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Ambient conditions during chicken embryogenesis, such as insufficient oxygen or changes in temperature, are expected to cause permanent phenotypic changes and affect their posthatch performance. Decades of genetic selection for high growth rate resulted with various physiological and morphological changes that can affect the broiler fitness under environmental stress. To evaluate the selection effect on responses to environmental challenge during embryonic development, and the long-term implications, we have used a unique genetic line, that was not selected for over 30 yr (since 1986), as control for the modern commercial genetic line. At embryonic day 5 (E5), broiler embryos from these 2 genetic lines were divided into 2 treatments: 1) control; 2) 15% O2 concentration for 12 h/day from E5 through E12 the embryonic period of chorioallantoic membrane formation. Embryos and hatched chicks were characterized for physiological and morphological parameters. Significant differences in relative embryo weight and yolk consumption were found between the 2 lines. The modern line was characterized by a higher metabolic rate and rapid growth, supported by higher hemoglobin levels and hematocrit concentrations, whereas the 1986 line had slower metabolism, lower levels of hematocrit and hemoglobin, higher oxygen volume per 1 g of embryonic tissue indicating higher oxygen availability. Both lines exhibited changes in heart rate, and blood parameters corresponding to cardiovascular system adaptation after hypoxic exposure, seemingly implemented to increase oxygen-carrying capacity to the embryo tissues. Our finding stand in agreement that the genetic selection for high growth rate that led to higher metabolism without a fit of the cardiovascular system, increased the imbalance between oxygen supply and demand.

Adequate ambient temperature and oxygenation are necessary to maintain normal embryonic development of broilers, however, hypoxia challenge during incubation can aid in improving regulatory plasticity and lead to different phenotypes later in life. This study aimed to examine the effects of moderate hypoxia (O2 17%) during the plateau phase on the embryonic physiological parameters and on post-hatch performance (growth rate, feed consumption and feed conversion) up to the age of poultry marketing. The study included examined embryos exposed to O2 17% for 12 h per day (h/d) from E16 through E18 (designated as 12H), or O2 17% continuously, from E16 through E17 (designated as 48H) and a standard incubation control group (21% O2). Physiological and morphological parameters of embryos and hatched chicks were measured. Male Chicks from all three treatment groups were raised under recommended temperature regime, and body weight, feed intake and FCR were recorded on a weekly basis. The intermittent hypoxia protocol (12H), allowed embryos to properly adapt to the shortage of oxygen, compensate for the gap in body mass that developed following the first exposure window, and hatch with characteristics similar to those of the control embryos. In contrast, while the 48H embryos were able to adapt to the hypoxic stress, the prolonged exposure prevented them from catching up with both control and 12H embryos. Broilers that were subjected to hypoxia showed hatchling body weights and growth rates similar to those of controls, throughout the entire growth phase. During the fifth week, lower feed consumption was observed in the 12H and 48H groups and became significantly lower than the control chicks in the sixth week of growth. Following hypoxia exposure, chicks managed to reach normal body weight with less feed, with the 12H group demonstrating lower and more efficient FCR during the last two weeks of growth. Broiler embryos reacted to plateau-phase hypoxia challenge with numerous physiological and metabolic modifications. The prudent alterations in metabolism and cardiovascular system during exposure to hypoxia and post-hatch, resulted in more efficient energy utilization in broilers, which may have a long-lasting enhancing effect on post-hatching thermotolerance and sustainability in chicks reared under sub-optimal environmental conditions.

