Hobbs Rebecca J, Upton Rose, Calatayud Natalie E, Silla Aimee J, Daly Jonathan, McFadden Michael S, O'Brien Justine K
Taronga Institute of Science and Learning, Taronga Conservation Society Australia, Mosman, NSW 2088, Australia.
Conservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Newcastle, NSW 2308, Australia.
Animals (Basel). 2023 Sep 25;13(19):3014. doi: 10.3390/ani13193014.
The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog () to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples ( = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (/) dimethyl sulfoxide + 1% (/) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (/) dimethylformamide + 10% (/) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.
配子的冷冻保存和储存（生物样本库）可为保护种群遗传多样性提供一种长期、低成本的选择，在应用于保护繁育计划（CBP）中的选择性育种管理时尤其具有影响力。本研究旨在为极度濒危的布鲁罗龙蛙（）制定一种精子冷冻保存方案，以在该物种最近建立的（2019年建立）CBP中捕获奠基者基因。使用既定方案实现激素诱导精子释放，并在6小时内收集含精子的尿液样本。将合并的含精子尿液样本（ = 3只雄性）在两种冷冻保护剂（CPA）处理之间平均分配，并以1:5（精子:CPA）的比例用简化两栖类林格氏液（SAR）中的15%（/）二甲基亚砜 + 1%（/）蔗糖（CPAA）或SAR中的10%（/）二甲基甲酰胺 + 10%（/）二水海藻糖（CPAB）进行稀释。样本使用程序降温仪（FrA）或改良的干运法（FrB）在0.25 mL细管中进行冷冻保存。解冻后的样本通过在水中稀释进行激活，并使用人工评估和计算机辅助精子分析（CASA；解冻后0小时、0.5小时）评估活力和运动性。激活后，无论CPA组成如何，使用FrB保存的精子冷冻保存悬浮液的存活率和运动性恢复（总运动性、前向运动和速度）均高于使用FrA保存的精子。这项工作支持了我们的长期目标，即率先将生物样本库冷冻保存的精子与种群遗传管理相结合，以最大限度地提高澳大利亚两栖类物种恢复计划的成果。
