Perinatal environmental conditions can shape neural and cognitive development in poultry, yet the combined effects of lighted-incubation and immediate post-hatch feed and water access remain unknown. In this exploratory study, layer chick embryos were exposed to continuous light during embryonic days 18-21 and provided with immediate post-hatch access to feed and water, as exemplified in on-farm hatching systems. Hippocampal expression of neural plasticity markers (i.e., BDNF, DCX, PCNA) was measured in left and right hemispheres at eight weeks of age to assess structural plasticity and lateralization, and its relationship to lateralized visuomotor responses within the same individual chicks. The different plasticity markers correlated highly with each other and were combined in a hippocampal plasticity index. Neither lighted incubation nor immediate feed and water access altered hippocampal plasticity or lateralization, suggesting that these traits may reflect intrinsic hemispheric organization rather than early-life modulatory effect by environmental conditions. We did, however, find a pronounced left-right asymmetry, particularly in the caudal hippocampus (p = 0.04). Behavioral observations from a multitasking and step detour test revealed potential hemisphere-specific associations in these exploratory analyses. Right-hemispheric plasticity negatively correlated with latency to detect a predator, consistent with right-hemispheric dominance in threat detection. Higher overall hippocampal plasticity was associated with faster return to foraging after predator exposure (p = 0.01), hinting at a link between hippocampal plasticity and stress resilience, as has been found in mice. Detouring behavior also correlated with opposite patterns of hippocampal plasticity (p = 0.002), suggesting interactions between structural asymmetrical plasticity and lateralized cognitive processing. These findings suggest that hippocampal plasticity may primarily reflect intrinsic developmental trajectories, with early-life environmental factors exerting only modest effects, if any. These preliminary findings highlight the potential importance of considering intrinsic neural organization alongside perinatal environmental conditions in shaping cognitive and behavioral traits.

Environmental stressors in commercial poultry systems can negatively affect bird welfare, although individuals vary considerably in their responses. Neuroplasticity within the hippocampus, measured through the density of doublecortin-positive (DCX+) neurons, provides a potential biomarker of stress experience in laying hens. However, the genetic basis underlying variation in this biomarker remains poorly understood. A total of 42 H&N and Hy-Line Brown hens housed in a multitier free range and enriched cage system, respectively, were genotyped using Genotyping by Sequencing, yielding over 200 000 SNP markers after initial filtering. Hippocampal tissue sections were immunostained for DCX to quantify the density of highly plastic neurons. A genome-wide association analysis identified 19 genomic regions across eight chromosomes within the top 1% of windows explaining the greatest proportion of genetic variance in the neuroplasticity phenotype. Within ±100 kb of these regions, 39 annotated genes were identified, several of which are involved in cellular regulation and genetic information processing pathways. Notably, PIK3R6, VPS37D, STX1A, BAZ1B, HGH1, MAF1, MAPK15, and PIT54 emerged as positional candidate genes potentially contributing to variation in stress responsiveness. These findings provide preliminary insight into the genetic architecture of hippocampal neuroplasticity in laying hens and highlight candidate genes that may contribute to individual differences in stress response, with potential implications for breeding strategies aimed at improving poultry welfare.

