Effects of different handling methods on the behavior of adult zebrafish

The zebrafish is an important biomedical research organism. In most research, zebrafish are removed from their home tank and subsequently their phenotype is measured. The method of handling the fish, however, may significantly affect a variety of phenotypes. This is particularly problematic for studies of brain function that measure behavioral or neuronal responses. Nevertheless, the potential effects of handling have not been analyzed, and in fact are usually ignored. Here, we explore the effects of two usual and two rarely or never-before employed handling methods on the behavior of adult zebrafish. We exposed each fish to one of four handling methods, a between subject experimental design: (1) net chasing followed by air-suspension, (2) gentle net catching (without chasing) followed by air-suspension, (3) gentle net catching followed by being placed in a beaker (no chasing and very short air-suspension), (4) transportation in home tank and pouring the fish directly into the test tank (no chasing, netting or air-suspension). With these handling methods, the fish were placed in a test tank and their swim path was videorecorded and analyzed. Handling significantly affected swim path parameters, duration and frequency of immobility, absolute turn angle and its temporal variance and velocity, but not the distance to bottom. The behavioral effects confirmed that chasing and netting induce robust behavioral changes, and that pouring the fish from its home to its test tank is least aversive for zebrafish. We recommend using this latter method to reduce experimental error variation and increase reproducibility of results.