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

本提案将导致与3R：REDUCTION：1）斑马鱼研究设施将需要生产更少的库存动物来满足实验数量，因为当水化学优化时，生产的动物将具有更高的质量。2）最佳的水化学也有望提高库存鱼质量的一致性（即个体间变异性较小）。这也将减少整体动物使用，因为：a）在检测治疗差异时，达到相同统计学把握度所需的研究内重复较少，以及B）研究和不同机构之间的重现性将得到改善。 3)优化水质还将延长成虫胚胎生产高峰期的时间，这反过来又将降低生产新亲鱼品系的频率。这个窗口期目前从4个月到9个月不等，因此始终在这个范围的上限工作可以使所需的新亲鱼品系数量减半。 改进：这项研究还将最大限度地减少与次优水质相关的疼痛，痛苦，痛苦或持久伤害。特别是，已知二氧化碳浓度升高会损害大脑功能和鱼类对感官刺激（嗅觉、听觉和视觉）作出适当反应的能力，这些刺激是鱼类正常生活和社会互动的一部分，会损害学习和认知功能，并可能引起焦虑。应对高二氧化碳和酸碱调节的成本也会减少用于健康生长、发育、生殖、免疫功能和抗病能力的能量。拟议的研究将提供证据，以优化二氧化碳本身，以及已知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相关。后者包括淡水Na+和Cl-水平，其限制鳃的酸和碱排泄速率，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CO2的耐受性，并恢复其酸碱平衡。因此，拟议的实验将提供机会，以显着减少水次优质量在世界各地的斑马鱼研究设施的不利影响。潜在3Rs影响的指标：在研究完成之前，很难精确量化预期的动物使用和痛苦减少。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3，250多个研究所进行斑马鱼研究。斑马鱼是动物研究中第二大最常用的脊椎动物物种，每年使用超过500万条鱼。因此，即使全球所有斑马鱼研究机构中只有1%采用了我们旨在制定的指导方针，那么每年至少有50，000条鱼将受益于改善的福利（包括生理稳态，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>4,000条斑马鱼受益。实际上，1%的数字可能是对全球斑马鱼福利改善的非常保守的估计。这将是由于更好的生产一致性（即更低的个体间变异性），以及研究和设施之间的重现性提高，导致每次研究所需的鱼更少。反过来，这将转化为关于研究结果的更有力和可靠的结论（例如药物治疗的功效等）。关于“完善”部分，如果制定和有效传播高质量的准则，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终身经验。

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.