Most sturgeon and paddlefish are critically endangered; therefore, effective measures to conserve these genetic resources are required. Cryopreservation of gonad tissues containing germline stem cells could be an effective strategy for long term preservation and restoration of fish species using germ cell transplantation procedure. The aim of this study was to develop an optimal procedure for long-term cryopreservation of American paddlefish gonads using a slow-freezing method. Through optimization of permeating cryoprotectants, nonpermeating cryoprotectants, and supplementation of proteins, gonad tissues were frozen with a cryomedium containing 1.3 M dimethyl sulfoxide, 0.1 M trehalose, and 10 % fetal bovine serum at a cooling rate of −1 °C/min. This method was also successfully utilized for the cryopreservation of Yangtze sturgeon testes. Viability of gonadal cells isolated from frozen gonads was not different from cells isolated from fresh gonadal tissues, while the number of gonadal cells dissociated from frozen gonads was less. Germline stem cells dissociated from long-term (1 year) cryopreserved gonads were labeled with PKH26 fluorescent dye and intraperitoneally transplanted into larvae of Yangtze sturgeon. The colonization of transplanted germline stem cells was confirmed by the presence of PKH26-labeled donor germline stem cells and donor-derived mtDNA sequence in the recipient gonads, providing evidence that germline stem cells from sturgeon and paddlefish gonads that had been preserved for a long period maintained their functions. The results of present study indicate the procedures used are effective for long-term preservation of critically endangered species within the Acipenseriformes order which can later be regenerated using surrogate broodstock technology.

大多数鲟鱼和白鲟都处于极度濒危状态；因此，需要采取有效措施保护这些遗传资源。冷冻保存含有生殖干细胞的性腺组织可能是使用生殖细胞移植程序长期保存和恢复鱼类物种的有效策略。本研究的目的是开发一种使用慢速冷冻方法长期冷冻保存美国白鲟性腺的最佳程序。通过优化渗透性冷冻保护剂、非渗透性冷冻保护剂和蛋白质的补充，性腺组织用含有 1.3 M 二甲基亚砜、0.1 M 海藻糖和 10% 胎牛血清的低温培养基以 -1 °C/min 的冷却速率进行冷冻。该方法也成功用于长江鲟睾丸的冷冻保存。从冷冻性腺中分离的性腺细胞的活力与从新鲜性腺组织中分离的细胞没有区别，而从冷冻性腺中分离出的性腺细胞数量较少。从长期（1 年）冷冻保存的性腺中分离出来的生殖干细胞用 PKH26 荧光染料标记，并腹腔内移植到长江鲟幼虫中。移植的生殖系干细胞的定殖通过在受体性腺中存在 PKH26 标记的​​供体生殖系干细胞和供体来源的 mtDNA 序列而得到证实，提供证据表明来自鲟鱼和白鲟性腺的生殖系干细胞已被长期保存维持了他们的职能。