# Transcriptome Analysis of the Hippocampus in Domestic Laying Hens with Different Fear Responses to the Tonic Immobility Test

## TL;DR

This study explores how fear responses in laying hens relate to changes in their brain's hippocampus, offering insights into improving animal welfare.

## Contribution

The study identifies specific hippocampal gene expression patterns and neuronal changes linked to fearfulness in laying hens.

## Key findings

- High-fear hens showed reduced Nissl bodies and altered expression of DCX and c-Fos proteins in hippocampal neurons.
- Transcriptomic analysis revealed 365 differentially expressed genes linked to neural and immune pathways in high-fear hens.
- Key pathways related to metabolism and stress regulation were enriched in high-fear hens, suggesting impaired neural function.

## Abstract

Fear behavior is associated with negative emotional responses, which can adversely affect animal welfare. A comprehensive understanding of the neurobiological mechanisms underlying fearfulness is essential for developing effective strategies to improve welfare. In this study, laying hens were classified into high- and low-fear groups based on tonic immobility tests, and hippocampal histological and transcriptomic characteristics were compared to explore the biological basis of individual differences in fear responses. The results revealed that hens with high-fear levels exhibited a significant reduction in Nissl bodies in hippocampal neurons, increased doublecortin (DCX) expression, and decreased c-Fos (Fos proto-oncogene) expression. Differentially expressed genes were enriched in several pathways related to neural function and immune regulation. This suggests that individuals with high fear may experience impaired neural plasticity and dysregulated stress responses. These findings provide molecular-level insights into the mechanisms underlying fearfulness differences and offer a theoretical foundation for future interventions aimed at experiencing less fear and enhancing welfare in laying hens.

Fear is a critical welfare concern in laying hens. Fearful behaviors in domestic chickens are influenced by both genetic and environmental factors, contributing to individual differences in stress responses. Tonic immobility (TI) duration is widely recognized as a reliable indicator of fear levels. The hippocampus, a critical brain region for emotional states, plays a pivotal role in associating fearful experiences with specific stimuli, enabling adaptive behavioral responses. This study investigated hippocampal histological characteristics and transcriptomic profiles in laying hens with different fear responses categorized based on TI duration. A total of 80 native Lindian hens (75 weeks old) were individually housed in modified conventional cages. At 76 weeks of age, hens exhibiting the longest and shortest TI durations were classified into the high-fear (TH) and low-fear (TL) groups, respectively. Whole hemibrains were collected for histological and immunohistochemical analyses, while hippocampal tissues underwent transcriptome sequencing. The results showed a significant reduction in Nissl body counts in hippocampal neurons of high-fear hens (p < 0.05), suggesting potential neuronal damage or functional impairment. Transcriptomic analysis revealed 365 differentially expressed genes (DEGs) between two groups, with 277 upregulated and 88 downregulated genes in TH chickens. KEGG pathway enrichment analysis identified seven significantly associated pathways (p < 0.01), including retinol metabolism, vitamin B6 metabolism, and nicotinate and nicotinamide metabolism, all of which are crucial for neuronal function and immune regulation. In addition, a significant increase in DCX protein expression (p < 0.05) and a decrease in c-Fos protein expression (p < 0.05) was noted in in high-fear hens, whereas PCNA levels remained unchanged (p > 0.05) under immunohistochemical validation. The neuronal alterations observed in high fear individuals suggest neural damage, while transcriptomic variations point to potential disruptions in neurogenesis, synaptic signaling, and stress-related pathways. Collectively, these results provide novel insights into the neurobiological basis of fear regulation in laying hens and may have implications for poultry welfare and management strategies.