{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:27:22Z","timestamp":1760059642484,"version":"build-2065373602"},"reference-count":41,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2025,6,26]],"date-time":"2025-06-26T00:00:00Z","timestamp":1750896000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Natural Science Foundation of China","award":["32272928","31972608","LH2022C032","LBH-Q21063"],"award-info":[{"award-number":["32272928","31972608","LH2022C032","LBH-Q21063"]}]},{"name":"Natural Science Foundation of Heilongjiang Province","award":["32272928","31972608","LH2022C032","LBH-Q21063"],"award-info":[{"award-number":["32272928","31972608","LH2022C032","LBH-Q21063"]}]},{"name":"Postdoctoral Research Development Fund of Heilongjiang Province","award":["32272928","31972608","LH2022C032","LBH-Q21063"],"award-info":[{"award-number":["32272928","31972608","LH2022C032","LBH-Q21063"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>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 &lt; 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 &lt; 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 &lt; 0.05) and a decrease in c-Fos protein expression (p &lt; 0.05) was noted in in high-fear hens, whereas PCNA levels remained unchanged (p &gt; 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.<\/jats:p>","DOI":"10.3390\/ani15131889","type":"journal-article","created":{"date-parts":[[2025,6,26]],"date-time":"2025-06-26T11:15:23Z","timestamp":1750936523000},"page":"1889","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Transcriptome Analysis of the Hippocampus in Domestic Laying Hens with Different Fear Responses to the Tonic Immobility Test"],"prefix":"10.3390","volume":"15","author":[{"given":"Jingyi","family":"Zhang","sequence":"first","affiliation":[{"name":"College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Min","family":"Li","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Shihezi University, Shihezi 832003, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liying","family":"Pan","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ye","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hui","family":"Yuan","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6266-4158","authenticated-orcid":false,"given":"Zhiwei","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Medicine, Shihezi University, Shihezi 832003, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chaochao","family":"Luo","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Shihezi University, Shihezi 832003, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6315-9399","authenticated-orcid":false,"given":"Runxiang","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1086\/418981","article-title":"Fear and Fearfulness in Animals","volume":"70","author":"Boissy","year":"1995","journal-title":"Q. Rev. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Acharya, R.Y., Hemsworth, P.H., Coleman, G.J., and Kinder, J.E. (2022). The Animal-Human Interface in Farm Animal Production: Animal Fear, Stress, Reproduction and Welfare. Animals, 12.","DOI":"10.3390\/ani12040487"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"859","DOI":"10.1007\/s40264-018-0664-0","article-title":"Vitamin B6 in Health Supplements and Neuropathy: Case Series Assessment of Spontaneously Reported Cases","volume":"41","author":"Kant","year":"2018","journal-title":"Drug Saf."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1079\/WPS19960013","article-title":"Fear and Adaptability in Poultry: Insights, Implications and Imperatives","volume":"52","author":"Jones","year":"1996","journal-title":"World\u2019s Poult. Sci. J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1013","DOI":"10.1071\/AN19680","article-title":"Non-Infectious Diseases and Laying Hen Welfare","volume":"61","author":"Groves","year":"2021","journal-title":"Anim. Prod. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2259","DOI":"10.3382\/ps.2012-02996","article-title":"Fear, Stress, and Feather Pecking in Commercial White and Brown Laying Hen Parent-Stock Flocks and Their Relationships with Production Parameters","volume":"92","author":"Kemp","year":"2013","journal-title":"Poult. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.beproc.2016.11.001","article-title":"Individual Differences in Personality in Laying Hens Are Related to Learning a Colour Cue Association","volume":"134","author":"Lee","year":"2017","journal-title":"Behav. Process."