In mammals, time-restricted feeding (TRF) with no caloric restriction provides health benefits and extends longevity, usually with a minor (∼3%) or no reduction in total food consumption. In the current study, a TRF regimen of 6 h free access to food (08:00–14:00 h) was applied to Leghorn chickens from 25 to 86 weeks of age; control birds ate freely during the light hours (06:00–20:00 h). Unexpectedly, the TRF-treated birds consumed, on average, 11.7% less food than the controls. This was manifested by an average reduction of 9.6% in body weight, 2.6-fold in visceral fat accumulation, and 6.5% in egg weight. Hen-housed egg production was reduced by 3.6% in the TRF group compared with the control, along the first 40 weeks of the follow-up (P < 0.05), and changed into a tendency of 0.7% higher egg production thereafter. Several parameters of egg quality showed significant improvement (P < 0.05) in the TRF group compared with the controls. A comparison of diurnal patterns of feed consumption revealed a higher rate of hourly consumption in the TRF group and increased consumption before dark in the control group. In conclusion, the reduced feed intake in response to the TRF treatment and loss in visceral fat accumulation supports the lack of a strong adipostat activity in chickens and different appetite regulation mechanisms compared with mammals. Therefore, future TRF studies in chickens should be adjusted by extending the ad libitum time window. The lower feed intake by the TRF-treated chickens compared with the ad libitum-fed controls seems to reduce the efficiency of egg production. Nevertheless, the improved egg quality and persistence of egg lay at the older age suggest that similarly to mammals, the TRF treatment delayed at least some of the negative impacts associated with advanced age.

Visual Abstract, Early life heat stress leads to either resilience or vulnerability to a similar stress later in life. We have previously shown that this tuning of the stress response depends on neural network organization in the preoptic anterior hypothalamus (PO/AH) thermal response center and is regulated by epigenetic mechanisms. Here, we expand our understanding of stress response establishment describing a role for epitranscriptomic regulation of the epigenetic machinery. Specifically, we explore the role of N6-methyladenosine (m6A) RNA methylation in long-term response to heat stress. Heat conditioning of 3-d-old chicks diminished m6A RNA methylation in the hypothalamus, simultaneously with an increase in the mRNA levels of the m6A demethylase, fat mass and obesity-associated protein (FTO). Moreover, a week later, methylation of two heat stress-related transcripts, histone 3 lysine 27 (H3K27) methyltransferase, enhancer of zeste homolog 2 (EZH2) and brain-derived neurotrophic factor (BDNF), were downregulated in harsh-heat-conditioned chicks. During heat challenge a week after conditioning, there was a reduction of m6A levels in mild-heat-conditioned chicks and an elevation in harsh-heat-conditioned ones. This increase in m6A modification was negatively correlated with the expression levels of both BDNF and EZH2. Antisense “knock-down” of FTO caused an elevation of global m6A RNA methylation, reduction of EZH2 and BDNF mRNA levels, and decrease in global H3K27 dimethylation as well as dimethyl H3K27 level along BDNF coding region, and, finally, led to heat vulnerability. These findings emphasize the multilevel regulation of gene expression, including both epigenetic and epitranscriptomic regulatory mechanisms, fine-tuning the neural network organization in a response to stress.

In all vertebrates, hypoxia plays an important role in fetal development, driving vasculogenesis, angiogenesis, hematopoiesis, and chondrogenesis. Therefore, the ability to sense and respond to changes in the availability of oxygen (O2) is crucial for normal embryonic development as well as for developmental plasticity. Moderate levels of hypoxia trigger a regulated process which leads to adaptive responses. Regulation of angiogenesis by hypoxia is an important component of homeostatic control mechanisms that link the cardio-pulmonary-vascular O2 supply to metabolic demands in local tissues. Hypoxia leads to the activation of genes that are important for cell and tissue adaptation to low O2 conditions, such as hypoxia-inducible factor 1. Previous studies have shown a dose-response effect to hypoxia in chicken embryos, with lower and/or prolonged O2 levels affecting multiple mechanisms and providing a spectrum of responses that facilitate the ability to maintain O2 demand despite environmental hypoxia. In chicken embryos, mild to extreme hypoxia during embryogenesis improves chorioallantoic membrane and cardiovascular development, resulting in an increase in O2 carrying capacity and leading to developmental plasticity that may affect post-hatch chick performance and improve adaptation to additional environmental stresses at suboptimal environmental conditions.

