{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T16:27:34Z","timestamp":1791217654933,"version":"4.1.0"},"reference-count":30,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,7,8]],"date-time":"2021-07-08T00:00:00Z","timestamp":1625702400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,7,8]],"date-time":"2021-07-08T00:00:00Z","timestamp":1625702400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100001700","name":"Ministry of Education, Culture, Sports, Science and Technology","doi-asserted-by":"publisher","award":["19H05758"],"award-info":[{"award-number":["19H05758"]}],"id":[{"id":"10.13039\/501100001700","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Cryopreservation of mouse spermatozoa is widely used for the efficient preservation and safe transport of valuable mouse strains. However, the current cryopreservation method requires special containers (plastic straws), undefined chemicals (e.g., skim milk), liquid nitrogen, and expertise when handling sperm suspensions. Here, we report an easy and quick (EQ) sperm freezing method. The main procedure consists of only one step: dissecting a single cauda epididymis in a microtube containing 20% raffinose solution, which is then stored in a \u221280\u00a0\u00b0C freezer. The frozen\u2013thawed spermatozoa retain practical fertilization rates after 1 (51%) or even 3\u00a0months (25%) with the C57BL\/6\u00a0J strain, the most sensitive strain for sperm freezing. More than half of the embryos thus obtained developed into offspring after embryo transfer. Importantly, spermatozoa stored at \u221280\u00a0\u00b0C can be transferred into liquid nitrogen for indefinite storage. As far as we know, our EQ method is the easiest and quickest method for mouse sperm freezing and should be applicable in all laboratories without expertise in sperm cryopreservation. This technique can help avoid the loss of irreplaceable strains because of closure of animal rooms in emergency situations such as unexpected microbiological contamination or social emergencies such as the COVID-19 threat.<\/jats:p>","DOI":"10.1038\/s41598-021-93604-y","type":"journal-article","created":{"date-parts":[[2021,7,8]],"date-time":"2021-07-08T10:04:40Z","timestamp":1625738680000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains"],"prefix":"10.1038","volume":"11","author":[{"given":"Keiji","family":"Mochida","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayumi","family":"Hasegawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daiki","family":"Shikata","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuhiko","family":"Itami","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masashi","family":"Hada","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naomi","family":"Watanabe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshiko","family":"Tomishima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Atsuo","family":"Ogura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,7,8]]},"reference":[{"key":"93604_CR1","doi-asserted-by":"publisher","first-page":"987","DOI":"10.1007\/s003359901145","volume":"10","author":"CE Thornton","year":"1999","unstructured":"Thornton, C. E., Brown, S. D. M. & Glenister, P. H. Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses. Mamm. Genome. 10, 987\u2013992 (1999).","journal-title":"Mamm. Genome."},{"key":"93604_CR2","doi-asserted-by":"publisher","first-page":"1653","DOI":"10.1016\/j.theriogenology.2012.08.012","volume":"78","author":"Y Agca","year":"2012","unstructured":"Agca, Y. Genome resource banking of biomedically important laboratory animals. Theriogenology 78, 1653\u20131665 (2012).","journal-title":"Theriogenology"},{"key":"93604_CR3","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1538\/expanim1978.40.4_493","volume":"40","author":"T Takeshima","year":"1991","unstructured":"Takeshima, T., Nakagata, N. & Ogawa, S. Cryopreservation of mouse spermatozoa. Exp. Anim. 40, 493\u2013497 (1991).","journal-title":"Exp. Anim."},{"key":"93604_CR4","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1538\/expanim.44.1","volume":"44","author":"N Nakagata","year":"1995","unstructured":"Nakagata, N. Studies on cryopreservation of embryos and gametes in mice. Exp. Anim. 44, 1\u20138 (1995).","journal-title":"Exp. Anim."},{"key":"93604_CR5","doi-asserted-by":"publisher","first-page":"1774","DOI":"10.1095\/biolreprod63.6.1774","volume":"63","author":"JM Sztein","year":"2000","unstructured":"Sztein, J. M., Farley, J. S. & Mobraaten, L. E. In vitro fertilization with cryopreserved inbred mouse sperm. Biol. Reprod. 63, 1774\u20131780 (2000).","journal-title":"Biol. Reprod."},{"key":"93604_CR6","doi-asserted-by":"publisher","first-page":"973","DOI":"10.1095\/biolreprod.103.024422","volume":"71","author":"H Nishizono","year":"2004","unstructured":"Nishizono, H., Shioda, M., Takeo, T., Irie, T. & Nakagata, N. Decrease of fertilizing ability of mouse spermatozoa after freezing and thawing is related to cellular injury. Biol. Reprod. 