Mammalian spermatozoa preservation now plays an important role in fertility treatments, generating hybrid animals and protecting endangered and extinct species. To date, the most common method of sperm preservation is freezing in liquid nitrogen (LN2). However, this method requires constant supplementation of LN2 and also presents some safety issues involved in transporting LN2. Here we describe a new sperm preservation method that does not involve freezing. Mouse spermatozoa were cultured in four basic media (HEPES–Chatot-Ziomele-Barister’s medium (HCZB), KSOM, K+-rich nuclear isolation medium (NIM), and PBS) with or without 10% BSA or 15% Ficoll as a cryoprotectant, and preserved in a refrigerator for up to 6 months. These preserved sperm were then injected into fresh oocytes and cultured to the blastocyst stage in vitro or transferred into recipient females to demonstrate their genetic integrity. Oocytes injected with 1-month-preserved spermatozoa in NIM and PBS showed significantly higher blastocyst rates (22.8% and 18.9%) than those in HEPES-CZB and KSOM (1-way ANOVA, P < 0.05). In embryos with 3-month-preserved spermatozoa in NIM or PBS with BSA or Ficoll, 5.3–24.0%; P < 0.05 of embryos, (n = 1056) developed to the blastocyst stage, and the developmental ratio was not decreased even for 6-month preservation (13.6–18.2%; P > 0.05). Surprisingly, 18 pups were obtained using spermatozoa stored in those mediums for 6 months. Moreover, this new method allowed easy production of healthy offspring even after transporting spermatozoa between two countries by aircraft at room temperature without any protection. In conclusion, this method allows for easy long-term preservation of mouse spermatozoa in a simple, modified medium at refrigerator temperature with very low cost and wide application.

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