Background

Zebrafish have been widely used to study anxiety-related phenotypes using the novel tank test (NTT). Although the NTT is well-characterized and commonly used by researchers, there is still a lack of information regarding how different experimental variables such as water quality can influence NTT performance. Zebrafish use different chemical cues and olfactory stimuli to communicate in water, so we predicted that water change frequency would affect cortisol, locomotion and anxiety-related parameters in the NTT.

New methods

After extensive literature research, we found that only about 18% of papers using NTT report partial or complete water changes between subjects. Here, we tested multiple zebrafish in the NTT using the same water up to 9 consecutive times (with no water change) and analyzed cortisol levels, as a stressrelated marker.

Results

We found that when using the same water for more than 4 trials, data variability is increased and a higher number of extreme values is observed for the time spent in the top zone and immobility. Moreover, after 4 trials with no water change, increased cortisol levels are observed, indicating that animals show increased stressrelated responses with the lack of water changes.

Conclusions

This study shows that lack of water change can significantly influence zebrafish stress-responses in the NTT. Altogether, behavioral experiments should avoid using the same water when testing multiple fish in the task, especially when looking at anxiety in the NTT.

中文翻译：

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水变化对斑马鱼 (Danio rerio) 焦虑相关任务中压力和受试者变异的影响

背景

斑马鱼已被广泛用于使用新型坦克测试 (NTT) 研究焦虑相关的表型。尽管 NTT 得到了很好的表征并被研究人员普遍使用，但仍然缺乏关于不同实验变量（如水质）如何影响 NTT 性能的信息。斑马鱼在水中使用不同的化学线索和嗅觉刺激进行交流，因此我们预测换水频率会影响 NTT 中的皮质醇、运动和焦虑相关参数。

新方法

经过广泛的文献研究，我们发现只有大约 18% 的使用 NTT 的论文报告了受试者之间部分或完全的水分变化。在这里，我们使用相同的水在 NTT 中测试了多条斑马鱼，最多连续 9 次（没有换水），并分析皮质醇水平，作为压力相关的标记。

结果

我们发现，当使用相同的水进行 4 次以上的试验时，数据可变性会增加，并且在顶部区域和静止状态中花费的时间会观察到更多的极值。此外，在不换水的 4 次试验后，观察到皮质醇水平升高，表明动物在不换水的情况下表现出与应激相关的反应增加。

结论

这项研究表明，缺乏换水可以显着影响 NTT 中斑马鱼的应激反应。总而言之，在任务中测试多条鱼时，行为实验应避免使用相同的水，尤其是在 NTT 中观察焦虑时。