71, 973\u2013978 (2004).","journal-title":"Biol. Reprod."},{"key":"93604_CR7","doi-asserted-by":"publisher","first-page":"117","DOI":"10.1262\/jrd.2017-148","volume":"64","author":"J Liu","year":"2018","unstructured":"Liu, J., Mochida, K., Hasegawa, A., Inoue, K. & Ogura, A. Identification of quantitative trait loci associated with the susceptibility of mouse spermatozoa to cryopreservation. J. Reprod. Dev. 64, 117\u2013127 (2018).","journal-title":"J. Reprod. Dev."},{"key":"93604_CR8","doi-asserted-by":"publisher","first-page":"1328","DOI":"10.1095\/biolreprod59.6.1328","volume":"59","author":"YH Choi","year":"1998","unstructured":"Choi, Y. H. & Toyoda, Y. Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol. Reprod. 59, 1328\u20131333 (1998).","journal-title":"Biol. Reprod."},{"key":"93604_CR9","doi-asserted-by":"publisher","first-page":"546","DOI":"10.1095\/biolreprod.107.065359","volume":"78","author":"T Takeo","year":"2008","unstructured":"Takeo, T. et al. Methyl-beta-cyclodextrin improves fertilizing ability of C57BL\/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells. Biol. Reprod. 78, 546\u2013551 (2008).","journal-title":"Biol. Reprod."},{"key":"93604_CR10","doi-asserted-by":"publisher","first-page":"9387","DOI":"10.1371\/journal.pone.0009387","volume":"5","author":"ML Bath","year":"2010","unstructured":"Bath, M. L. Inhibition of in vitro fertilizing capacity of cryopreserved mouse sperm by factors released by damaged sperm, and stimulation by glutathione. PLoS ONE 5, 9387 (2010).","journal-title":"PLoS ONE"},{"key":"93604_CR11","doi-asserted-by":"publisher","first-page":"1066","DOI":"10.1095\/biolreprod.111.092536","volume":"85","author":"T Takeo","year":"2011","unstructured":"Takeo, T. & Nakagata, N. Reduced glutathione enhances fertility of frozen\/thawed C57BL\/6 mouse sperm after exposure to methyl-\u03b2-cyclodextrin. Biol. Reprod. 85, 1066\u20131072 (2011).","journal-title":"Biol. Reprod."},{"key":"93604_CR12","doi-asserted-by":"publisher","first-page":"156","DOI":"10.1262\/jrd.11-097N","volume":"58","author":"A Hasegawa","year":"2012","unstructured":"Hasegawa, A., Yonezawa, K., Ohta, A., Mochida, K. & Ogura, A. Optimization of a protocol for cryopreservation of mouse spermatozoa using cryotubes. J. Reprod. Dev. 58, 156\u2013161 (2012).","journal-title":"J. Reprod. Dev."},{"key":"93604_CR13","doi-asserted-by":"publisher","first-page":"52","DOI":"10.1016\/j.theriogenology.2018.06.020","volume":"119","author":"M Raspa","year":"2018","unstructured":"Raspa, M. et al. A new, simple and efficient liquid nitrogen free method to cryopreserve mouse spermatozoa at \u201380 \u00b0C. Theriogenology 119, 52\u201359 (2018).","journal-title":"Theriogenology"},{"key":"93604_CR14","doi-asserted-by":"publisher","first-page":"13098","DOI":"10.1073\/pnas.0605755103","volume":"103","author":"N Ogonuki","year":"2006","unstructured":"Ogonuki, N. et al. Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring. Proc. Natl. Acad. Sci. USA 103, 13098\u201313103 (2006).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"93604_CR15","doi-asserted-by":"publisher","first-page":"204","DOI":"10.1006\/cryo.2000.2281","volume":"41","author":"C Koshimoto","year":"2000","unstructured":"Koshimoto, C., Gamliel, E. & Masur, P. Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose. Cryobiology 41, 204\u2013231 (2000).","journal-title":"Cryobiology"},{"key":"93604_CR16","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1016\/S0015-0282(16)48918-1","volume":"44","author":"P Quinn","year":"1985","unstructured":"Quinn, P., Kerin, J. F. & Warnes, G. M. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil. Steril. 44, 493\u2013498 (1985).","journal-title":"Fertil. Steril."},{"key":"93604_CR17","first-page":"1","volume":"94","author":"A Hasegawa","year":"2017","unstructured":"Hasegawa, A. et al. High-yield superovulation in adult mice by anti-inhibin serum treatment combined with estrous cycle synchronization. Biol. Reprod. 94, 1\u20138 (2017).","journal-title":"Biol. Reprod."},{"key":"93604_CR18","doi-asserted-by":"publisher","first-page":"539","DOI":"10.1262\/jrd.2017-068","volume":"63","author":"A Hasegawa","year":"2017","unstructured":"Hasegawa, A. et al. Efficient and scheduled production of pseudopregnant female mice for embryo transfer by estrous cycle synchronization. J. Reprod. Dev. 63, 539\u2013545 (2017).","journal-title":"J. Reprod. Dev."},{"key":"93604_CR19","doi-asserted-by":"publisher","first-page":"679","DOI":"10.1530\/jrf.0.0860679","volume":"86","author":"CL Chatot","year":"1989","unstructured":"Chatot, C. L., Ziomek, C. A., Bavister, B. D., Lewis, J. L. & Torres, I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J. Reprod. Fertil. 