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1016\/bs.pbr.2022.02.009","article-title":"Chapter 9\u2014Autonomic Correlates of Defense Responses, Including Tonic Immobility (TI)","volume":"Volume 271","author":"Carli","year":"2022","journal-title":"Progress in Brain Research"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e14703","DOI":"10.7717\/peerj.14703","article-title":"Differences among Domestic Chicken Breeds in Tonic Immobility Responses as a Measure of Fearfulness","volume":"11","author":"Tiemann","year":"2023","journal-title":"PeerJ"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1016\/j.yhbeh.2008.07.002","article-title":"Genetic Differences in Coping Strategies in Response to Prolonged and Repeated Restraint in Japanese Quail Divergently Selected for Long or Short Tonic Immobility","volume":"54","author":"Hazard","year":"2008","journal-title":"Horm. Behav."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1202","DOI":"10.1093\/brain\/awg103","article-title":"Role of the Hippocampal System in Associative Learning beyond the Spatial Domain","volume":"126","author":"Brasted","year":"2003","journal-title":"Brain"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Jin, J., and Maren, S. (2015). Prefrontal-Hippocampal Interactions in Memory and Emotion. Front. Syst. Neurosci., 9.","DOI":"10.3389\/fnsys.2015.00170"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1038\/npp.2015.171","article-title":"Stress Effects on Neuronal Structure: Hippocampus, Amygdala, and Prefrontal Cortex","volume":"41","author":"McEwen","year":"2016","journal-title":"Neuropsychopharmacology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1186\/s13041-020-00652-y","article-title":"A Critical Role of Hippocampus for Formation of Remote Cued Fear Memory","volume":"13","author":"Oh","year":"2020","journal-title":"Mol. Brain"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/S0014-2999(03)01283-4","article-title":"The Place of the Hippocampus in Fear Conditioning","volume":"463","author":"Sanders","year":"2003","journal-title":"Eur. J. Pharmacol."},{"key":"ref_16","first-page":"7","article-title":"Genetic Analysis on Rose-Comb, Fishy Taint and Dwarf Traits in Lindian Chicken Population","volume":"37","author":"Qiu","year":"2015","journal-title":"China Poult."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1080\/00071668.2018.1544414","article-title":"Comparative Transcriptome Profiles of Lindian Chicken Eyelids Identify Melanin Genes Controlling Eyelid Pigmentation","volume":"60","author":"Yuan","year":"2019","journal-title":"Br. Poult. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1016\/0003-3472(63)90011-3","article-title":"Imprinting and the Immobility Reactions of Domestic Fowl","volume":"11","author":"Salzen","year":"1963","journal-title":"Anim. Behav."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"103816","DOI":"10.1016\/j.psj.2024.103816","article-title":"The Inflammatory Immunity and Gut Microbiota Are Associated with Fear Response Differences in Laying Hens","volume":"103","author":"Wang","year":"2024","journal-title":"Poult. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"912","DOI":"10.1016\/j.tins.2023.08.003","article-title":"The Role of the Hippocampus in the Consolidation of Emotional Memories during Sleep","volume":"46","author":"Pronier","year":"2023","journal-title":"Trends Neurosci."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Danieli, K., Guyon, A., and Bethus, I. (2023). Episodic Memory Formation: A Review of Complex Hippocampus Input Pathways. Prog. Neuropsychopharmacol. Biol. Psychiatry, 126.","DOI":"10.1016\/j.pnpbp.2023.110757"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1083\/jcb.1.1.69","article-title":"The Fine Structure of Neurons","volume":"1","author":"Palay","year":"1955","journal-title":"J. Biophys. Biochem. Cytol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1007\/s00401-006-0108-2","article-title":"The Fate of Nissl-Stained Dark Neurons Following Traumatic Brain Injury in Rats: Difference between Neocortex and Hippocampus Regarding Survival Rate","volume":"112","author":"Ooigawa","year":"2006","journal-title":"Acta Neuropathol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"13001","DOI":"10.1074\/jbc.R500027200","article-title":"Understanding Retinol Metabolism: Structure and Function of Retinol Dehydrogenases","volume":"281","author":"Eriksson","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"E672","DOI":"10.1152\/ajpendo.00280.2001","article-title":"Retinoic Acid Is Detected at Relatively High Levels in the CNS of Adult Rats","volume":"282","author":"Werner","year":"2002","journal-title":"Am. J. Physiol. Endocrinol. Metab."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1007\/s00429-011-0359-0","article-title":"High Expression of Retinoic Acid Receptors and Synthetic Enzymes in the Human Hippocampus","volume":"217","author":"Fragoso","year":"2012","journal-title":"Brain Struct. Funct."