Impact of acute handling stress, anaesthesia, and euthanasia on fish plasma biochemistry: implications for veterinary screening and metabolomic sampling

Impacts of pre-sampling practices on fish plasma biochemistry may bias the outcome of a study if not considered within the general sampling strategy. Acute handling stresses can be imposed on fish during capture, and it is common practice to immobilise fish via sedation prior to obtaining blood samples for non-lethal extraction purposes, and/or to reduce stress, pain, or suffering before being euthanised. We investigated these potential influences using a Chinook salmon model (Oncorhynchus tshawytscha) by measuring levels of 119 biochemical targets comprising ions, metabolites, and enzymes in plasma. Multivariate analyses showed that 2 min of confinement with mild handling manipulation led to a significant departure from baseline metabolism, which was further exasperated during a prolonged 5-min challenge. These changes were characterised by a disruption in osmoregulation, a switch towards anaerobic metabolism, and shifts in ammonia recycling, among others. Sedation of fish with clove oil and AQUI-S® had major impacts on plasma biochemical profiles, with alterations signalling changes in glycolytic metabolism, respiratory modes, carbon flux through the TCA cycle, and lipid compartmentalisation. Sedation also enhanced levels of plasma amino acids, revealing a key difference between responses to handling stress and sedation. These results demonstrate that pre-harvest practices should be carefully managed during fish sampling for biochemical/metabolomic-based analyses, and if manipulations are essential, they should be standardised.