The number of conspecific alarm substance donors notably influences the behavioural responses of zebrafish subjected to a traumatic stress procedure.

Zebrafish (Danio rerio) represents a complementary pre-clinical model in stress and anxiety research. Conspecific alarm substance (CAS), an alarm pheromone secreted by injured fish, acts as a warning signal and modulates fear responses. Given their schooling nature and that injury precedes CAS release, varying fresh CAS concentrations extracted from different numbers of CAS-donating zebrafish may uniquely influence trauma-related behaviours. Thus, we investigated the behaviour of juvenile and adult zebrafish exposed to traumatic stress protocols, in the presence of CAS extracted from varying numbers of donating zebrafish. Juveniles were assessed for anxiety and boldness in the light-dark and open field tests (LDT and OFT), while adults were assessed in the novel tank test (NTT) and novel OFT (nOFT). We found that (1) trauma minimally impacted juvenile behaviour regardless of donor-derived CAS concentrations, (2) trauma-exposed adults displayed reduced exploration and heightened risk-taking behaviours in the NTT and nOFT compared to control-exposed fish, (3) NTT and nOFT freezing behaviours were distinctly emulated in adult fish and (4) post-trauma behaviour in adults was influenced by the number of donors. Therefore, CAS concentration as determined by donor number has age-related effects on anxiety- and risk-taking behaviours in trauma-exposed zebrafish, a valuable finding for studies utilising fresh CAS as a stress trigger. While we did not directly investigate CAS concentration through serial dilution, our data are of significant translational and ethological relevance, highlighting the importance of in-house method standardization in stress-related studies utilizing fresh CAS as an alarm cue.