Notice à reprendre car pas de clé UT au 19 Septembre; In mammals, leptin and tumor-necrosis factor (TNF) are prominent interacting adipokinesmediating appetite control and insulin sensitivity. While TNF pleiotropically functions in immunedefense and cell survival, leptin is largely confined to signaling energy stores in adipocytes. Knowledgeabout the function of avian leptin and TNF is limited and they are absent or lowly expressed inadipose, respectively. Employing radiation-hybrid mapping and FISH-TSA, we mapped TNF andits syntenic genes to chicken chromosome 16 within the major histocompatibility complex (MHC)region. This mapping position suggests that avian TNF has a role in regulating immune response.To test its possible interaction with leptin within the immune system and beyond, we comparedthe transcription patterns of TNF, leptin and their cognate receptors obtained by meta-analysis ofGenBank RNA-seq data. While expression of leptin and its receptor (LEPR) were detected in thebrain and digestive tract, TNF and its receptor mRNAs were primarily found in viral-infected andLPS-treated leukocytes. We confirmed leptin expression in the duodenum by immunohistochemistrystaining. Altogether, we suggest that whereas leptin and TNF interact as adipokines in mammals,in birds, they have distinct roles. Thus, the interaction between leptin and TNF may be uniqueto mammals.

In mammals, leptin and tumor-necrosis factor (TNF) are prominent interacting adipokines mediating appetite control and insulin sensitivity. While TNF pleiotropically functions in immune defense and cell survival, leptin is largely confined to signaling energy stores in adipocytes. Knowledge about the function of avian leptin and TNF is limited and they are absent or lowly expressed in adipose, respectively. Employing radiation-hybrid mapping and FISH-TSA, we mapped TNF and its syntenic genes to chicken chromosome 16 within the major histocompatibility complex (MHC) region. This mapping position suggests that avian TNF has a role in regulating immune response. To test its possible interaction with leptin within the immune system and beyond, we compared the transcription patterns of TNF, leptin and their cognate receptors obtained by meta-analysis of GenBank RNA-seq data. While expression of leptin and its receptor (LEPR) were detected in the brain and digestive tract, TNF and its receptor mRNAs were primarily found in viral-infected and LPS-treated leukocytes. We confirmed leptin expression in the duodenum by immunohistochemistry staining. Altogether, we suggest that whereas leptin and TNF interact as adipokines in mammals, in birds, they have distinct roles. Thus, the interaction between leptin and TNF may be unique to mammals.

BACKGROUND: The mammalian adipose tissue plays a central role in energy-balance control, whereas the avian visceral fat hardly expresses leptin, the key adipokine in mammals. Therefore, to assess the endocrine role of adipose tissue in birds, we compared the transcriptome and proteome between two metabolically different types of chickens, broilers and layers, bred towards efficient meat and egg production, respectively. RESULTS: Broilers and layer hens, grown up to sexual maturation under free-feeding conditions, differed 4.0-fold in weight and 1.6-fold in ovarian-follicle counts, yet the relative accumulation of visceral fat was comparable. RNA-seq and mass-spectrometry (MS) analyses of visceral fat revealed differentially expressed genes between broilers and layers, 1106 at the mRNA level (FDR ≤ 0.05), and 203 at the protein level (P ≤ 0.05). In broilers, Ingenuity Pathway Analysis revealed activation of the PTEN-pathway, and in layers increased response to external signals. The expression pattern of genes encoding fat-secreted proteins in broilers and layers was characterized in the RNA-seq and MS data, as well as by qPCR on visceral fat under free feeding and 24 h-feed deprivation. This characterization was expanded using available RNA-seq data of tissues from red junglefowl, and of visceral fat from broilers of different types. These comparisons revealed expression of new adipokines and secreted proteins (LCAT, LECT2, SERPINE2, SFTP1, ZP1, ZP3, APOV1, VTG1 and VTG2) at the mRNA and/or protein levels, with dynamic gene expression patterns in the selected chicken lines (except for ZP1; FDR/P ≤ 0.05) and feed deprivation (NAMPT, SFTPA1 and ZP3) (P ≤ 0.05). In contrast, some of the most prominent adipokines in mammals, leptin, TNF, IFNG, and IL6 were expressed at a low level (FPKM/RPKM< 1) and did not show differential mRNA expression neither between broiler and layer lines nor between fed vs. feed-deprived chickens. CONCLUSIONS: Our study revealed that RNA and protein expression in visceral fat changes with selective breeding, suggesting endocrine roles of visceral fat in the selected phenotypes. In comparison to gene expression in visceral fat of mammals, our findings points to a more direct cross talk of the chicken visceral fat with the reproductive system and lower involvement in the regulation of appetite, inflammation and insulin resistance., The study was supported by the Israel Academy of Sciences grants no. 876/ 14 and 1294/17, and Chief Scientist of the Israeli Ministry of Agriculture 0469/14 (to MFE and ES).

