ObjectiveTo compare the freezing effect of three cryoprotectants usevitrification-cryopreservation methods on immature C57BL/6J mouse testicular tissues.MethodTestis from five-day-old mouse were cryopreserved using DMSO、PROH and EFS cryopreservation solution and thawed respectively. Cryodamage of seminiferous cords was H&E stained and semi-quantitatively determined, establishing a scoring of alterations. The function of testicular tissues after xenografting was evaluated by Real Time quantitive PCR and assisted reproduction. ResultsThe morphology of the fresh and frozen–thawed samples was significant different(P﹤0.05). After eight-week xenografting, mature sperms were achieved from all tissues and both methods resulted in pregnancies.The number of progenies from DMSO, PROH and EFS were 5, 2 and 8 respectively, after micromanipulation and embryos implanting. Moreover, the tissue-specific genes were expressed normally. ConclusionsThe threefreezing protocols are able to maintain mouse immature testicular tissue architecture and functionality. The preference is EFScryoprotactants solution.