Thermal conditioning of quail embryos has transgenerational and reversible long-term effects.

BACKGROUND: In the current context of global warming, thermal manipulation of avian embryos has received increasing attention as a strategy to promote heat tolerance in avian species by simply increasing the egg incubation temperature. However, because of their likely epigenetic origin, thermal manipulation effects may last more than one generation with consequences for the poultry industry. In this work, a multigenerational and transgenerational analysis of thermal manipulation during embryogenesis was performed to uncover the long-term effects of such procedure. RESULTS: Thermal manipulation repeated during 4 generations had an effect on hatchability, body weight, and weight of eggs laid in Japanese quails, with some effects increasing in importance over generations. Moreover, the effects on body weight and egg weight could be transmitted transgenerationally, suggesting non-genetic inheritance mechanisms. This hypothesis is reinforced by the observed reversion of the effect on growth after five unexposed generations. Interestingly, a beneficial effect of thermal manipulation on heat tolerance was observed a few days after hatching, but this effect was not transgenerational. CONCLUSIONS: Our multigenerational study showed that thermal conditioning of quail embryos has a beneficial effect on post-hatch heat tolerance hampered by transgenerational but reversible defects on growth. Assuming that no genetic variability underlies these changes, this study provides the first demonstration of epigenetic inheritance of traits induced by environmental temperature modification associated with long-term impacts in an avian species.

Chronic dietary exposure to a glyphosate-based herbicide results in reversible increase early embryo mortality in chicken.

Glyphosate (Gly) is the active molecule of non-selective herbicides used in conventional agriculture. Some evidence shows that exposure to Glyphosate-Based Herbicides (GBH) can affect both male and female fertility in animal models. However, few data exist on birds that can be easily exposed through their cereal-based diet. To our knowledge, there are no current studies on the effects of chronic dietary exposure to GBH and the potential reversibility on the fertility and embryo development in chickens. In our protocol, hens (32 weeks-old) were exposed to GBH (47 mg kg-1/day-1 glyphosate equivalent corresponding to half of the No-Observed-Adverse-Effect-Level (NOAEL) as defined by European Food Safety Authority in birds, GBH group (GBH), n = 75) or not (Control group (CT), n = 75) for 6 weeks. Then, both CT and GBH groups were fed for 5 more weeks without GBH exposure. During these two periods, we investigated the consequences on the egg performance and quality, fertilization rate, embryo development, and viability of offspring. Despite the accumulation of Gly and its metabolite aminomethylphosphonic acid (AMPA) in the hen blood plasma, the body weight and laying rate were similar in GBH and CT animals. We observed from the 4th day of exposure an accumulation of Gly (but not AMPA) only in the yolk of the eggs produced by the exposed hens. After artificial insemination of the hens followed by eggs incubation, we showed a strong significant early embryonic mortality level in GBH compared to CT animals (78 ± 2 % vs 2.5 ± 0.3 %, p < 0.0001) with embryo death mainly occurring on the third day of incubation. By using computed tomography (CT) and magnetic resonance imaging (MRI) tools, we noted a significant delay in the embryo development of GBH survivors at 15 days with a reduction by half of the embryo volume and some disturbances in the calculated volumes of the embryonic annexes. At 20 days of incubation, we showed a reduction in the length of the tibia and in the volume of the soft tissues whereas the skeleton volume was increased in GBH chicks. The vast majority of these phenotypes disappeared two weeks after an arrest of the GBH maternal dietary exposure. Taken together, the dietary chronic exposure of broiler hens to GBH at a Gly equivalent concentration lower than NOAEL induces an accumulation of Gly in the egg yolk resulting in severe early embryonic mortality and a delayed embryonic development in survivors that were abolished two weeks after the end of GBH exposure.

Chemerin is secreted by the chicken oviduct, accumulates in egg albumen and could promote embryo development.

