain axis participates in chronic cerebral hypoperfusion by disrupting the metabolism of short-chain fatty acids","volume":"10","author":"Xiao","year":"2022","journal-title":"Microbiome."},{"key":"ref53","doi-asserted-by":"publisher","first-page":"237","DOI":"10.3389\/fcimb.2017.00237","article-title":"Reducing viability bias in analysis of gut microbiota in preterm infants at risk of NEC and sepsis","volume":"7","author":"Young","year":"2017","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref54","doi-asserted-by":"publisher","first-page":"2872","DOI":"10.1038\/s41467-018-05336-9","article-title":"Antibiotic-induced microbiome depletion alters metabolic homeostasis by affecting gut signaling and colonic metabolism","volume":"9","author":"Zarrinpar","year":"2018","journal-title":"Nat. Commun."},{"key":"ref55","doi-asserted-by":"publisher","first-page":"182","DOI":"10.1016\/j.aninu.2024.08.008","article-title":"Improving fatty liver hemorrhagic syndrome in laying hens through gut microbiota and oxylipin metabolism by Bacteroides fragilis: a potential involvement of arachidonic acid","volume":"20","author":"Zhang","year":"2025","journal-title":"Anim. Nutr."},{"key":"ref56","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1186\/s40104-025-01319-1","article-title":"Metabolite-mediated crosstalk: unraveling the interactions between gut microbiota and host in fatty liver hemorrhagic syndrome of laying hens","volume":"17","author":"Zhang","year":"2026","journal-title":"J. Anim. Sci. 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