Development of germplasm cryopreservation and transplantation techniques for sturgeons

鲟鱼种质超低温保存与移植技术的发展

• 批准号: RGPIN-2019-07138
• 负责人: Litvak, Matthew
• 金额: $ 3.42万
• 依托单位: Mount Allison University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=689457
• 关键词: Development; germplasm; cryopreservation; transplantation; techniques

Sturgeon are among the most valuable fish in the world. They are also the most endangered vertebrate group in the world. This is a direct result of human activities including pollution, damming, river fragmentation, climate change, and fisheries persecution, through directed fisheries, by-catch and illegal activities such as poaching. While efforts are being made to stop these activities, they are not fast enough or on a scale large enough to ensure sturgeon survival. Fortunately, advances in cryobiology have allowed us to expand the potential for preservation of wild and domestic stock variability through cryobanks. I propose to develop germplasm cryobanking protocols for 3 species of Canadian sturgeons that are of both aquaculture and conservation interest: shortnose (Acipenser brevirostrum), Lake (A. fulvescens), and White (A. transmontanus) sturgeons. First, we will develop sperm cryopreservation protocols for these species. Sperm cryopreservation procedures can be used to: synchronize gamete availability, conserve sperm volume, resolve space issues needed to meet genetic diversity targets by reducing number of broodstock held in hatcheries, reduce the risk of disease transfer, aid in spawning multiple males with several females throughout the spawning season, extend generation interval (gametes outlive the donor), provide insurance against extinction, and conserve genetic variability to reduce the risk of inbreeding in domestic and wild populations. Second, we will develop protocols for germplasm collection. Current germplasm collection protocols rely on sacrificing donors. This is not good when trying to generate rescue programs for endangered fish. Based on previous work in our lab, we will develop a preselection protocol based on sturgeon morphology and ultrasonography to select suitable candidates for non-lethal laparoscopic removal of small amounts of gonadal material for development of germplasm cryobanks. Third, we will develop cryopreservation procedures for oogonial cells (OC) from female sturgeon. In sturgeon, females are the heterogametic sex (ZW); in order to preserve genetic diversity we must cryopreserve OCs. Fourth, I propose to develop protocols to generate offspring directly from cryopreserved germplasm. We will develop procedures to transplant cryopreserved OCs into a sterile triploid surrogate, the blue tilapia (Oreochromis aureus). Blue tilapia were chosen because of the ease in generating sterile triploids and their very short time to maturation. They mature in under six months, unlike sturgeons that take 6-25 years to mature. Donor OCs will develop as either male or female gonads based on the sex of the surrogate (blue tilapia). When the surrogates mature, they will produce eggs and sperm of the donor species (sturgeon). This program will provide a roadmap for development of germplasm cryobanks for sturgeons and other fishes in Canada. One PhD, 3 MSc and 10 Honours students will be trained during this research program.

鲟鱼是世界上最有价值的鱼类之一。它们也是世界上最濒危的脊椎动物。这是人类活动的直接结果，包括污染、筑坝、河流破碎化、气候变化，以及通过定向捕捞、副渔获物和偷猎等非法活动对渔业的迫害。虽然人们正在努力阻止这些活动，但它们的速度不够快，规模也不够大，无法确保鲟鱼的生存。幸运的是，低温生物学的进步使我们能够通过冷冻银行扩大保护野生和国内种群多样性的潜力。我建议为3种具有养殖和保护价值的加拿大鲟鱼制定种质冷冻库协议：短鼻鲟（Acipenser brevirostrum），湖鲟（A. fulvescens）和白鲟（A. transmontanus）。首先，我们将为这些物种制定精子冷冻保存方案。精子冷冻保存程序可用于：同步配子可用性，保存精子数量，通过减少孵化场中饲养的亲鱼数量来解决满足遗传多样性目标所需的空间问题，减少疾病转移的风险，在整个产卵季节帮助多个雄性和几个雌性产卵，延长世代间隔（配子比供体寿命长），提供防止灭绝的保险，并保存遗传变异性以减少家养和野生种群近亲繁殖的风险。二是制定种质资源采集方案。目前的种质收集方案依赖于牺牲供体。当试图为濒危鱼类制定救援计划时，这并不好。基于我们实验室之前的工作，我们将基于鲟鱼形态学和超声检查制定一项预选方案，以选择合适的候选人，进行非致命的腹腔镜切除少量性腺材料，用于种质冷冻库的开发。第三，我们将开发雌性鲟鱼卵母细胞（OC）的冷冻保存程序。在鲟鱼中，雌性是异种交配的性别（ZW）；为了保护遗传多样性，我们必须冷冻保存oc。第四，我建议制定方案，直接从冷冻保存的种质产生后代。我们将开发将冷冻保存的OCs移植到不育的三倍体替代物蓝色罗非鱼（Oreochromis aureus）中的程序。选择蓝罗非鱼是因为容易产生不育的三倍体和它们的成熟时间很短。它们在六个月内成熟，不像鲟鱼需要6-25年才能成熟。供体OCs将根据代孕者（蓝色罗非鱼）的性别发育为雄性或雌性性腺。当替代物成熟时，它们会产生供体物种（鲟鱼）的卵子和精子。该计划将为加拿大鲟鱼和其他鱼类种质冷冻库的发展提供路线图。该项目将培养1名博士、3名硕士和10名荣誉学生。