Age differences in fear response of laying hens exposed to isolation test in light and dark environments

The isolation fear test is a welfare-based test used to assess fear in poultry. Yet the impacts of isolation on the birds' neurobiology associated with fear is unknown. This study examines how isolation in the light or dark affects vocalizations, corticosterone, and brain neurotransmitters. Thirty-six W-36 laying hens at 12 days of age, and 12, 25, and 70 weeks of age were tested. Two treatments (LIGHT and DARK; n = 12/group/age) were isolated in an anechoic chamber for 90 min and vocalizations were recorded. Both groups were then euthanized. Controls (CON; n = 12/age) were removed from their home pens and euthanized immediately. Mass spectrometry was used to measure dopamine (DA) and serotonin (5-HT) with their metabolites and respective turnover rates and melatonin. Neurotransmitter and corticosterone were analyzed by ANOVA, and vocalizations with a Wilcoxon signed-rank test. LIGHT chicks had a shorter duration to (p = 0.0020) and frequency of (p = 0.0039) vocalizing compared to DARK. In chicks there was an increase in 5-HT turnover in the diencephalon (DI) (p = 0.0866) and the lateral telencephalon (LT) (p = 0.0560), suggesting a negative affective state. There were no significant differences found in DA and 5-HT activity in pullets. Peak lay LIGHT hens had a shorter duration to first vocalization (p = 0.0005) and frequency of vocalizations (p = 0.0039) than peak lay DARK hens. DARK geriatric hens did not vocalize much, while LIGHT geriatric hens vocalized infrequently but more than DARK (p = 0.0370). We found that peak lay LIGHT hens had higher 5-HT turnover in the caudal mesencephalon (CM) (p = 0.0004), rostral mesencephalon (RM) (p = 0.0100), and LT (p = 0.0002) compared to DARK and CON. Geriatric DARK hens had a significantly higher turnover in the LT (p = 0.0170) and the medial telencephalon (MT) (p = 0.0170) compared to CON. These data suggest age differences in behavioral and physiological responses that need to be considered when evaluating fear in poultry.