Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture and Rural Affairs, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China.
Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture and Rural Affairs, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China.
Anim Reprod Sci. 2021 Jan;224:106667. doi: 10.1016/j.anireprosci.2020.106667. Epub 2020 Dec 3.
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.
大多数鲟鱼和匙吻鲟都处于极度濒危状态；因此，需要采取有效的措施来保护这些遗传资源。通过冷冻保存含有生殖干细胞的性腺组织，可以利用生殖细胞移植程序，成为鱼类物种长期保存和恢复的有效策略。本研究的目的是开发一种使用慢速冻法长期冷冻保存美洲匙吻鲟性腺的最佳程序。通过优化渗透保护剂、非渗透保护剂和蛋白质补充剂，使用含有 1.3 M 二甲基亚砜、0.1 M 海藻糖和 10%胎牛血清的冷冻培养基以-1°C/min 的冷却速率冷冻性腺组织。该方法还成功地应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的生殖干细胞用 PKH26 荧光染料标记，并通过腹腔内移植到长江鲟幼鱼体内。在受体性腺中存在 PKH26 标记的供体生殖干细胞和供体衍生的 mtDNA 序列，证实了移植的生殖干细胞的定植，这为长期保存的鲟鱼和匙吻鲟性腺中的生殖干细胞保持其功能提供了证据。本研究的结果表明，所使用的程序可有效保存极度濒危的鲟形目物种，以后可利用代用亲鱼技术进行再生。
