Anastas Zara M, Silla Aimee J, Byrne Phillip G, Hobbs Rebecca J, McFadden Michael S, Daly Jonathan, O'Brien Justine K
Environmental Futures, School of Earth, Atmospheric and Life Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
Taronga Institute of Science and Learning, Taronga Conservation Society Australia, Mosman, NSW 2088, Australia.
Vet Sci. 2025 Jan 8;12(1):30. doi: 10.3390/vetsci12010030.
Reproductive technologies, including sperm cryopreservation, offer conservationists enhanced capacity to genetically manage populations and improve the outcomes of conservation breeding programs (CBPs). Despite this potential, the post-thaw quality of amphibian sperm is highly variable following cryopreservation, and research focused on protocol refinement is needed. The aim of this study was twofold: (1) to investigate the effect of the addition of bovine serum albumin (BSA) to the cryopreservation medium (pre-freeze), and (2) the effect of the addition of caffeine to the activation medium (post-thaw), on post-thaw sperm characteristics in the critically endangered Booroolong frog (). Spermic urine samples were collected from 14 male frogs following hormonal induction of spermiation, and each sample was split among three cryopreservation treatments, where the cryopreservation medium contained either 0 (control), 0.5, or 1% BSA (/). Samples were cryopreserved and thawed, and sperm motility was then activated in one of two activation treatments: Milli-Q water (control) or Milli-Q water plus 4.5 mM caffeine. Sperm viability (proportion live/dead) was assessed using fluorescent microscopy, and sperm motility metrics were evaluated using computer-assisted sperm analysis (CASA). Results from this study showed that BSA concentration had no effect on post-thaw sperm viability. Additionally, neither BSA concentration nor activation in caffeine influenced post-thaw sperm motility characteristics (total motility, forward progressive motility, and velocity). Assessment time of sperm motility varied from 5 to 13 min post-activation and was significantly correlated with each motility measure, with motility and velocity metrics decreasing as time post-activation increased. The results reported herein provide no evidence for an effect of BSA or caffeine at the concentrations tested on post-thaw sperm characteristics in the Booroolong frog, but they highlight the time-sensitive nature of sperm assessment post-thaw and implications for the timing of sperm handling during assisted fertilisation efforts.
包括精子冷冻保存在内的生殖技术，为保护主义者提供了更强的基因管理种群能力，并改善了保护育种计划（CBPs）的成果。尽管有这种潜力，但两栖动物精子冷冻保存后的解冻后质量差异很大，因此需要专注于优化方案的研究。本研究的目的有两个：（1）研究在冷冻保存培养基（冷冻前）中添加牛血清白蛋白（BSA）的效果，以及（2）在激活培养基（解冻后）中添加咖啡因的效果，对极度濒危的布罗龙蛙（）解冻后精子特征的影响。在激素诱导精子形成后，从14只雄蛙收集精液样本，每个样本在三种冷冻保存处理之间进行分配，其中冷冻保存培养基含有0（对照）、0.5或1%的BSA（/）。样本进行冷冻保存和解冻，然后在两种激活处理之一中激活精子活力：超纯水（对照）或超纯水加4.5 mM咖啡因。使用荧光显微镜评估精子活力（活/死比例），并使用计算机辅助精子分析（CASA）评估精子活力指标。本研究结果表明，BSA浓度对解冻后精子活力没有影响。此外，BSA浓度和咖啡因激活均未影响解冻后精子活力特征（总活力、向前运动活力和速度）。精子活力评估时间在激活后5至13分钟之间变化，并且与每个活力测量值显著相关，随着激活后时间的增加，活力和速度指标降低。本文报道的结果没有提供证据表明在所测试的浓度下，BSA或咖啡因对布罗龙蛙解冻后精子特征有影响，但它们突出了解冻后精子评估的时间敏感性以及对辅助受精过程中精子处理时间的影响。