The prenatal circulatory system is adaptive and capable of plasticity designed for the needs of the growing tissue. When a broiler embryo is faced with hypoxic stress, the process of angiogenesis in tissues begins. Exposure to hypoxic conditions of 17% oxygen during the chorioallantoic membrane (CAM) development (E5 to E12) affected the circulatory system and contributed to an increase in the blood oxygen carrying capacity. The present study aimed to evaluate the effects of hypoxic exposure during CAM development on post-hatch performance of broilers and to examine whether hypoxic exposure improved sustainability of birds exposed to acute heat stress. Two consecutive trials, with male broilers from each of the incubation treatments—optimal conditions and exposure to hypoxia of 15 or 17% oxygen, for 12 h/day, during CAM development—were conducted. In experiment 1, 60 male chicks from each group were raised in individual cages. In experiment 2, 160 male chicks from each group were raised in 40-chick pens until marketing. On d 35, 20 birds from each group were transferred to individual cages kept at a temperature of 23°C for 72 h, and then birds were exposed to 35°C for 5 hours. Body temperatures were measured at 0, 2, and 5 h of the heat exposure. In both experiments BW, feed intake, and FCR were recorded. At marketing, chicks were slaughtered, and relative weights of breast muscle, abdominal fat pad, heart, and liver were calculated. Hypoxia treatment resulted in a FCR advantage. Food intake was similar in all treatments, but groups exposed to hypoxia grew better than controls until the age of 35 days. Hypoxia-treated groups had higher relative breast, heart, and liver weights than controls. Body temperatures of hypoxia-treated chickens remained lower during heat stress exposure, and their mortality rate was lower as well. Intermittent exposure to moderate hypoxia during CAM development confers advantages to broilers in feed utilization efficiency and in coping with heat stress. It may be considered as a mitigating step in incubation to facilitate broilers in achieving their full growth potential.

Storing eggs at low temperature prior to incubation is common practice in the broiler hatchery industry; however, prolonged storage (beyond 7 d) is known to increase early embryonic mortality and reduce chick quality and performance. To better understand the basis of this mortality, we previously published milestone criteria to evaluate morphological and cellular properties of the freshly laid embryo. Using these criteria, in the present study we checked the effects of storage at 18°C and 12°C for up to 28 d on hatchability and chick quality. Furthermore, using a 3D high-resolution episcopic microscopy (HREM) imaging system combined with standard and confocal microscopy and cell viability markers, we analyzed the effects of the different storage conditions on embryonic developmental stage, cytoarchitectural properties, mitotic index and cell survival. A total of 1,483 eggs from a young flock were divided in 2 groups, 18°C and 12°C, and stored for 7, 14, 21, and 28 d. Following storage, randomly selected 1,222 eggs were incubated, and the hatched chicks were evaluated for chick quality parameters. Nonhatched eggs were also analyzed to determine the stage of embryonic mortality. The remaining 261 eggs were isolated and analyzed for developmental stage, cytoarchitecture, mitotic index, and cell death following storage. Hatchability rates beyond 7 d of storage at 12°C were significantly improved compared to 18°C, and chick quality remained high. Similar results were obtained for an old flock's eggs (n = 1,350). Analyzing the embryos, at each time point, we found that at 12°C, the developmental progression during storage slows significantly, mitotic index-which at this temperature may indicate mitotic arrest-increases and the rate of early apoptosis is half than at 18°C. Moreover, the HREM system and histological sections showed that embryos stored at 18°C for prolonged times undergo dramatic cytoarchitectural changes that may be maladaptive to resuming normal development after diapause. We thus demonstrate the usefulness of the milestone criteria for predicting and studying the storage conditions that will allow for better performance in hatchery practice.

