The aim of this study was to investigate the differences in fearfulness between two Japanese native chicken breeds, Tosa‐Kukin (TOK) and Yakido (YKD). In a tonic immobility test, TOK breed chicks showed longer duration and lower induction number compared with YKD. The number of peeps in YKD in an isolation test was lower than that in TOK, whereas there were more bouts of peeping in YKD than in TOK. In a manual restraint test, YKD showed more active responses and initiated peeps and struggling earlier than TOK. The three behavioral tests all indicated that YKD are less fearful than TOK chickens. A latent structures discriminant (OPLS‐DA) analysis was used to identify behavioral parameters that contributed to the differences between the breeds. The major parameters were duration and number of inductions in the tonic immobility test and number of struggle in the manual restraint test. These results suggest that three behavioral tests can be used together to evaluate fearfulness of Japanese native breeds of chickens.

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