{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T14:29:11Z","timestamp":1785248951039,"version":"3.55.0"},"reference-count":40,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T00:00:00Z","timestamp":1780099200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T00:00:00Z","timestamp":1780099200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100008335","name":"Arizona Biomedical Research Commission","doi-asserted-by":"publisher","award":["RFGA 2023\u2010008\u201029"],"award-info":[{"award-number":["RFGA 2023\u2010008\u201029"]}],"id":[{"id":"10.13039\/100008335","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000055","name":"National Institute on Deafness and Other Communication Disorders","doi-asserted-by":"publisher","award":["R01DC020889"],"award-info":[{"award-number":["R01DC020889"]}],"id":[{"id":"10.13039\/100000055","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000050","name":"National Heart, Lung, and Blood Institute","doi-asserted-by":"publisher","award":["R00HL164973"],"award-info":[{"award-number":["R00HL164973"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000050","name":"National Heart, Lung, and Blood Institute","doi-asserted-by":"publisher","award":["HL 141249"],"award-info":[{"award-number":["HL 141249"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000050","name":"National Heart, Lung, and Blood Institute","doi-asserted-by":"publisher","award":["T32 HL007249"],"award-info":[{"award-number":["T32 HL007249"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["physoc.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The Journal of Physiology"],"abstract":"\n Abstract<\/jats:title>\n \n This retrospective study challenges the assumption that physiological variance among neonates is greater between litters than within litters of multiparous species like\u00a0rats. Conventional wisdom holds that litters \u2013 not individual pups \u2013 should be the unit of replication due to shared perinatal environment and genetics. We provide empirical evidence contradicting this view. We measured within\u2010\n versus<\/jats:italic>\n between\u2010litter variance in body weight of outbred Sprague\u2013Dawley (SD) neonatal rats across three cohorts: (1) pups from 24\u00a0mothers weighed on postnatal day 4 (P4); (2) P4 pups from 10 untreated and seven nicotine\u2010exposed litters; and (3) breathing frequency in pups aged P3\u2013P4 from six litters. We used Welch's ANOVA and the intraclass correlation coefficient (ICC) as primary outcome measures. Importantly, ICC quantifies raw variance distribution, unlike ANOVA's mean\u2010square calculation. Despite significant \u2018litter effects\u2019 detected by conventional ANOVA for body weight, ICC analysis revealed that \u223c40\u201375% of total variance in cohort 1 originated within litters. In cohort 2, variance was distributed more equally, but between\u2010litter variance (43%) remained less than within\u2010litter variance (57%). Remarkably, for breathing frequency, nearly 100% of total variance originated within litters. These findings align with the genetics and developmental biology of outbred SD rats: each fetus is genetically unique and possesses its own placenta with distinct blood flow, hormone levels and nutrient delivery. We therefore argue against uniform policies mandating how many offspring per litter to use experimentally. Instead, researchers should base this decision on their specific research question, outcome variables, rat genetics, and careful consideration of the raw variance both within and between litters.\n \n \n image<\/jats:alt-text>\n <\/jats:graphic>\n <\/jats:boxed-text>\n <\/jats:p>\n <\/jats:sec>\n \n Key points<\/jats:title>\n \n \n \n Because rat littermates share a genetic background and perinatal environment, conventional wisdom assumes variance is greater between litters than within litters, which has led statisticians and reviewers to conclude that the litter, not each animal within the litter, is the true unit of replication.<\/jats:p>\n <\/jats:list-item>\n \n Our retrospective analysis of outbred Sprague\u2013Dawley rat pups revealed the opposite: within\u2010litter variance significantly exceeded between\u2010litter variance for body weight (40\u201375% within) and breathing frequency (nearly 100% within).<\/jats:p>\n <\/jats:list-item>\n \n Even in a cohort that included pups from nicotine\u2010exposed litters, within\u2010litter variance (57%) exceeded between\u2010litter variance (43%).<\/jats:p>\n <\/jats:list-item>\n \n Each outbred fetus is genetically unique with its own placenta, umbilical cord, and uterine position \u2013 making substantial within\u2010litter variation expected, not surprising.<\/jats:p>\n <\/jats:list-item>\n \n These findings challenge conventional wisdom about littermate homogeneity and necessitate reconsideration of experimental design practices in developmental research in outbred rat strains.<\/jats:p>\n <\/jats:list-item>\n <\/jats:list>\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1113\/jp289210","type":"journal-article","created":{"date-parts":[[2026,5,31]],"date-time":"2026-05-31T03:01:07Z","timestamp":1780196467000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Challenging conventional wisdom: A fresh look at the litter effect in rats"],"prefix":"10.1113","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8745-2231","authenticated-orcid":false,"given":"Adrianne G.","family":"Huxtable","sequence":"first","affiliation":[{"name":"Department of Human Physiology University of Oregon Eugene OR USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily G.","family":"Flanigan","sequence":"additional","affiliation":[{"name":"Department of Physiology University of Arizona Tucson AZ USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8775-7692","authenticated-orcid":false,"given":"Michael L.","family":"Frazure","sequence":"additional","affiliation":[{"name":"Department of Physiology University of Arizona Tucson AZ USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lila B.","family":"Wollman","sequence":"additional","affiliation":[{"name":"Department of Human Physiology University of Oregon Eugene OR USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenbo","family":"Sun","sequence":"additional","affiliation":[{"name":"The University of Arizona Cancer Center, University of Arizona Tucson AZ USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ritu","family":"Pandey","sequence":"additional","affiliation":[{"name":"The University of Arizona Cancer Center, University of Arizona Tucson AZ USA"},{"name":"Department of Cellular & Molecular Medicine University of Arizona Tucson AZ USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0052-3285","authenticated-orcid":false,"given":"Linda L.","family":"Restifo","sequence":"additional","affiliation":[{"name":"Department of Cellular & Molecular Medicine University of Arizona Tucson AZ USA"},{"name":"Department of Neuroscience University of Arizona Tucson AZ USA"},{"name":"Department of Neurology University of Arizona Tucson AZ USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6849-7530","authenticated-orcid":false,"given":"Ralph F.","family":"Fregosi","sequence":"additional","affiliation":[{"name":"Department of Physiology University of Arizona Tucson AZ USA"},{"name":"Department of Neuroscience University of Arizona Tucson AZ USA"},{"name":"Department of Anesthesiology University of Arizona Tucson AZ USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,5,30]]},"reference":[{"issue":"4","key":"e_1_2_5_2_1","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1002\/dev.420060406","article-title":"Statistical procedure in developmental studies on species with multiple offspring","volume":"6","author":"Abbey H.","year":"1973","journal-title":"Developmental Psychobiology"},{"issue":"1","key":"e_1_2_5_3_1","doi-asserted-by":"crossref","DOI":"10.1590\/1984-3143-ar2021-0134","article-title":"Rodent models in placental research. 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