In this extract from Conservation Physiology, Lachlan Campbell et al. address the lacking research on reproductive technologies for non-mammalian species that can aid conservation efforts and even reverse species extinction. The authors look to the Australian lizard Varanus panoptes to investigate and develop an optimised protocol for reptile sperm cryopreservation.
在《 保护生理学》的 摘录中 ，拉克兰·坎贝尔(Lachlan Campbell)等人。 解决了缺乏对非哺乳动物物种生殖技术的研究，这些研究可以帮助保护工作甚至逆转物种灭绝。 作者期望澳大利亚蜥蜴Varanus能够研究和开发优化的爬行动物精子冷冻保存方案。
The collection and storage of genomes through sperm cryopreservation, combined with associated assisted reproductive technologies (ARTs) for restoring animals and genes using the genetic material, could play a large role in threatened species conservation (Browne et al., 2011; Clulow and Clulow, 2016; Howard et al., 2015). Despite the potential to apply these technologies to all vertebrate taxa, reptiles are a relatively neglected taxon, with few successful sperm cryopreservation protocols developed for this group despite some attempts and typically much lower rates of successful post-thaw recovery (Browne, et al., 2011; Clulow and Clulow, 2016; Molinia et al., 2010). This is important because up to 25% of reptilian species are threatened with extinction globally and a further 25% are classed as data deficient (Böhm et al., 2013). Indeed, some reptile orders such as the Testudines and Crocodilia may be as high as 50% threatened with extinction globally (Böhm, et al., 2013; Grigg and Kirshner, 2015).
通过精子冷冻保存来收集和存储基因组，再结合相关的辅助生殖技术(ART)，利用遗传物质来恢复动物和基因，可以在受威胁物种的保护中发挥重要作用(Browne等，2011； Clulow和Clulow， 2016;霍华德等人，2015)。 尽管有可能将这些技术应用于所有脊椎动物类群，但爬行动物是一个相对被忽略的类群，尽管进行了一些尝试并且成功地将解冻后的恢复率降低了，但针对该组的成功的精子冷冻保存方案却很少(Browne等人， 2011； Clulow和Clulow，2016； Molinia等人，2010)。 这很重要，因为全球多达25％的爬虫类物种面临灭绝的威胁，另外25％被归类为数据不足的物种(Böhm等，2013)。 确实，全球某些物种的爬行动物纲目(如睾丸类和鳄科)可能面临高达50％的灭绝威胁(Böhm等，2013； Grigg和Kirshner，2015)。
Given the limited number of reports and species investigated across the reptiles, whose major lineages have evolved very different reproductive systems, there is a strong imperative to understand general principles and shared aspects of sperm cryopreservation protocols in one or more model reptile species. Ideally, these studies would serve as a prelude for the development of optimized protocols within and between reptilian groups. Lizards are one of the most diverse reptile groups, yet sadly, of the known approximately 6100 lizard species globally, the International Union for Conservation of Nature (IUCN) has classified at least one third as threatened with extinction (Böhm, et al., 2013; Gibbons et al., 2000). Given their significance and diversity within squamate reptiles, lizards are an appropriate group to focus on for the development of an optimized sperm cryopreservation protocol, notwithstanding the success reported for one species (Young et al., 2017). One such lizard that is currently suffering large population declines is the yellow-spotted monitor (Varanus panoptes), an Australian species that has been severely impacted by the presence of the invasive cane toad (Rhinella marina) (Doody et al., 2009; Doody et al., 2014; Doody et al., 2017). Affected populations decline through increased mortality rates resulting from lethal toxic ingestion when the toad is consumed as prey (Doody, et al., 2009; Doody, et al., 2017; Ujvari and Madsen, 2009). Since its introduction to Australia in 1935 (Doody, et al., 2019; Lever, 2006), the cane toad has caused population crashes of up to 97% in V. panoptes (Doody, et al., 2009) as well as extirpations of this and at least one other monitor species in some parts of northern Australia (Doody, et al., 2017), which in turn has resulted in rippling effects throughout ecosystems via trophic cascades (Doody, et al., 2017; Doody et al., 2015). Varanus panoptes is thus an ideal model species for the development of sperm cryostorage and ARTs, because it is abundant ahead of the toad front for conducting experiments, but is threatened behind the invasion vanguard and exerts a disproportionately large influence over its ecosystem, thus warranting urgent conservation attention.
由于整个爬行动物的报告和物种数量有限，其主要血统进化了非常不同的生殖系统，因此有必要了解一种或多种模型爬行动物物种的精子冷冻保存方案的一般原理和共享方面。 理想情况下，这些研究将作为开发爬虫类群内部和之间的优化方案的前奏。 蜥蜴是最多样化的爬行动物群体之一，但可悲的是，在全球已知的约6100种蜥蜴物种中，国际自然保护联盟(IUCN)至少将三分之一归类为濒临灭绝的物种(Böhm等，2013)。 ； Gibbons等，2000)。 鉴于蜥蜴在鳞状爬行动物中的重要性和多样性，尽管已报道了一种物种的成功，但蜥蜴仍是适合于专注于优化精子冷冻保存方案开发的合适人群(Young等人，2017)。 一种目前正遭受大量种群减少的蜥蜴是黄斑监测器( Varanus panoptes )，这是一种澳大利亚物种，受到入侵性蟾蜍蟾蜍(Rhinella marina)的严重影响(Doody等，2009； Doody)。等人，2014； Doody等人，2017)。 当蟾蜍被当作猎物食用时，由于致命的毒性摄入而导致死亡率上升，受影响的人口减少(Doody等，2009； Doody等，2017； Ujvari和Madsen，2009)。 自1935年引入澳大利亚以来(Doody等人，2019年； Lever，2006年)，甘蔗蟾蜍已使泛盲弧菌的种群崩溃率高达97％(Doody等人，2009年)以及灭绝澳大利亚北部某些地区的这种和至少一种其他监测物种(Doody等，2017)，进而通过营养级联导致整个生态系统的涟漪效应(Doody等，2017; Doody等。，2015)。 因此， 天牛座是精子冷冻和ARTs发展的理想模式物种，因为它在蟾蜍前部进行实验的数量丰富，但在入侵先锋的后面受到威胁，对其生态系统产生不成比例的巨大影响，因此迫在眉睫。养护注意。
The addition of caffeine could thus potentially be a novel and simple means by which to stimulate post-thaw motility of lizard sperm, although to the best of our knowledge this principle has never been tested.
