Development of germplasm cryopreservation and transplantation techniques for sturgeons

鲟鱼是世界上最有价值的鱼之一。它们也是世界上最濒危的脊椎动物群体。这是人类活动的直接结果，包括污染、筑坝、河流碎裂、气候变化和渔业迫害，通过直接渔业、副渔获物和非法活动，如偷猎。虽然人们正在努力阻止这些活动，但它们的速度还不够快，规模也不足以确保鲟鱼的生存。幸运的是，低温生物学的进步使我们能够通过冷藏库扩大保存野生和家畜可变性的可能性。我建议为三种既具有水产养殖价值又具有保护价值的加拿大鲟鱼制定种质冷藏库协议：短吻鲟鱼(Acipenser Brevirostrum)、莱克鲟鱼(A.fulvescens)和白鲟鱼(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：同步配子可获得性，保存精子体积，通过减少孵化室中的亲代数量来解决实现遗传多样性目标所需的空间问题，降低疾病传播风险，帮助在整个产卵季节与几个雌性繁殖多个雄性，延长世代间隔(配子比供体活得更长)，提供防止灭绝的保险，以及保存遗传变异以降低家养和野生种群近亲繁殖的风险。第二，我们将制定种质收集协议。目前的种质收集协议依赖于牺牲捐赠者。当试图为濒危鱼类制定救援计划时，这并不是一件好事。在本实验室前期工作的基础上，我们将开发一种基于鲟鱼形态和超声检查的预选方案，以选择合适的候选方案，用于在非致命性腹腔镜下切除少量性腺材料，以开发种质冷藏库。第三，我们将开发雌性鲟卵原细胞(OC)的冷冻保存程序。在鲟鱼中，雌性是异性恋的性别(ZW)；为了保存遗传多样性，我们必须冷冻保存OCS。第四，我建议制定协议，直接从超低温保存的种质中产生后代。我们将开发程序，将冷冻保存的OCs移植到无菌的三倍体代用蓝罗非鱼(Oreochromis Aureus)。选择蓝色罗非鱼是因为它容易产生不育的三倍体，而且成熟时间很短。它们在不到6个月的时间内成熟，不像鲟鱼需要6-25年才能成熟。捐赠者OCS将根据代孕(蓝色罗非鱼)的性别发育为雄性或雌性性腺。当代孕体成熟时，它们将产生捐赠者物种(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藏库的开发提供路线图。1名博士、3名硕士和10名优秀生将在本研究计划期间接受培训。

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.