{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T07:14:05Z","timestamp":1788506045597,"version":"build-2803163510"},"reference-count":42,"publisher":"SAGE Publications","issue":"2-3","license":[{"start":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T00:00:00Z","timestamp":1776124800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.sagepub.com\/page\/policies\/text-and-data-mining-license"}],"funder":[{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"crossref","award":["RGPIN-2014-04980\/RGPIN-2019-07138"],"award-info":[{"award-number":["RGPIN-2014-04980\/RGPIN-2019-07138"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["journals.sagepub.com"],"crossmark-restriction":true},"short-container-title":["Zebrafish"],"published-print":{"date-parts":[[2026,6,1]]},"abstract":"<jats:p>\n                    Cryopreservation enables the long-term storage of viable biological material at ultra-low temperatures and forms the foundation for germplasm cryobanks that maintain valuable genetic lines of model organisms such as zebrafish (\n                    <jats:italic toggle=\"yes\">Danio rerio<\/jats:italic>\n                    ). However, reliable and reproducible cryopreservation protocols for fish germline stem cells remain difficult to develop, partly because key steps such as equilibration are often overlooked or assigned arbitrarily. Here, we optimized equilibration time for cryopreservation of zebrafish ovarian tissue. Ovarian fragments were equilibrated in 2 M methanol + 0.1 M glucose + 10% egg yolk for varying durations (15\u2013120 min) before controlled slow cooling and storage in liquid nitrogen. Post-thaw viability was assessed using a Trypan Blue exclusion assay. A 60-min equilibration yielded the highest viability of ovarian cells in Experiment 1 (55.7 \u00b1 1.7%), whereas a 30-min equilibration yielded the highest viability in Experiment 2 (75.9 \u00b1 2.4%), but was not significantly different from the 60-min equilibration in Experiment 2 (75.6 \u00b1 2.0%) (\n                    <jats:italic toggle=\"yes\">p<\/jats:italic>\n                    = 0.998). Equilibration alone accounted for a 48.7% increase in post-thaw viability relative to controls. The framework presented here provides a reproducible method for determining species-specific equilibration optima and supports the development of effective germplasm cryobanks for both model and endangered fish species.\n                  <\/jats:p>","DOI":"10.1177\/15458547261441744","type":"journal-article","created":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T04:48:39Z","timestamp":1776228519000},"page":"64-70","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimizing Equilibration Time to Enhance Post-Thaw Viability of Cryopreserved Zebrafish (\n                    <i>Danio rerio<\/i>\n                    ) Ovarian Fragments"],"prefix":"10.1177","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-5995-7168","authenticated-orcid":false,"given":"Maya L.","family":"Wade","sequence":"first","affiliation":[{"name":"Department of Biology, Mount Allison University, New Brunswick, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew K.","family":"Litvak","sequence":"additional","affiliation":[{"name":"Department of Biology, Mount Allison University, New Brunswick, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"179","published-online":{"date-parts":[[2026,4,14]]},"reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1089\/zeb.2009.0592"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1590\/S1516-89132012000300014"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2011.09.042"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1546-5098(08)60306-4"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10695-018-0506-0"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.theriogenology.2019.03.032"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2016.05.042"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2016.03.004"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0006132"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1111\/raq.70057"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1533\/9780857097460.1.76"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ygcen.2016.06.019"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2013.10.006"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0509218103"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1242\/dev.044982"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4020-6235-3_14"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-50169-1"},{"issue":"2","key":"e_1_3_3_19_2","first-page":"148","article-title":"Effects on cell viability of three zebrafish testicular cell or tissue cryopreservation methods","volume":"30","author":"Bono-Mestre C","year":"2009","unstructured":"Bono-Mestre C, , Cardona-Costa J, , Garc\u00eda-Xim\u00e9nez F. Effects on cell viability of three zebrafish testicular cell or tissue cryopreservation methods. Cryo Letters, 2009; 30(2):148\u2013152.","journal-title":"Cryo Letters"},{"key":"e_1_3_3_20_2","unstructured":"K\u00e1sa E. Vitrification of Fish Sperm and Spermatogonia. PhD thesis. Szent Istv\u00e1n University; 2017."