因此，添加咖啡因可能是刺激蜥蜴精子解冻后运动的一种新颖而简单的方法，尽管据我们所知，这一原理从未得到验证。
Sperm cryopreservation involves a series of steps that must be optimized to minimize the cell damage associated with deep freezing (Dinnyes et al., 2007). Dimethyl sulfoxide (DMSO) and glycerol are perhaps the two most commonly investigated cryoprotectants for the spermatozoa of wildlife species, although the relative success of each often varies depending on the species (Comizzoli et al., 2012). Cryoprotective agents (CPAs) also become increasingly toxic as the concentration increases (Best, 2015), which can result from non-specific toxicity via water molecule interference with the cell membrane or specific toxicity derived from the CPA type and concentration (Fahy, 1986; Fahy, 2010). It is important to understand the interplay of these factors in order to develop appropriate conditions for sperm cryopreservation.
精子冷冻保存涉及一系列步骤，必须对其进行优化以最大程度地减少与深度冷冻相关的细胞损伤(Dinnyes等，2007)。 二甲基亚砜(DMSO)和甘油也许是研究野生动植物物种精子的两种最常用的冷冻保护剂，尽管每种物种的相对成功率通常取决于物种(Comizzoli等，2012)。 冷冻保护剂(CPA)随着浓度的增加也变得越来越有毒(Best，2015)，这可能是由于水分子干扰细胞膜而引起的非特异性毒性或源自CPA类型和浓度的特异性毒性(Fahy，1986; Fahy，2010年)。 重要的是要了解这些因素的相互作用，以便为精子冷冻保存开发适当的条件。
In addition to optimizing cryoprotectant, media and cooling rates as a part of the freeze-thaw protocol, other factors may aid in increasing the viability of sperm and thus improve their motility characteristics in preparation for ARTs such as in vitro fertilization (IVF) or artificial insemination. For example, the addition of the phosphodiesterase inhibitor caffeine has been shown to increase intracellular levels of the second messenger, cyclic-adenosine monophosphate (cAMP) (Pukazhenthi et al., 2005; Saragusty, 2012), resulting in a consequential increase in forward progressive motility in unfrozen epididymal sperm of the lizard, Lacerta vivipara (Depeiges and Dacheux, 1985). In a similar context, cryopreserved mammalian spermatozoa have also been shown to display elevated levels of post-thaw motility following incubation in caffeine (Mbizvo et al., 1993; Rota et al., 2018; Schill et al., 1979; Stachecki et al., 1994). The addition of caffeine could thus potentially be a novel and simple means by which to stimulate post-thaw motility of lizard sperm, although to the best of our knowledge this principle has never been tested. Therefore, optimizing cryopreservation protocols requires attention to the separate components of cryoprotectant, media and cooling/thawing rates, but also may be enhanced by additional post-thaw treatments.
除了优化冷冻保护剂，培养基和冷却速率作为冻融方案的一部分外，其他因素还可以帮助提高精子的生存力，从而改善其活力特性，从而为进行抗逆转录病毒疗法或人工授精等抗逆转录病毒疗法做准备授精。 例如，磷酸二酯酶抑制剂咖啡因的添加已显示可增加第二信使环状单磷酸腺苷(cAMP)的细胞内水平(Pukazhenthi等，2005； Saragusty，2012)，从而导致正向进行性增加蜥蜴的未冷冻附睾精子中的蠕动(Visara)(Depeiges and Dacheux，1985)。 在类似的情况下，在咖啡因中孵育后，冷冻保存的哺乳动物精子也显示出高水平的解冻后运动能力(Mbizvo等人，1993; Rota等人，2018; Schill等人，1979; Stachecki等人) (1994年)。 因此，添加咖啡因可能是刺激蜥蜴精子解冻后运动的一种新颖而简单的方法，尽管据我们所知，这一原理从未得到验证。 因此，优化冷冻保存方案需要注意冷冻保护剂，培养基和冷却/解冻速率的单独组成部分，但也可以通过附加的解冻后处理来增强。
Conservation Physiology is an online only, fully open access journal published on behalf of the Society for Experimental Biology. Biodiversity across the globe faces a growing number of threats associated with human activities. Conservation Physiology publishes research on all taxa (microbes, plants and animals) focused on understanding and predicting how organisms, populations, ecosystems and natural resources respond to environmental change and stressors. Lachlan Campbell, Shenae L Cafe, Rose Upton, J Sean Doody, Brett Nixon, John Clulow, Simon Clulow contributed to the above excerpt.
保护生理学是仅在线的，完全开放的期刊，代表实验生物学学会出版。 全球的生物多样性面临着与人类活动有关的越来越多的威胁。 保护生理学发表了关于所有分类单元(微生物，植物和动物)的研究，其重点是理解和预测生物体，种群，生态系统和自然资源如何应对环境变化和压力。 Lachlan Campbell，Shenae L Cafe，Rose Upton，J Sean Doody，Brett Nixon，John Clulow，Simon Clulow促成了上述摘录。
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