Birds dissipate considerable heat through respiratory-evaporative and cutaneous-evaporative mechanisms and sensible heat loss (SHL) via radiation, convection, and conduction. The significance of SHL in laying hens is still to be confirmed. This study aimed to elucidate the effect of ventilation on egg production and quality during exposure to high ambient temperature. Lohman laying hens were raised outdoors up to age 35 wk, and 300 hens with similar egg production were divided among 5 treatments each comprising 4 replicates of 15 hens. Birds in 4 treatments were kept in computerized controlled-environment rooms acclimated to 35°C and 50% RH, with ventilation flow rates of 0.5, 1.5, 2.0, and 3.0 m/s, respectively, and those in the control were kept outdoors. Hens were acclimated to the controlled environment rooms for 1 wk and to the targeted environmental conditions for another week, and then were subjected to measurements for 2 wk. Egg production, mass, and shell density, and feed and water consumption were monitored. Body temperature, SHL, and plasma thyroid hormone concentrations were measured at the end of the experiment. The high environmental temperature impaired egg production and quality: whereas exposure of hens to ventilation flows of 2.0 and 3.0 m/s elicited significant recovery of these parameters with time, exposure to a rate of 0.5 m/s negatively affected these parameters throughout the experimental period. The highest feed intake and water consumption were observed in hens exposed to 2.0 and 3.0 m/s, respectively, and the highest SHL was observed in those exposed to 3.0 m/s. It can be concluded that ventilation rate significantly affected hens exposed to high ambient temperature: high ventilation (3.0 m/s) improved egg production whereas low ventilation (0.5 m/s) negatively affected production and quality.

The aim of this study was to fine-tune previous acute cold exposure treatments of broiler embryos during late embryogenesis to improve lifelong cold resistance and performance. Six hundred Cobb hatching eggs were incubated under standard conditions and then exposed to 3 treatments: control; cold treatment in which embryos were exposed to 15°C for 30 min on d 18 and 19 of incubation (30 × 2); and cold treatment similar to 30 × 2 but with 60-min exposures (60 × 2). Egg shell temperature (T(egg)) and heart rate (HR) were monitored pre- and posttreatment. Upon hatching, hatchability, body weight, and body temperature were recorded. From 14 to 35 d of age, three quarters of the chickens in each treatment were raised under ascites-inducing conditions (AIC) and the remaining birds were raised under standard brooding conditions (SBC). The T(egg) and HR decreased significantly in response to increased exposure time on d 18 of incubation. On d 19 of incubation, before the second cold exposure, the 30 × 2 group showed greater T(egg) and HR than the controls, and during the second exposure they maintained these parameters better than the 60 × 2 embryos. No treatment effect on hatchability was observed. At 35 d of age ascites incidence among 30 × 2 chickens under AIC was significantly less than that among the controls (P < 0.01), and body weight of these chickens under either SBC or AIC was significantly higher than that of the controls. Under SBC relative breast muscle weight was significantly higher in 60 × 2 chickens, whereas the relative heart weight was higher in both cold-treated groups than in the controls. It can be concluded that repeated short acute cold exposures during late embryogenesis significantly reduced ascites incidence and improved growth rate under either SBC or AIC. These results may be related to a prenatal epigenetic adaptation of the thermoregulatory and cardiovascular systems to low ambient temperature.