},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.3791\/56118"},{"key":"e_1_3_3_22_2","unstructured":"Anil S. Development of In-Vitro Culture and Cryopreservation Protocol for Zebrafish (Danio Rerio) Ovarian Tissue Fragments. PhD thesis. University of Bedfordshire; 2013."},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2008.03.001"},{"key":"e_1_3_3_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.theriogenology.2009.01.014"},{"key":"e_1_3_3_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-51696-7"},{"issue":"6","key":"e_1_3_3_26_2","first-page":"525","article-title":"Impact of cryoprotectants and cryopreservation on metabolic activity and cytoskeleton proteins of zebrafish (Danio rerio) ovarian fragments","volume":"32","author":"Zampolla T","year":"2011","unstructured":"Zampolla T, , Spikings E, , Srirangarajah S, et al. Impact of cryoprotectants and cryopreservation on metabolic activity and cytoskeleton proteins of zebrafish (Danio rerio) ovarian fragments. Cryo Letters, 2011; 32(6):525\u2013536.","journal-title":"Cryo Letters"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2025.105251"},{"key":"e_1_3_3_28_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2013.10.002"},{"key":"e_1_3_3_29_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2015.09.004"},{"key":"e_1_3_3_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2005.09.016"},{"key":"e_1_3_3_31_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2016.01.004"},{"key":"e_1_3_3_32_2","doi-asserted-by":"publisher","DOI":"10.1093\/jhered\/90.1.231"},{"key":"e_1_3_3_33_2","doi-asserted-by":"publisher","DOI":"10.1534\/genetics.114.169284"},{"key":"e_1_3_3_34_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1469-185X.2007.00030.x"},{"key":"e_1_3_3_35_2","unstructured":"COSEWIC (Committee on the Status of Endangered Wildlife in Canada). COSEWIC Assessment and Status Report on the Shortnose Sturgeon Acipenser Brevirostrum in Canada. 2015."},{"issue":"6","key":"e_1_3_3_36_2","first-page":"477","article-title":"Studies on cryoprotectant toxicity to early stage zebrafish (Danio rerio) ovarian follicles","volume":"29","author":"Tsai S","year":"2008","unstructured":"Tsai S, , Rawson DM, , Zhang T. Studies on cryoprotectant toxicity to early stage zebrafish (Danio rerio) ovarian follicles. Cryo Letters, 2008; 29(6):477\u2013483.","journal-title":"Cryo Letters"},{"key":"e_1_3_3_37_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2017.04.004"},{"key":"e_1_3_3_38_2","doi-asserted-by":"publisher","DOI":"10.1089\/zeb.2020.1942"},{"key":"e_1_3_3_39_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2019.01.016"},{"key":"e_1_3_3_40_2","volume-title":"Discovering Statistics Using R","author":"Field AP","year":"2012","unstructured":"Field AP, , Miles J, , Field Z. Discovering Statistics Using R. Sage: London; Thousand Oaks, Calif; 2012."},{"key":"e_1_3_3_41_2","doi-asserted-by":"publisher","DOI":"10.1002\/bimj.200810425"},{"key":"e_1_3_3_42_2","doi-asserted-by":"publisher","DOI":"10.1071\/RD19457"},{"key":"e_1_3_3_43_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cryobiol.2009.02.002"}],"container-title":["Zebrafish"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.sagepub.com\/doi\/pdf\/10.1177\/15458547261441744","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.sagepub.com\/doi\/full-xml\/10.1177\/15458547261441744","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.sagepub.com\/doi\/pdf\/10.1177\/15458547261441744","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T06:51:23Z","timestamp":1788504683000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.sagepub.com\/doi\/10.1177\/15458547261441744"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,14]]},"references-count":42,"journal-issue":{"issue":"2-3","published-print":{"date-parts":[[2026,6,1]]}},"alternative-id":["10.1177\/15458547261441744"],"URL":"https:\/\/doi.org\/10.1177\/15458547261441744","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2025.10.28.685221","asserted-by":"object"}]},"ISSN":["1545-8547","1557-8542"],"issn-type":[{"value":"1545-8547","type":"print"},{"value":"1557-8542","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,14]]}}}