Relating Post-Mortem Hippocampal Neurogenesis to Longitudinally Recorded Severity of Keel Bone Fractures in Adult Laying Hens

In commercial flocks of laying hens, keel bone fractures (KBFs) are prevalent and associated with signs of poor welfare, including behavioural indicators of pain. However, whether their impact is severe enough to induce a depressive-like state of chronic stress is unknown. As levels of adult hippocampal neurogenesis (AHN) in the brains of mammals and birds are suppressed by the chronic experience of stress, this measure may be applicable as a neural biomarker of subjective welfare state. Radiographs obtained longitudinally (at 11 timepoints) from Lohmann Brown laying hens housed in a commercial multi-tier aviary were used to score the severity of naturally occurring KBFs between the ages of 21 and 62 weeks. Individual mobility data was collected using an infrared tracking system to record transitions between aviary zones. Immunohistochemistry was used to stain immature neurons expressing the protein marker doublecortin (DCX). Focal hens with severe KBFs at the final timepoint (3-4 weeks prior to sampling, n=15) had lower densities of immature DCX+ multipolar and bipolar neurons in the hippocampal formation (HF) than focal hens with minimal fractures at this time (n=9). KBF severity scores at this time also negatively predicted DCX+ cell numbers across the whole HF on an individual level, while the duration of time since a hen had acquired their first fracture tended to negatively correlate with numbers of DCX+ neurons in the caudal HF. Activity levels at the final timepoint (3-4 weeks prior to sampling) were not associated with AHN. KBFs thus appear to present a source for chronic stress to laying hens, inducing a negative affective state that lasts at least 3-4 weeks. Management steps to reduce their occurrence are therefore likely to have significant benefits for welfare. Ethical approval for the study was obtained from the Veterinary Office of the Canton of Bern in Switzerland (approval number BE31/15) and the the Animal Welfare and Ethical Review Body at Newcastle University (Project ID #549). The experiment complied with Swiss regulations regarding the treatment of experimental animals.在商业规模化养殖的蛋鸡群中，龙骨骨折（keel bone fractures, KBFs）极为普遍，且与动物福利不佳的诸多表征相关，包括疼痛相关的行为学指标。然而目前尚不明确，龙骨骨折的影响是否严重到足以诱发慢性应激类抑郁状态。由于哺乳动物与鸟类脑中的成年海马神经发生（adult hippocampal neurogenesis, AHN）会被慢性应激经历所抑制，因此该指标可作为个体主观福利状态的神经生物标志物。研究人员对饲养于商业多层式鸡舍的罗曼褐（Lohmann Brown）蛋鸡进行了纵向X光拍摄（共11个时间点），以此对21至62周龄间自然发生的龙骨骨折严重程度进行评分。通过红外追踪系统收集个体活动数据，记录蛋鸡在鸡舍不同区域间的移动转换情况。采用免疫组织化学染色法，对表达双皮质素（doublecortin, DCX）这一蛋白标志物的未成熟神经元进行染色标记。在最终采样前3至4周的时间点被确诊为重度龙骨骨折的目标个体（n=15），其海马结构（hippocampal formation, HF）内未成熟DCX阳性多极神经元与双极神经元的密度，低于同期仅存在轻微骨折的目标个体（n=9）。此时的龙骨骨折严重程度评分，同样可在个体水平上负向预测整个海马结构内的DCX阳性细胞数量；而蛋鸡首次发生骨折后的时长，与尾侧海马结构内的DCX阳性神经元数量呈负相关趋势。最终采样前3至4周的活动水平与成年海马神经发生并无显著关联。由此可见，龙骨骨折似乎是蛋鸡慢性应激的潜在诱因之一，可诱发至少持续3至4周的负面情感状态。因此，采取管理措施降低龙骨骨折的发生率，有望为蛋鸡福利带来显著改善。本研究已获得瑞士伯尔尼州兽医办公室的伦理审批（审批号：BE31/15），以及英国纽卡斯尔大学动物福利与伦理审查委员会的批准（项目编号：#549）。本实验严格遵循瑞士关于实验动物处理的相关法规要求。