Upton Rose, Clulow Simon, Calatayud Natalie E, Colyvas Kim, Seeto Rebecca G Y, Wong Lesley A M, Mahony Michael J, Clulow John
The Conservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia; and FAUNA Research Alliance, PO Box 5092, Kahibah, NSW 2290, Australia; and Corresponding author.
FAUNA Research Alliance, PO Box 5092, Kahibah, NSW 2290, Australia; and Centre for Conservation Ecology and Genomics, Institute for Applied Ecology, University of Canberra, Bruce, ACT 2617, Australia.
Reprod Fertil Dev. 2021 Apr;33:562-572. doi: 10.1071/RD20296.
Amphibians are becoming increasingly reliant on captive breeding programs for continued survival. Assisted reproductive technologies including gamete cryopreservation and IVF can help reduce costs of breeding programs, provide insurance against extinction and assist genetic rescue in wild populations. However, the use of these technologies to produce reproductively mature offspring has only been demonstrated in a few non-model species. We aimed to optimise sperm cryopreservation in the threatened frog Litoria aurea and generate mature offspring from frozen-thawed spermatozoa by IVF. We tested three concentrations (1.4, 2.1 and 2.8M) of the cryoprotectants dimethylsulfoxide (DMSO) and glycerol with 0.3M sucrose. Using DMSO was more likely to result in recovery of sperm motility, vitality and acrosome integrity than glycerol, regardless of concentration, with forward progressive motility being most sensitive to damage. The lowest concentrations of 1.4 and 2.1M provided the best protection regardless of cryoprotectant type. Spermatozoa cryopreserved in 2.1M DMSO outperformed spermatozoa cryopreserved in equivalent concentrations of glycerol in terms of their ability to fertilise ova, resulting in higher rates of embryos hatching and several individuals reaching sexual maturity. We have demonstrated that sperm cryopreservation and subsequent offspring generation via IVF is a feasible conservation tool for L. aurea and other threatened amphibians.
两栖动物越来越依赖圈养繁殖计划来维持生存。包括配子冷冻保存和体外受精在内的辅助生殖技术有助于降低繁殖计划的成本，为防止灭绝提供保障，并协助野生种群的基因拯救。然而，这些技术用于产生生殖成熟后代的情况仅在少数非模式物种中得到证实。我们旨在优化濒危蛙类绿金雨滨蛙的精子冷冻保存，并通过体外受精从冻融精子中产生成熟后代。我们测试了三种浓度（1.4、2.1和2.8M）的冷冻保护剂二甲基亚砜（DMSO）和甘油与0.3M蔗糖的组合。无论浓度如何，使用DMSO比甘油更有可能使精子活力、活力和顶体完整性恢复，其中向前运动性对损伤最敏感。无论冷冻保护剂类型如何，1.4和2.1M的最低浓度提供了最佳保护。在使卵子受精的能力方面，保存在2.1M DMSO中的精子优于保存在同等浓度甘油中的精子，从而导致更高的胚胎孵化率和几只个体达到性成熟。我们已经证明，精子冷冻保存以及随后通过体外受精产生后代是绿金雨滨蛙和其他濒危两栖动物可行的保护工具。
