In commercial flocks of laying hens, keel bone fractures (KBFs) are prevalent and associated with behavioural indicators of pain. However, whether their impact is severe enough to induce a depressive-like state of chronic stress is unknown. As chronic stress downregulates adult hippocampal neurogenesis (AHN) in mammals and birds, we employ this measure as a neural biomarker of subjective welfare state. Radiographs obtained longitudinally 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 21–62 weeks. Individual birds’ transitions between aviary zones were also recorded. Focal hens with severe KBFs at 3–4 weeks prior to sampling (n = 15) had lower densities of immature doublecortin-positive (DCX⁺) multipolar and bipolar neurons in the hippocampal formation than focal hens with minimal fractures (n = 9). KBF severity scores at this time also negatively predicted DCX⁺cell numbers on an individual level, while hens that acquired fractures earlier in their lives had fewer DCX⁺neurons in the caudal hippocampal formation. Activity levels 3–4 weeks prior to sampling were not associated with AHN. KBFs thus lead to a negative affective state lasting at least 3–4 weeks, and management steps to reduce their occurrence are likely to have significant welfare benefits.

Adult hippocampal neurogenesis (AHN) in the dentate gyrus is known to respond to environmental enrichment, chronic stress, and many other factors. The function of AHN may vary across the septo-temporal axis of the hippocampus, as different subdivisions are responsible for different functions. The dorsal pole regulates cognitive-related behaviors, while the ventral pole mediates mood-related responses through the hypothalamic–pituitary–adrenal (HPA) axis. In this study, we investigate different methods of quantifying the effect of environmental enrichment on adult hippocampal neurogenesis in the dorsal and ventral parts of the dentate gyrus (dDG and vDG). To this purpose, 11-week-old female CD-1 mice were assigned for 8 days to one of two conditions: the environmental Enrichment (E) group received i) running wheels, ii) larger cages, iii) plastic tunnels, and iv) bedding with male urine, while the Control (C) group received standard housing. Dorsal CA (Cornu Ammonis) and DG regions were larger in the E than the C animals. Distance run linearly predicted the volume of the dorsal hippocampus, as well as of the intermediate and ventral CA regions. In the dDG, the amount of Doublecortin (DCX) immunoreactivity was significantly higher in E than in C mice. Surprisingly, this pattern was the opposite in the vDG (C > E). Real-time PCR measurement of Dcx mRNA and DCX protein analysis using ELISA showed the same pattern. Brain derived neurotrophic factor (BDNF) immunoreactivity and mRNA displayed no difference between E and C, suggesting that upregulation of DCX was not caused by changes in BDNF levels. BDNF levels were higher in vDG than in dDG, as measured by both methods. Bdnf expression in vDG correlated positively with the distance run by individual E mice. The similarity in the patterns of immunoreactivity, mRNA and protein for differential DCX expression and for BDNF distribution suggests that the latter two methods might be effective tools for more rapid quantification of adult hippocampal neurogenesis.

Progress in improving the welfare of captive animals has been hindered by a lack of objective indicators to assess the quality of lifetime experience, often called cumulative affective experience. Recent developments in stress biology and psychiatry have shed new light on the role of the mammalian hippocampus in affective processes. Here we review these findings and argue that structural hippocampal biomarkers demonstrate criterion, construct and content validity as indicators of cumulative affective experience in mammals. We also briefly review emerging findings in birds and fish, which have promising implications for applying the hippocampal approach to these taxa, but require further validation. We hope that this review will motivate welfare researchers and neuroscientists to explore the potential of hippocampal biomarkers of cumulative affective experience.

In the mammalian brain, adult hippocampal neurogenesis (AHN) is suppressed by chronic stress, primarily at the ventral pole of the hippocampus. Based upon anatomy, we hypothesise that the caudal pole of the avian Hippocampal Formation (HF) presents a homologous subregion. We thus investigated whether AHN is preferentially suppressed in the caudal chicken HF by unpredictable chronic mild stress (UCMS). Adult hens were kept in control conditions or exposed to UCMS for 8 weeks. Hens experiencing UCMS had significantly fewer doublecortin-positive multipolar neurons (p < 0.001) and beaded axons (p = 0.021) at the caudal pole of the HF than controls. UCMS birds also had smaller spleens and lower baseline plasma corticosterone levels compared to controls. There were no differences in AHN at the rostral pole, nor were there differences in expression of genetic mediators of the HPA stress response in the pituitary or adrenal glands. Duration of tonic immobility and heterophil/lymphocyte (H/L) ratios were also not responsive to our UCMS treatment. These results support the hypothesised homology of the caudal pole of the avian HF to the ventral pole of the rodent hippocampus. Furthermore, quantifying neurogenesis in the caudal HF post-mortem may provide an objective, integrative measure of welfare in poultry, which may be more sensitive than current welfare measures.

Access to outdoor areas is provided as a means of enhancing welfare in commercial systems for laying hens (Gallus gallus domesticus), but substantial individual differences exist in their proportional use. Baseline cell proliferation levels of Adult Hippocampal Neurogenesis (AHN) have been associated with individual differences in reactive vs. proactive coping style, and in both mammals and birds, AHN is upregulated by positive experiences including environmental enrichment and exercise. We thus sought to explore whether individual differences in use of outdoor areas and in tonic immobility responses (indicative of fearfulness) were associated with hippocampal cell proliferation and neuronal differentiation. Radio frequency identification technology was used to track the ranging behavior of 440 individual focal hens within a commercially-relevant system over a 72-days period, after which tonic immobility durations were measured. Following hippocampal tissue collection from 58 focal hens, proliferation and neuronal differentiation were measured through quantitative PCR for proliferating cell nuclear antigen (PCNA) and doublecortin mRNA, respectively. Individual differences in tonic immobility duration positively correlated with PCNA expression over the whole hippocampal formation, while greater time spent in outdoor areas (the grassy range and stone yard) was associated with higher proliferation in the rostral subregion. Basal proliferation in the chicken hippocampal formation may thus relate to reactivity, while levels in the rostral region may be stimulated by ranging experience. Doublecortin expression in the caudal hippocampus negatively co-varied with time on the grassy range, but was not associated with tonic immobility duration. This suggests that ranging outside may be associated with stress. Within laying hen flocks, individual differences in hippocampal plasticity thus relate to coping style and use of external areas.

It is still unclear which commercial housing system provides the best quality of life for laying hens. In addition, there are large individual differences in stress levels within a system. Hippocampal neurogenesis or plasticity may provide an integrated biomarker of the stressors experienced by an individual. We selected 12 adult hens each with good and poor body condition (based on body size, degree of feather cover and redness of the comb) from a multi-tier free range system containing H&N strain hens, and from an enriched cage system containing Hy-Line hens (n = 48 total). Immature neurons expressing doublecortin (DCX) were quantified in the hippocampus, contents of the caecal microbiome were sequenced, and expression of inflammatory cytokines was measured in the spleen. DCX⁺cell densities did not differ between the housing systems. In both systems, poor condition hens had lower DCX⁺cell densities, exhibited elevated splenic expression of interleukin-6 (IL6) mRNA, and had a higher relative caecal abundance of methanogenic archeaMethanomethylophilaceae. The findings suggest poor body condition is an indicator that individual hens have experienced a comparatively greater degree of cumulative chronic stress, and that a survey of the proportion of hens with poor body conditions might be one way to evaluate the impact of housing systems on hen welfare.