{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T23:45:32Z","timestamp":1777419932293,"version":"3.51.4"},"reference-count":33,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2021,2,17]],"date-time":"2021-02-17T00:00:00Z","timestamp":1613520000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Andrology"],"published-print":{"date-parts":[[2021,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a \u221280\u00b0C freezer, maintaining their ability to fertilize mouse eggs.<\/jats:p><\/jats:sec><jats:sec><jats:title>Objectives<\/jats:title><jats:p>The main objective here was to evaluate the effects of five years at \u221280\u00b0C compared to liquid nitrogen storage (LN<jats:sub>2<\/jats:sub>, control condition) on mouse sperm viability, physiological parameters, and fertilization capacity.<\/jats:p><\/jats:sec><jats:sec><jats:title>Materials and methods<\/jats:title><jats:p>Three different strains were used: C57BL\/6N, C57BL\/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR\u201014 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3\u2010AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The <jats:italic>in vitro fertilization<\/jats:italic> (IVF) was used to evaluate the sperm fertilizing ability.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at \u221280\u00b0C for 5\u00a0years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN<jats:sub>2<\/jats:sub>.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>Long\u2010term mouse sperm storage at \u221280\u00b0C (five years) could be considered an ideal alternative to the most common LN<jats:sub>2<\/jats:sub> approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a \u221280\u00b0C freezer with no significant differences compared to the storage in LN<jats:sub>2<\/jats:sub> in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity.<\/jats:p><\/jats:sec>","DOI":"10.1111\/andr.12971","type":"journal-article","created":{"date-parts":[[2021,1,15]],"date-time":"2021-01-15T05:51:13Z","timestamp":1610689873000},"page":"989-999","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["The impact of five years storage\/biobanking at \u221280\u00b0C on mouse spermatozoa fertility, 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