The aim of this study is to further advance our understanding of the physiology of the stress response. We plan to utilize the successful methodological setup that has already enabled us to publish two notable studies (Madaro et al., 2022 and 2024) on the acute stress response in Atlantic salmon. The current study aims to characterize the response time and peak of interrenal activation following acute stress, specifically measuring catecholamines (adrenaline and noradrenaline) alongside cortisol metabolites in post-smolt Atlantic salmon. We plan to expose a group of post-smolt salmon to an acute stressor, involving netting and transferring fish to several new holding tanks. We will document the time-course response to stress by sampling groups of fish before (T0) and at 5, 10, 15, 30, 45, 60, 90, 180 and 360 minutes post-stress. Subsequently, all individuals involved will be euthanized with an overdose of anesthetics after 5-360 minutes. Descriptions of the temporal development of the sympathetic stress response have been lacking in fish, including Atlantic salmon. Understanding the release and peak timing will enhance our knowledge of stress regulatory mechanisms and the fight-or-flight response. The 3Rs principle guides our experimental design as follows: Replacement is not feasible in this context. Reduction is achieved by limiting samples to the first two hours post-stress, with the exception of one-hour later (3-hour) sample point to assess recovery. We aim for a minimum of ten killed fish per sample point, recognizing that a proportion of samples may need to be rejected due to outliers or hemolysis in plasma, resulting in a lower number available for statistical analysis. Refinement strategies include keeping the fish in their home tank until stressed and then transferring them to similar tanks at the same temperature, where they are left undisturbed until sampled. Netting and transferring fish to several new holding tanks are chosen as stressors to ensure a clear stress response while minimizing severe stress that could result from prolonged air exposure durations. (Madaro et al 2024; doi.org/10.1016/j.aqrep.2024.102041) (Madaro et al 2023; doi.org/10.1007/s10695-022-01163-4)

When the experiment start, 5 fish/tank (3x15) are netted out of the stock tank. These are killed immediately for baseline (T0) by an overdose of Finquel (1g/L). Thereafter, the water level in the tank will be reduced and fish will be netted and transferred in a transport tank simultaneously so that we can be sure that they alle will be treated in the same manner. Then fish will be then splitted equally between 9 tanks before being sampled after 5, 10, 15, 30, 45, 60, 90, 180 and 360 minutes post-stress. When fish are to be sampled, they will be wet-netted into a separate vessel containing overdose sedation to euthanise them. They will have blood samples taken immediately once dead, and subsequently body measures and finally dissected.

It is not possible to describe the temporal stress response or difference between fish without the use of live fish. This is even more true in this specific case where we aim to see interaction between different axes: the sympathetic and HPI axis. Reduction