Before migration, birds express hyperphagia leading to deposition of fuel in support of long‐distance flight. Long days in spring stimulate a photoperiodic neuroendocrine cascade leading to heightened food intake. A major component of the response of the reproductive system to increased daylength in birds is the local conversion of thyroxine (T4) to triiodothyronine (T3) in the medial basal hypothalamus. However, mechanisms of photostimulation regulating hyperphagia in migratory birds have yet to be resolved. We report results from two studies of Gambel's White‐crowned Sparrow ( Zonotrichia leucophrys gambelii ), a long‐distance migrant. We used quantitative PCR to measure basal hypothalamic gene expression of several neuropeptides, glucocorticoid receptors, type II and type III iodothyronine deiodinase enzymes (DIO2 and DIO3), and α1 and α2 subunits of the cellular energy sensor AMP‐activated protein kinase (AMPKα1 and AMPKα2). The first study involved birds on short days of 9L:15D exposed to 18 h food deprivation. In the second study, birds were photostimulated by one or two long days of 20L:4D. We observed no significant effects of food deprivation on hypothalamic gene expression. However, photostimulation significantly increased food intake on the first and second long days and was associated with significant increases in agouti‐related protein (AGRP) and AMPKα2 mRNAs and in the ratio of DIO2/DIO3 expression. The pattern of increased DIO2 and decreased DIO3 gene expression is likely to have increased basal hypothalamic T3 content. This, in turn, may lead to altered local AMPK signaling to increase AGRP biosynthesis and thereby promote photostimulated hyperphagia.

INTRODUCTION:Different functional domains can be identified along the longitudinal axis of the mammalian hippocampus. We have recently hypothesized that a similar functional gradient may exist along the longitudinal axis of the avian hippocampal formation (HF) as well. If the 2 gradients are homologous, we would expect the caudal HF to be more responsive to acute stress than the rostral HF. METHODS:We restrained 8 adult Dekalb White hens in a bag for 30 min under red-light conditions and compared FOS-immunoreactive (FOS-ir) cell densities in different hippocampal subdivisions to control hens. RESULTS:Although we could find no evidence of an activated stress response in the hypothalamic-pituitary-adrenal axis of the restrained birds, we did find a significant increase in FOS-ir cell densities in the rostral HF of the restrained birds compared to controls. CONCLUSION:We speculate that the HF response is not due to an acute stress response, but instead, it is related to the change in spatial context that was part of taking the birds and restraining them in a different room. We see no activation in the caudal HF. This would be consistent with our hypothesis that the longitudinal axis of the avian HF is homologous to the long axis of the mammalian hippocampus.

Abstract Recurring stress, which can be termed a negative cumulative affective experience has numerous detrimental phenotypic impacts on poultry welfare. Consequently, this negative experience could lead to reduced productivity. Biomarkers that serve as indicators of cumulative affective experience have been found through the density of highly plastic neurons in the hippocampus. There is limited published research on biomarkers of cumulative affective experience in poultry. Therefore, identifying genomic regions associated with the biomarkers of poultry susceptible or resistant to the negative affective experience could aid in creating more efficient and sustainable farming practices. For this study, H&N and Hy-Line Brown hens (n = 42) housed in a multi-tier free range and enriched cage system, respectively, were available. All hens were genotyped with a proprietary 200K SNP panel and phenotypic data were collected from brain tissue sections stained using an antibody to doublecortin (DCX), which was used to quantify densities of highly plastic neurons in the hippocampus. A genome-wide association study (GWAS) was used to detect genomic regions associated with biomarkers of cumulative affective experience in poultry. The GWAS results found a total of 19 regions from 8 chromosomes with a comparatively high proportion of genetic variance explained, which is associated with the cumulative affective experience. From these regions, 39 different genes were found to be within 100 kb upstream and downstream of the identified regions. These genes were examined for their biological function and role in the cumulative affective experience. The identified genes are associated with cellular processes and genetic information processing. Of the genes, PIKR6, VPS37D, STX1A, BAZ1B, HGH1, MAF, MAPK15, PIT54 were identified as important candidate genes for the cumulative affective experience. Notably, the identified gene, PIK3R6, overlapped with previously reported genes for the stress response in chickens. These identified genes with association to the hippocampal neural density serve as potential candidate genes underlying the cumulative affective experience in poultry. This study shows that genomic regions associated with cumulative affective experience can be detected and help to provide insight into the genetic architecture of the trait.

