{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T07:02:15Z","timestamp":1775545335901,"version":"3.50.1"},"reference-count":40,"publisher":"Public Library of Science (PLoS)","issue":"10","license":[{"start":{"date-parts":[[2021,10,28]],"date-time":"2021-10-28T00:00:00Z","timestamp":1635379200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"deutsche forschungsgemeinschaft","doi-asserted-by":"publisher","award":["KFO 329"],"award-info":[{"award-number":["KFO 329"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"deutsche forschungsgemeinschaft","doi-asserted-by":"publisher","award":["EXC 229"],"award-info":[{"award-number":["EXC 229"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"deutsche forschungsgemeinschaft","doi-asserted-by":"publisher","award":["EXC 2030\/1 \u2013 390661388"],"award-info":[{"award-number":["EXC 2030\/1 \u2013 390661388"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>The advent of genome editing tools like CRISPR\/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The <jats:italic>in vitro<\/jats:italic> fertilization using cryopreserved sperm from the most widely used C57BL\/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for <jats:italic>in vitro<\/jats:italic> fertilization using cryopreserved C57BL\/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. Large-scale comparison of fertilization rates from independent lines of genetically engineered C57BL\/6 mice upon cryopreservation and <jats:italic>in vitro<\/jats:italic> fertilization with our approach demonstrated equal or significantly superior fertilization rates to current protocols. Our novel SEcuRe (<jats:underline>S<\/jats:underline>imple <jats:underline>Ec<\/jats:underline>onomical set-<jats:underline>u<\/jats:underline>p for <jats:underline>Re<\/jats:underline>derivation) method provides an affordable, easily adaptable and harmonized protocol for highly efficient rederivation using cryopreserved C57BL\/6 sperm for a broad application of colony management in the sense of the 3Rs.<\/jats:p>","DOI":"10.1371\/journal.pone.0259202","type":"journal-article","created":{"date-parts":[[2021,10,28]],"date-time":"2021-10-28T17:39:08Z","timestamp":1635442748000},"page":"e0259202","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":9,"title":["A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm"],"prefix":"10.1371","volume":"16","author":[{"given":"Magdalena","family":"Wigger","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1156-2976","authenticated-orcid":true,"given":"Simon E.","family":"Tr\u00f6der","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7845-9522","authenticated-orcid":true,"given":"Branko","family":"Zevnik","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2021,10,28]]},"reference":[{"key":"pone.0259202.ref001","first-page":"23677221994613","article-title":"History of genome editing: From meganucleases to CRISPR","volume":"2021","author":"SE Tr\u00f6der","journal-title":"Lab Anim"},{"key":"pone.0259202.ref002","first-page":"236772211007306","article-title":"Archiving genetically altered animals: a review of cryopreservation and recovery methods for genome edited animals","volume":"2021","author":"S Hart-Johnson","journal-title":"Lab Anim"},{"key":"pone.0259202.ref003","first-page":"116","article-title":"In vitro fertilization (IVF) and artificial insemination (AI) by cryopreserved spermatozoa in mouse","volume":"7","author":"IS Okuyama M","year":"1990","journal-title":"J Fertil Implant"},{"issue":"3","key":"pone.0259202.ref004","first-page":"317","article-title":"Cryopreservation of mouse spermatozoa from inbred and F1 hybrid strains","volume":"42","author":"N Nakagata","year":"1993","journal-title":"Jikken Dobutsu"},{"issue":"6","key":"pone.0259202.ref005","doi-asserted-by":"crossref","first-page":"1774","DOI":"10.1095\/biolreprod63.6.1774","article-title":"In vitro fertilization with cryopreserved inbred mouse sperm","volume":"63","author":"JM Sztein","year":"2000","journal-title":"Biol Reprod"},{"issue":"3","key":"pone.0259202.ref006","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1006\/cryo.1997.2047","article-title":"Cryopreservation of Mouse Spermatozoa","volume":"35","author":"N Songsasen","year":"1997","journal-title":"Cryobiology"},{"issue":"7","key":"pone.0259202.ref007","doi-asserted-by":"crossref","first-page":"e2792","DOI":"10.1371\/journal.pone.0002792","article-title":"Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation","volume":"3","author":"GC Ostermeier","year":"2008","journal-title":"PLoS One"},{"issue":"2","key":"pone.0259202.ref008","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1258\/la.2009.009074","article-title":"Combination medium of cryoprotective agents containing L-glutamine and methyl-\u03b2-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL\/6J mouse sperm","volume":"44","author":"T Takeo","year":"2010","journal-title":"Lab Anim"},{"issue":"4","key":"pone.0259202.ref009","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1071\/BI9510581","article-title":"Observations on the penetration of the sperm in the mammalian egg","volume":"4","author":"CR Austin","year":"1951","journal-title":"Aust J Sci Res B"},{"issue":"4277","key":"pone.0259202.ref010","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1038\/168697b0","article-title":"Fertilizing capacity of spermatozoa deposited into the fallopian tubes","volume":"168","author":"MC Chang","year":"1951","journal-title":"Nature"},{"issue":"4","key":"pone.0259202.ref011","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1262\/jrd1955.16.147","article-title":"Studies on the fertilization of mouse eggs in vitro. 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