Investigating Non-invasive Measures of Stress in Ornamental Fish

Abstract

The transport of ornamental marine fish may cause stress, which to date has been the subject of limited research. The present study aimed to characterise the behavioural and physiological responses to simulated transport stress in the common clownfish Amphiprion ocellaris (Cuvier, 1830) with the additional goal of validating non-invasive measures of water cortisol in a marine teleost for the first time. Behaviour and physiology of the animals was measured at different stages of transport (from initial capture and handling up to 72 hours transport time) and water quality measurements were taken from the transport water at key sampling points. In a second experiment biological filtration materials (“Bioballs” with denitrifying bacteria) were added to the transport bag to determine if stress was reduced when water quality was improved. The results of the study suggest that capture, handling and transport are stressful for clownfish, and the stress response appears to peak between 24 and 48 hours after the onset of the stressor. Water-borne cortisol was found to be a valid alternative to invasive methods of sampling, although only an average of 53% cortisol was recovered from sea water. Although handling and confinement appeared to be highly significant factors in eliciting the stress response water quality measurements revealed that fish are temporarily subjected to relatively high concentrations of ammonia as transport time increased, which may contribute to long-term effects on the health of the animals. This was reflected in an increased latency to feed and reduced social behaviours in fish transported for 24 hours or longer. Improving water quality did reduce the concentration of ammonia present; however, fish still exhibited elevated cortisol excretion suggesting that water quality is not the primary stressor associated with transport. Thus, the duration of transport should be restricted to a maximum of 24 hours to reduce the stress associated with this practice. A separate study investigated the potential for beauty treatment ‘fish spas’ to elicit stress in the freshwater cyprinid fish Garra rufa (Heckel, 1843). Water cortisol was measured non-invasively to determine if stress was reduced through the provision of environmental enrichment / furnishings, and whether stocking density influenced stress. Water quality was monitored to determine the effects of stocking density on environmental parameters (pH, ammonia, nitrite and nitrate). Finally, the effect of ii human hands being placed into the aquarium was investigated, to determine whether this influenced stress. Three hundred G. rufa were used in total, with groups being allocated randomly to one of four treatment groups: OP/B (optimum stocking density / barren tank – i.e. no enrichment); OP/E (optimum stocking density / enriched tank); OS/B (overstocked / barren tank); and OS/E (overstocked / enriched tank). Human hands were placed in each tank, and water samples were collected before and after for measurement of cortisol by ELISA, and to determine water quality parameters. The results revealed that overstocking tanks with G. rufa produced relatively higher baseline cortisol levels, suggesting that stocking density may have a significant effect on stress levels. The addition of furnishings into the aquarium did not reduce baseline cortisol levels in the fish prior to the introduction of human hands. However, fish maintained under enriched conditions exhibited a greater cortisol response when compared to individuals in barren tanks. It is hypothesised that the provision of enrichment reduces the available space for fish following the introduction of human hands, thereby increasing stress. Further studies are required to attempt to determine the effect of enrichment based upon the results of the present study. Feeding on human hands resulted in an elevated cortisol response from three out of the four treatment groups (with the exception of OP/B), with the results suggesting that either 1) the lower (i.e. optimal) stocking density and lack of enrichment in holding tanks is preferable for G. rufa fish welfare, or 2) the elevated cortisol reflected a response to a rewarding stimulus and is linked to increased foraging. Overall, the results of these studies have shown that water cortisol measurements are a valid means of assessing physiological stress in two species of fish in different contexts. This negates the need for invasive sampling and is an important refinement to existing protocols where fish are killed for plasma or whole body samples. The results also highlight the welfare impacts of transport and overstocking of ornamental fish providing valuable evidence that may be relevant to improving the husbandry and guidelines with respect to the ornamental fish industry.