Peer Review #1 of "An attention bias test to assess anxiety states in laying hens (v0.2)"

Abstract

Fear is a response to a known threat, anxiety is a response to a perceived threat. Both of these affective states can be detrimental to animal welfare in modern housing environments. In comparison to the well-validated tests for assessing fear in laying hens, tests for measuring anxiety are less developed. Perception of a threat can result in an attention bias that may indicate anxious affective states in individual hens following playback of an alarm call. In Experiment 1, an attention bias test was applied to hens that differed in their range access to show that hens that never ranged were more vigilant (stretching of the neck and looking around: P < 0.001) and slower to feed following the second alarm call playback (P = 0.01) compared with hens that ranged daily. All hens showed a reduction in comb temperature following the first alarm call (P < 0.001). In Experiment 2, an open field test was used to determine an effective dose of 2mg/kg for the anxiogenic drug meta-Chlorophenylpiperazine (m-CPP) in adult laying hens. Hens dosed with 2mg/kg showed reduced locomotion compared with a saline solution (P < 0.05). In Experiment 3, 2mg/kg m-CPP or saline was administered to adult hens previously habituated to the open field arena to pharmacologically validate an attention bias test as a measure of anxiety. Hens dosed with m-CPP were slower to feed (P = 0.02) and faster to vocalize following a second alarm call playback (P = 0.03) but these hens did not exhibit the same vigilance behavior as documented in Experiment 1. The m-CPP hens also spent more time stepping and vocalizing (both P < 0.001) than the saline hens. An attention bias test could be used to assess anxiety. However, behavioral responses of hens may vary depending on their age or test environment familiarity, thus further refinement of the test is required. In these tests, 2mg/kg of m-CPP resulted in freezing when the environment was novel, but more movement and vocalizing when the environment was familiar. The extreme behavioral phenotypes exhibited by individually-tested birds may both be indicators of negative states.