86, 679\u2013688 (1989).","journal-title":"J. Reprod. Fertil."},{"key":"93604_CR20","doi-asserted-by":"publisher","first-page":"533","DOI":"10.1530\/REP-09-0434","volume":"139","author":"K Yoshida","year":"2010","unstructured":"Yoshida, K. et al. A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin. Reproduction 139, 533\u2013544 (2010).","journal-title":"Reproduction"},{"key":"93604_CR21","doi-asserted-by":"publisher","first-page":"1011","DOI":"10.1002\/cyto.a.20484","volume":"71A","author":"D Dom\u0131nguez-Fandos","year":"2007","unstructured":"Dom\u0131nguez-Fandos, D., Camejo, M. I., Ballesc\u00e0, J. L. & Oliva, R. Human sperm DNA fragmentation: Correlation of TUNEL results as assessed by flow cytometry and optical microscopy. Cytometry 71A, 1011\u20131018 (2007).","journal-title":"Cytometry"},{"key":"93604_CR22","first-page":"109","volume":"21","author":"M Muratori","year":"2015","unstructured":"Muratori, M. et al. Investigation on the origin of sperm DNA fragmentation: Role of apoptosis, immaturity and oxidative stress Mol. Med. 21, 109\u2013122 (2015).","journal-title":"Med."},{"key":"93604_CR23","first-page":"100157","volume":"4","author":"MBR Alves","year":"2015","unstructured":"Alves, M. B. R. et al. An efficient technique to detect sperm reactive oxygen species: The Cell Rox Deep Red\u00ae fluorescent probe. Biochem. Physiol. 4, 100157 (2015).","journal-title":"Biochem. Physiol."},{"key":"93604_CR24","doi-asserted-by":"publisher","first-page":"265","DOI":"10.1262\/jrd.20056","volume":"56","author":"B Jin","year":"2008","unstructured":"Jin, B. et al. The mechanism by which mouse spermatozoa are injured during freezing. J. Reprod. Dev. 56, 265\u2013269 (2008).","journal-title":"J. Reprod. Dev."},{"key":"93604_CR25","doi-asserted-by":"publisher","first-page":"41","DOI":"10.1016\/j.theriogenology.2017.10.036","volume":"107","author":"M Raspa","year":"2018","unstructured":"Raspa, M. et al. Long term maintenance of frozen mouse spermatozoa at \u221280 \u00b0C. Theriogenology 107, 41\u201349 (2018).","journal-title":"Theriogenology"},{"key":"93604_CR26","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1016\/S0011-2240(02)00105-0","volume":"45","author":"C Koshimoto","year":"2002","unstructured":"Koshimoto, C. & Mazur, P. Effects of warming rate, temperature, and antifreeze proteins on the survival of mouse spermatozoa frozen at an optimal rate. Cryobiology 45, 49\u201359 (2002).","journal-title":"Cryobiology"},{"key":"93604_CR27","doi-asserted-by":"publisher","first-page":"193","DOI":"10.1016\/j.cryobiol.2005.01.004","volume":"50","author":"EM Walters","year":"2005","unstructured":"Walters, E. M. et al. Osmotic tolerance of mouse spermatozoa from various genetic backgrounds: acrosome integrity, membrane integrity, and maintenance of motility. Cryobiology 50, 193\u2013205 (2005).","journal-title":"Cryobiology"},{"key":"93604_CR28","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1530\/jrf.0.0810077","volume":"81","author":"LR Fraser","year":"1987","unstructured":"Fraser, L. R. Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro. J. Reprod. Fertil. 81, 77\u201389 (1987).","journal-title":"J. Reprod. Fertil."},{"key":"93604_CR29","doi-asserted-by":"publisher","first-page":"363","DOI":"10.1530\/jrf.0.0960363","volume":"96","author":"LR Fraser","year":"1992","unstructured":"Fraser, L. R. & McDermott, C. A. Ca2+-related changes in the mouse sperm capacitation state: a possible role for Ca2+-ATPase. J. Reprod. Fertil. 96, 363\u2013377 (1992).","journal-title":"J. Reprod. Fertil."},{"key":"93604_CR30","doi-asserted-by":"publisher","first-page":"259","DOI":"10.1017\/S0967199408004619","volume":"16","author":"S Kito","year":"2008","unstructured":"Kito, S. & Ohta, Y. In vitro fertilization in inbred BALB\/c mice II: effects of lactate, osmolarity and calcium on in vitro capacitation. Zygote 16, 259\u2013270 (2008).","journal-title":"Zygote"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-93604-y.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-93604-y","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-93604-y.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,3]],"date-time":"2022-12-03T11:04:45Z","timestamp":1670065485000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-93604-y"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,8]]},"references-count":30,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2021,12]]}},"alternative-id":["93604"],"URL":"https:\/\/doi.org\/10.1038\/s41598-021-93604-y","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,8]]},"assertion":[{"value":"2 February 2021","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 June 2021","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"8 July 2021","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"14149"}}