The role of water chemistry in zebrafish welfare and reproducibility of research studies

目前的建议将在减少和改进3R部分方面带来改进：1)斑马鱼研究设施将需要生产更少的种群动物来满足实验数量，因为当水化学得到优化时，所产生的动物将具有更高的质量。2)最优的水化学还有望提高种群鱼类质量的一致性(即较少的个体间变异性)。这也将从整体上减少动物的使用，因为：a)在检测处理差异方面达到相同的统计能力所需的研究内的重复性更少，以及b)研究和不同设施之间的重复性将得到改善。3)优化水质还将延长成虫胚胎生产高峰期的时间范围，这反过来又将减少产生新亲鱼系的频率。这一窗口期目前从4个月到9个月不等，因此持续在这一范围的上端工作可能会使所需的新亲鱼系数量减半。改进：这项研究还将最大限度地减少与次优水质相关的痛苦、痛苦、痛苦或持久的伤害。特别是，已知二氧化碳浓度升高会损害鱼类的大脑功能和对感官刺激(嗅觉、听觉和视觉)做出适当反应的能力，这些刺激是鱼类正常生活和社会互动的一部分，还会损害学习和认知功能，并可能导致焦虑。应对高二氧化碳和酸碱调节的成本也减少了用于健康生长、发育、生殖、免疫功能和抗病的能量。这项拟议的研究将提供优化二氧化碳本身的证据，以及已知的限制鱼类实现酸碱平衡能力的变量，这些变量与消化/运动/饲养压力和高二氧化碳的日常挑战有关。后者包括淡水的Na+和Cl-水平，它们限制了鳃的酸和碱排泄速度，以及直接调节皮肤和鳃对所有分子的渗透性的钙。水碳酸氢盐还决定了鱼类对高二氧化碳的耐受性，并恢复了它们的酸碱平衡。因此，拟议的实验将提供机会，大大减少世界各地斑马鱼研究设施中水质不佳的水的有害影响。潜在3RS影响的指标：在研究完成之前，很难准确量化预期使用的动物减少和经历的痛苦。然而，斑马鱼是全球鱼类研究中使用的头号模式物种，估计有100个国家的3250多个机构进行斑马鱼研究。在动物研究中，斑马鱼是第二大最常用的脊椎动物物种，每年使用500万条鱼。因此，即使只有1%的全球斑马鱼研究设施采用了我们计划生产的指导方针，那么每年至少有5万条斑马鱼将受益于改善的福利(应包括生理动态平衡、生长、行为和免疫功能)。即使这1%的数字只适用于英国，那么每年仍有4000条斑马鱼受益。事实上，1%的数字很可能是对全球斑马鱼福利改善的非常保守的估计。这将是因为每项研究需要更少的鱼，因为生产的一致性更好(即个体间变异性更低)，以及由于研究和设施之间的重复性提高。反过来，这将转化为关于研究结果的更有力和更可靠的结论(例如，药物治疗的效果等)。关于“改进”部分，如果有效地编制和传播高质量的准则，就有可能在真正的全球范围内减少动物的痛苦，并改善每年数百万动物的累积生活体验。

The present proposal will result in improvements relating to the Reduction and Refinement components of the 3Rs: REDUCTION: 1) Zebrafish research facilities will need to produce fewer stock animals to fulfil experimental numbers as the animals produced will be higher quality when water chemistry is optimised.2) Optimal water chemistry is also expected to improve the consistency of the quality of stock fish (i.e. less inter-individual variability). This will also reduce animal use overall because: a) less replication within studies required to achieve the same statistical power in detecting treatment differences, and b) the reproducibility between studies and different facilities will be improved. 3) Optimising water quality will also extend the timeframe of peak embryo production by adults, which in turn will reduce the frequency of producing new broodstock lines. This window currently varies from 4 to 9 months, so consistently working at the upper end of this range could halve the number of new broodstock lines required. REFINEMENT: This study will also minimise pain, suffering, distress or lasting harm associated with sub-optimal water quality. In particular elevated CO2 is known to impair brain function and the ability of fish to respond appropriately to sensory stimuli (olfactory, auditory and visual) that are part of their normal life and social interactions, and impairs learning and cognitive function, and can induce anxiety. The costs of coping with high CO2 and acid-base regulation also detract from energy spent on healthy growth, development, reproduction, immune function and disease resistance. The proposed study will provide evidence to optimise CO2 itself, as well as variables known to limit the ability of fish to achieve acid-base homeostasis associated everyday challenges of digestion/exercise/husbandry stresses and high CO2. The latter includes freshwater Na+ and Cl- levels which limit the rate of acid and base excretion by the gills, and calcium that directly regulates skin and gill permeability to all molecules. Water bicarbonate also determines the tolerance of fish to high CO2, and recovering their acid-base balance. The proposed experiments will therefore offer the opportunity to significantly reduce the detrimental impacts of water sub-optimal quality in zebrafish research facilities throughout the world. METRICS ON POTENTIAL 3Rs IMPACT: Until the research is completed it is difficult to precisely quantify the anticipated reduction of animals used and suffering experienced. However, zebrafish are the number one model species used in fish studies globally, with more than 3,250 institutes in 100 countries estimated to conduct zebrafish research. Zebrafish represent the 2nd most used species of any vertebrate in animal research, with >5 million fish used per annum. Therefore, even if only 1 % of all the global zebrafish research facilities adopted the guidelines we aim to produce, then at least 50,000 fish per year would benefit from improved welfare (which should include physiological homeostasis, growth, behaviour and immune function). Even if this 1 % figure only applied to the UK, then >4,000 zebrafish would still benefit per year. In reality the 1 % figure is likely to be a very conservative estimate of improved welfare for zebrafish globally. This will be due to fewer fish being required per study due to better consistency of production (i.e. lower inter-individual variability), and due to improved reproducibility between studies and facilities. In turn, this will translate into more robust and reliable conclusions regarding the outcomes of research (e.g. efficacy of drug treatments etc.). Regarding the "Refinement" component, if high quality guidelines are produced and disseminated effectively there is potential to reduce animal suffering on a truly global scale and improve the cumulative lifetime experience of millions of animals per year.