The parents of broiler (meat) chickens (ie, broiler breeders) are food-restricted until sexual maturity, ensuring good health and reproduction, but resulting in hunger. We investigated whether diets with added insoluble fiber promote satiety and reduce behavioral, motivational, and physiological signs of hunger. Ninety-six broiler breeders were fed 1 of 4 feed treatments (n = 24 per diet) from 6 to 12 wk of age: 1) a commercial diet fed to the recommended ration (R) or 2) ad libitum (AL), the same diet as R but mixed with oat hulls at 3) 20% (OH20%) or 4) 40% (OH40%). The R, OH20% and OH40% diets were approximately iso-energetic and resulted in mean 12 wk of age weights within 2.5% of each other (1.21 kg), while AL birds weighed 221% as much (2.67kg). At 12 wk of age, agouti-related protein (AGRP) expression, was, on average, more than 12 times lower in AL birds (P P < 0.001) but did not differ between the fiber diet treatments and R. Pro-opiomelanocortin (POMC) expression, was, on average, over 1.5 times higher in AL birds, but was not statistically significantly affected by feed treatments (P = 0.33). In their home pens, AL birds stood/sat more, foraged less and fed more in total (P < 0.001) and OH40% birds spent longer feeding than R (P = 0.001). Motivation to forage tested by willingness to walk through water to access an area of wood shavings (without food) was not significantly affected by diet (P = 0.33). However, restricted birds were willing to cross in only 7.3% to 12.5% of tests. Mostly birds stayed on the start platform, where AL birds sat more than other treatments and OH40% birds reduced walking relative to R birds (P P = 0.016). Across the behavioral and physiological measurements there was a dichotomy of effects in response to approximately isoenergetic diets differing in fiber. There were some potentially beneficial behavioral effects related to reduced foraging and walking. However, there was no evidence that these diets significantly improved physiological measures of satiety of broiler breeders.

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 archea Methanomethylophilaceae. 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.

Broiler breeder chickens are commercially feed restricted to slow their growth and improve their health and production, however, there is research demonstrating that this leads to chronic hunger resulting in poor welfare. A challenge in these studies is to account for possible daily rhythms or the effects of time since last meal on measures relating hunger. To address this, we used 3 feed treatments: AL (ad libitum fed), Ram (restricted, fed in the morning), and Rpm (restricted, fed in the afternoon) to control for diurnal effects. We then conducted foraging motivation tests and collected home pen behavior and physiological samples at 4 times relative to feeding throughout a 24-h period. The feed treatment had the largest influence on the data, with AL birds weighing more, having lower concentrations of plasma NEFA, and mRNA expression of AGRP and NPY alongside higher expression of POMC in the basal hypothalamus than Ram or Rpm birds (P < 0.001). R birds were more successful at and had a shorter latency to complete the motivation test, and did more walking and less feeding than AL birds in the home pen (P < 0.01). There was little effect of time since last meal on many measures (P > 0.05) but AGRP expression was highest in the basal hypothalamus shortly after a meal (P < 0.05), blood plasma NEFA was higher in R birds just before feeding (P < 0.001) and glucose was higher in Ram birds just after feeding (P < 0.001), and the latency to complete the motivation test was shortest before the next meal (P < 0.05). Time of day effects were mainly found in the difference in activity levels in the home pen when during lights on and lights off periods. In conclusion, many behavioral and physiological hunger measures were not significantly influenced by time of day or time since the last meal. For the measures that do change, future studies should be designed so that sampling is balanced in such a way as to minimize bias due to these effects.

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.

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.

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.

Mechanisms regulating food intake in vertebrates involve complex interactions between neuronal networks in the brain and endocrine and metabolic signals from the body. Metabolic and reproductive hormones provide a coordinating role to match the expression of feeding behavior to an animal's nutritional state and to environmental cues. The neuroendocrine circuitry is known in great detail in laboratory rodents. Although information from other vertebrate classes is limited, the signaling mechanisms involved have generally been conserved during evolution. The challenge is to understand how the neuroendocrine mechanisms controlling food intake are adjusted in relation to the complex life histories of free-living vertebrates.

