Forsøksdyr: Temporal developement of interrenal activation after stress in Atlantic salmon


Godkjenningsdato 31.08.2020

Godkjenningsperiode 01.10.2020-31.10.2020

I need to repeat an experiment already approved in 2018 (id15228;sakn18/35409). The former was performed successfully but the nucleic acid material has been lost during analyses due to a bad reagents batch. The parr cortisol data will be reported in the ppt as example (annex)
The experiment is described below as for the former approved application with minor changes.
The original study aimed to describe the response time and peak of the interrenal activation, including plasma cortisol levels and activation of several genes in parr and post-smolts of Atlantic salmon. This time we would like to add a transcriptome study for the same tissue to enquire also the up/down regulation and timing of the stress response and immune system. The experiment will involve a standard stress regime of 60 s air exposure in a dip net and euthanization with an overdose of anesthetics after 5-120 min for all individuals involved. Descriptions of the temporal development of the stress response have been done for several species including Atlantic salmon, but with low temporal resolution. In the stress trials with salmonids blood and other tissues are often sampled at the same time, usually around 30-60 min post-stress, although different stress measures probably peak at different times post-stress, and the temporal development likely varies with life stage in addition environmental factor such as temperature. Sampling at the peak response would give higher quality of the data and less variation between studies. Salmon will be netted out from a larger group and held in air for 60 s before they are transferred in groups of 10 to new tanks for sampling after 5, 10, 20, 30, 45, 60, 120 or 360 min post-stress. An extra group of 10 fish will hold and stressed again (same as described before) 6 hr after the first stress and sampled 1 h after. This fish group will give valuable information to better understand fish physiological capacity and regulatory mechanisms triggered in respons to addistional stressors (cumulative stress). 10 fish will be sampled without air exposure in order to estimate baseline levels. 10 sampling point involving 10 fish each, and 2 different life stages the total number of fish will be 200. The 3Rs: Replacement: not possible. Reduction: restricting samples to the first two hour post-stress, except for one later (6 h) sample point to evaluate recovery. Ten killed fish per sample point is regarded a minimum as a proportion of the samples often have to be rejected, for instance due to outliers or hemolysis in plasma, leaving a lower number for statistical analysis. Refinement: The fish will be held in their home tank until stressed, and thereafter transferred to similar tanks at the same temperature and left undisturbed until sampled. 1 min air exposure as stressor is chosen to secure a clear stress response but still avoid sever stress that could arise from longer air exposure durations.