Continuous on-line non-invasive cortisol measurement in aquaculture

The work described in this thesis is derived from the need to establish good welfare for fish in aquafarms. Sea food has become an integral part of the quest to meet the protein demands of an ever-growing world. When the world scrambles towards higher production rates, both sustainability and the welfare of the farmed animals are jeopardised. The work presented in this thesis focuses on a particular type of highdensity fish framing called the recirculating aquaculture system (RAS), posed to have the potential for meeting the increasing demands for fish and allied products.

The stress hormone cortisol is an apt indicator of non-optimal conditions in (RAS). Nevertheless, it is not straightforward to measure the cortisol level of fish without going for invasive procedures. However, a breakthrough was made by researchers in the Centre for Environment Fisheries and Aquaculture Science (CEFAS), UK, when they establish beyond doubt that fish release cortisol into the surrounding water and can be measured by collecting that water sample. The drawback of the method was that it was laborious and not suitable for continuous measurement.

It was clear that cortisol had to be continuously monitored to use the data for welfare assessment since variation from circadian rhythm needs to be established to determine the change in cortisol level. The way ahead was to develop a system that could measure cortisol (i) non-invasively, (ii) continuously, (iii) in the pM range and (iv) automatically. The solution was to create a flow injection analysis system that could take samples from the fish tank without disturbing the fish, measure the cortisol level and keep repeating the same every day. This thesis presents how such an automated non-invasive flow system for cortisol measurement was developed.

The system comprised multiple peristaltic pumps and injection valves controlled through a custom-made LabView operated automation circuit. Two different immunoreaction chamber (IRC) formats were tested, where cortisol antibody was bound to Protein G on either a gold surface or superparamagnetic beads. They provided an IC50 and LOD of 0.91 nM and 0.6 pM (gold), 7.8 nM and 1.3 pM (beads), respectively.

Actual water samples were tested from a model RAS at DTU Aqua. The IRC with magnetic beads was modified to incorporate an immuno-supported liquid membrane (ISLM) unit for sample cleaning and enrichment. Despite the higher IC50 and LOD, the loaded Protein G modified magnetic beads render the system highly versatile for use. After 900 s of sample flow through the ISLM donor phase, the antibody binding sites in the acceptor phase were fully occupied by cortisol, showing that continuous cortisol detection in water is feasible. The automation system has been designed to accommodate more pumps and valves if the need arises. Also, the design and fabrication methods have been adapted for industrial manufacturing practices.

The signal output from the system can be integrated into the central control system of the aquafarm to facilitate a better idea regarding the welfare situation in the aquafarm. Corrective actions can then be initiated to maintain a good level of welfare. The developed system shows excellent promise in the aquafarming sector.