This research aims to describe the effects of a variable number of Danio rerio fish subjects, ranging from one to eight, in the light/dark box preference test. Four hundred eighty adult male short-finned phenotype zebrafish were tested in the light/dark box. There were four groups in this experiment and a different number of subjects was used in each group: the control group had only one subject, whereas the experimental groups had either two, four, or eight subjects simultaneously inside the apparatus in every session. The average occurrence (AO) of subjects in the white side of the aquarium and the first choice average (FC) were recorded. The AO revealed no difference between the control group and test groups with two and four subjects. The results for the test group with eight subjects showed significant difference when compared to the control group and from the test group with two subjects. The FC also showed no difference between the control group and test groups with two and four subjects. There was significant variation between the control and the test group with eight subjects. The results reflect a conflict between the animal's preference for dark places and the innate drive to explore new environments. Zebrafish are highly social animals, exhibiting preference for swimming in groups and other patterns of social cohesion. The reduced white avoidance behavior in the test group of eight subjects may possibly reflect the role of shoaling, which is a defensive behavior, in reducing anxiety and stress. On the other hand, the absence of difference between the control group and test groups with two and four subjects suggest that it is feasible to run the light/dark test with up to four subjects, becoming an alternative to streamline and simplify data collection and test analysis.
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