Anselme & Gunturkun propose a novel mechanism to explain the increase in foraging motivation when experiencing an unpredictable food supply. However, the physiological mechanisms that maintain energy homeostasis already control foraging intensity in response to changes in energy balance. Therefore, unpredictability may just be one of many factors that feeds into the same dopaminergic "wanting" system to control foraging intensity.

A variety of neuropeptides has been reported to stimulate or inhibit feeding behavior in mammals but few studies have addressed the specific effects of neuropeptides on feeding behavior in ground squirrels. A shorter duration of the feeding response to galanin injection in comparison to that of neuropeptide Y (NPY) has been reported for direct hypothalamic microinjections of the neuropeptides in rats. If NPY and galanin play a physiological role in the regulation of food intake in ground squirrels, it would be expected that genes encoding the neuropeptides are expressed in the hypothalamus. The chapter summarizes the findings on the role of NPY and galanin in seasonal changes in energy balance of the goldenmantled ground squirrel Spermophilus saturatus. It addresses the hypothesis that the synthesis of neuropeptides in the hypothalamus that control feeding is subject to long-term regulatory influences that are independent of short-term regulation imposed by metabolic hormones.

Background: Research into energy balance and growth has infrequently considered genetic sex, yet there is sexual dimorphism for growth across the animal kingdom. We test the hypothesis that in the chicken, there is a sex difference in arcuate nucleus neuropeptide gene expression, since previous research indicates hypothalamic AGRP expression is correlated with growth potential and that males grow faster than females. Because growth has been heavily selected in some chicken lines, food restriction is necessary to improve reproductive performance and welfare, but this increases hunger. Dietary dilution has been proposed to ameliorate this undesirable effect. We aimed to distinguish the effects of gut fullness from nutritional feedback on hypothalamic gene expression and its interaction with sex. Methods: Twelve-week-old male and female fast-growing chickens were either released from restriction and fed ad libitum or a restricted diet plus 15% w/w ispaghula husk, a non-nutritive bulking agent, for 2 days. A control group remained on quantitative restriction. Hypothalamic arcuate nucleus neuropeptides were measured using real-time PCR. To confirm observed sex differences, the experiment was repeated using only ad libitum and restricted fed fast-growing chickens and in a genetically distinct breed of ad libitum fed male and female chickens. Linear mixed models (Genstat 18) were used for statistical analysis with transformation where appropriate. Results: There were pronounced sex differences: expression of the orexigenic genes AGRP (P < 0.001) and NPY (P < 0.002) was higher in males of the fast-growing strain. In genetically distinct chickens, males had higher AGRP mRNA (P = 0.002) expression than females, suggesting sex difference was not restricted to a fast-growing strain. AGRP (P < 0.001) expression was significantly decreased in ad libitum fed birds but was high and indistinguishable between birds on a quantitative versus qualitative restricted diet. Inversely, gene expression of the anorectic genes POMC and CART was significantly higher in ad libitum fed birds but no consistent sex differences were observed. Conclusion: Expression of orexigenic peptides in the avian hypothalamus are significantly different between sexes. This could be useful starting point of investigating further if AGRP is an indicator of growth potential. Results also demonstrate that gut fill alone does not reduce orexigenic gene expression.

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 birds little is known about the hormonal signals that communicate nutritional state to the brain and regulate appetitive behaviours. In mammals, the peptide hormones ghrelin and leptin elevate and inhibit consumption and food hoarding, respectively. But in birds, administration of both ghrelin and leptin inhibit food consumption. The role of these hormones in the regulation of food hoarding in avian species has not been examined. To investigate this, we injected wild caught coal tits (Periparus ater) with leptin, high-dose ghrelin, low-dose ghrelin and a saline control in the laboratory. We then measured food hoarding and mass gain, as a proxy of food consumption, every 20 min for 2 h post-injection. Both high-dose ghrelin and leptin injections significantly reduced hoarding and mass gain compared with controls. Our results provide the first evidence that hoarding behaviour can be reduced by both leptin and ghrelin in a wild bird. These findings add to evidence that the hormonal control of food consumption and hoarding in avian species differs from that in mammals. Food hoarding and consumptive behaviours consistently show the same response to peripheral signals of nutritional state, suggesting that the hormonal regulation of food hoarding has evolved from the consumption regulatory system.