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1079\/WPS19760010","article-title":"Vitamin B6 in Poultry Nutrition A Review","volume":"32","author":"Daghir","year":"1976","journal-title":"World\u2019s Poult. Sci. J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1038\/s41398-021-01381-z","article-title":"Vitamin B6 Deficiency Hyperactivates the Noradrenergic System, Leading to Social Deficits and Cognitive Impairment","volume":"11","author":"Toriumi","year":"2021","journal-title":"Transl. Psychiatry"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"544","DOI":"10.1056\/NEJMoa1616361","article-title":"NAD Deficiency, Congenital Malformations, and Niacin Supplementation","volume":"377","author":"Shi","year":"2017","journal-title":"N. Engl. J. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1178646918776658","DOI":"10.1177\/1178646918776658","article-title":"The Influence of Nicotinamide on Health and Disease in the Central Nervous System","volume":"11","author":"Fricker","year":"2018","journal-title":"Int. J. Tryptophan Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1016\/j.jad.2019.11.147","article-title":"Nicotinamide Mononucleotide Ameliorates the Depression-like Behaviors and Is Associated with Attenuating the Disruption of Mitochondrial Bioenergetics in Depressed Mice","volume":"263","author":"Xie","year":"2020","journal-title":"J. Affect. Disord."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Luan, Y., Zhang, H., Ma, K., Liu, Y., Lu, H., Chen, X., Liu, Y., and Zhang, Z. (2023). CCN3\/NOV Regulates Proliferation and Neuronal Differentiation in Mouse Hippocampal Neural Stem Cells via the Activation of the Notch\/PTEN\/AKT Pathway. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241210324"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Hou, W., Li, G.-S., Gao, L., Lu, H.-P., Zhou, H.-F., Kong, J.-L., Chen, G., Xia, S., and Wei, H.-Y. (2022). SYNJ2 Is a Novel and Potential Biomarker for the Prediction and Treatment of Cancers: From Lung Squamous Cell Carcinoma to Pan-Cancer. BMC Med. Genom., 15.","DOI":"10.1186\/s12920-022-01266-0"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/j.gene.2008.08.015","article-title":"Odd-Skipped Related 1 Gene Expression Is Regulated by Runx2 and Ikzf1 Transcription Factors","volume":"426","author":"Yamauchi","year":"2008","journal-title":"Gene"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1093","DOI":"10.1037\/0735-7044.107.6.1093","article-title":"Effects of Amygdala, Hippocampus, and Periaqueductal Gray Lesions on Short- and Long-Term Contextual Fear","volume":"107","author":"Kim","year":"1993","journal-title":"Behav. Neurosci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1966","DOI":"10.1002\/cne.23514","article-title":"DCX-Expressing Cells in the Vicinity of the Hypothalamic Neurogenic Niche: A Comparative Study between Mouse, Sheep, and Human Tissues","volume":"522","author":"Batailler","year":"2014","journal-title":"J. Comp. Neurol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"956","DOI":"10.1038\/s41380-021-01353-1","article-title":"Amygdala DCX and Blood Cdk14 Are Implicated as Cross-Species Indicators of Individual Differences in Fear, Extinction, and Resilience to Trauma Exposure","volume":"27","author":"Maheu","year":"2022","journal-title":"Mol. Psychiatry"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3343","DOI":"10.1038\/s41380-023-01966-8","article-title":"Age, Sex, and Apolipoprotein E Isoform Alter Contextual Fear Learning, Neuronal Activation, and Baseline DNA Damage in the Hippocampus","volume":"28","author":"Boutros","year":"2023","journal-title":"Mol. Psychiatry"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/0006-8993(91)90890-8","article-title":"Effects of Early Experience on C-Fos Gene Expression in the Chick Forebrain","volume":"544","author":"Anokhin","year":"1991","journal-title":"Brain Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1016\/0306-4522(94)00355-9","article-title":"Pattern and Time Course of Immediate Early Gene Expression in Rat Brain Following Acute Stress","volume":"64","author":"Cullinan","year":"1995","journal-title":"Neuroscience"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1080\/102538902900012369","article-title":"Mapping Brain Response to Social Stress in Rodents With C-Fos Expression: A Review","volume":"5","author":"Martinez","year":"2002","journal-title":"Stress"}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/15\/13\/1889\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:59:25Z","timestamp":1760032765000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/15\/13\/1889"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,26]]},"references-count":41,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2025,7]]}},"alternative-id":["ani15131889"],"URL":"https:\/\/doi.org\/10.3390\/ani15131889","relation":{},"ISSN":["2076-2615"],"issn-type":[{"type":"electronic","value":"2076-2615"}],"subject":[],"published":{"date-parts":[[2025,6,26]]}}}