The arcuate nucleus is generally conserved across vertebrate taxa in its neuroanatomy and neuropeptide expression. Gene expression of agouti-related protein (AGRP), neuropeptide Y (NPY), pro-opiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) has been established in the arcuate nucleus of several bird species and co-localization demonstrated for AGRP and NPY. The proteins encoded by these genes exert comparable effects on food intake in birds after central administration to those seen in other vertebrates, with AGRP and NPY being orexigenic and CART and α-melanocyte-stimulating hormone anorexigenic. We have focused on the measurement of arcuate nucleus AGRP and POMC expression in several avian models in relation to the regulation of energy balance, incubation, stress, and growth. AGRP mRNA and POMC mRNA are, respectively, up- and downregulated after energy deprivation and restriction. This suggests that coordinated changes in the activity of AGRP and POMC neurons help to drive the homeostatic response to replace depleted energy stores in birds as in other vertebrates. While AGRP and POMC expression are generally positively and negatively correlated with food intake, respectively, we review here situations in some avian models in which AGRP gene expression is dissociated from the level of food intake and may have an influence on growth independent of changes in appetite. This suggests the possibility that the central melanocortin system exerts more pleiotropic functions in birds. While the neuroanatomical arrangement of AGRP and POMC neurons and the sensitivity of their activity to nutritional state appear generally conserved with other vertebrates, detailed knowledge is lacking of the key nutritional feedback signals acting on the avian arcuate nucleus and there appear to be significant differences between birds and mammals. In particular, recently identified avian leptin genes show differences between bird species in their tissue expression patterns and appear less closely linked in their expression to nutritional state. It is presently uncertain how the regulation of the central melanocortin system in birds is brought about in the situation of the apparently reduced importance of leptin and ghrelin compared to mammals.

The mammalian hippocampus is particularly vulnerable to chronic stress. Adult neurogenesis in the dentate gyrus is suppressed by chronic stress and by administration of glucocorticoid hormones. Post-natal and adult neurogenesis are present in the avian hippocampal formation as well, but much less is known about its sensitivity to chronic stressors. In this study, we investigate this question in a commercial bird model: the broiler breeder chicken. Commercial broiler breeders are food restricted during development to manipulate their growth curve and to avoid negative health outcomes, including obesity and poor reproductive performance. Beyond knowing that these chickens are healthier than fully-fed birds and that they have a high motivation to eat, little is known about how food restriction impacts the animals' physiology. Chickens were kept on a commercial food-restricted diet during the first 12 weeks of life, or released from this restriction by feeding them ad libitum from weeks 7-12 of life. To test the hypothesis that chronic food restriction decreases the production of new neurons (neurogenesis) in the hippocampal formation, the cell proliferation marker bromodeoxyuridine was injected one week prior to tissue collection. Corticosterone levels in blood plasma were elevated during food restriction, even though molecular markers of hypothalamic- pituitary-adrenal axis activation did not differ between the treatments. The density of new hippocampal neurons was significantly reduced in the food-restricted condition, as compared to chickens fed ad libitum, similar to findings in rats at a similar developmental stage. Food restriction did not affect hippocampal volume or the total number of neurons. These findings indicate that in birds, like in mammals, reduction in hippocampal neurogenesis is associated with chronically elevated corticosterone levels, and therefore potentially with chronic stress in general. This finding is consistent with the hypothesis that the response to stressors in the avian hippocampal formation is homologous to that of the mammalian hippocampus.