Understanding of the distribution of chemerin and its receptors, Chemokine-like Receptor 1 (CMKLR1), G Protein-coupled Receptor 1 (GPR1) and Chemokine (C-C motif) receptor-like 2 (CCRL2), in the egg and the embryonic annexes is currently lacking, and their role during embryogenesis remains unknown. By immunoblot using monoclonal anti-chicken antibodies and Enzyme Linked Immunosorbent Assays (ELISA), we found that chemerin is expressed 10 times higher in albumen eggs than in blood plasma, and it is also abundant in the perivitelline membrane but undetectable in yolk. Chicken chemerin can inhibit bacterial growth. By Reverse Transcription-quantitative Polymerisation Chain Reaction (RT-qPCR), western-blot, and immunofluorescence, we show that chemerin is locally produced by the oviduct magnum that participates in albumen formation. Using cultures of magnum explants, we demonstrate that progesterone (P4) and oestradiol (E2) treatment increases chemerin secretion into cultured media and expression in magnum. Chemerin and its three receptors are present in amniotic and Chorio Allantoic Membranes (CAM). Only CMKLR1 expression decreased from embryonic day (ED) 7 to ED11 and remained low until ED18. Chemerin concentrations strongly increased in amniotic fluid at D14 when egg albumen crossed the amniotic membrane. In ovo injections of neutralising chemerin and CMKLR1 antibodies (0.01, 0.1 and 1 µg) increased embryo mortality, which occurred mainly at ED12-13, in a dose-dependent manner. Chemerin treatment increased primary CAM viability. Finally, chemerin and CMKLR1 inhibition within the CAM led to a decrease in blood vessel development and associated angiogenic gene expression. Our results show an important function of the chemerin system during embryo development in chickens, suggesting the potential use of this adipokine as a predictive marker for egg fertility or hatchability.

Early Growth and Protein-Energy Metabolism in Chicken Lines Divergently Selected on Ultimate pH.

In chickens, a divergent selection on the Pectoralis major pHu allowed the creation of the pHu+ and pHu- lines, which represent a unique model for studying the biological control of carbohydrate storage in muscle. The present study aimed to describe the early mechanisms involved in the establishment of pHu+ and pHu- phenotypes. At hatching, pHu+ chicks were slightly heavier but exhibited lower plasma glucose and triglyceride and higher uric acid. After 5 days, pHu+ chicks exhibited higher breast meat yield compared to pHu- while their body weight was no different. At both ages, in vivo muscle glycogen content was lower in pHu+ than in pHu- muscles. The lower ability of pHu+ chicks to store carbohydrate in their muscle was associated with the increased expression of SLC2A1 and SLC2A3 genes coding glucose transporters 1 and 3, and of CS and LDHα coding key enzymes of oxidative and glycolytic pathways, respectively. Reduced muscle glycogen content at hatching of the pHu+ was concomitant with higher activation by phosphorylation of S6 kinase 1/ribosomal protein S6 pathway, known to activate protein synthesis in chicken muscle. In conclusion, differences observed in muscle at slaughter age in the pHu+ and pHu- lines are already present at hatching. They are associated with several changes related to both carbohydrate and protein metabolism, which are likely to affect their ability to use eggs or exogenous nutrients for muscle growth or energy storage.

Adipokines expression profile in liver, adipose tissue and muscle during chicken embryo development.

In broiler chickens, the intense genetic selection for rapid growth has resulted in an increase in growth rate and fat deposition. Adipose tissue is now recognized as an important endocrine organ that secretes a variety of factors including adipokines. However, the expression pattern of these adipokines is unclear in chicken embryo development. In the present study, we determined the expression profile of three novel adipokines, NAMPT, RARRES2 and ADIPOQ, and their cognate receptors in metabolic tissues (liver, muscles and adipose tissue) of chicken embryo/chicks from 15 days of incubation (E15) to hatching (D0). From E15 to hatching, embryos gradually gained weight and started to develop subcutaneous adipose tissue at E15. We conducted western blot and RT-qPCR tests and found that ADIPOQ expression increased over time and was positively correlated with adipose tissue weight. In addition, NAMPT expression increased only in muscles. By using a new homemade chicken RARRES2 specific antibody we showed that RARRES2 protein levels increased specifically at hatching in adipose tissue, liver and pectoralis major and this was associated with an increase in the weight of embryo. Taken together, these results support a potential involvement of adipokines in metabolic regulation during chicken embryo development.

Incubation temperature affects the expression of young precocial birds' fear-related behaviours and neuroendocrine correlates.

The influence of embryonic microclimate on the behavioural development of birds remains unexplored. In this study, we experimentally tested whether chronic exposure to suboptimal temperatures engendered plasticity in the expression of fear-related behaviours and in the expression of the corticotropin-releasing factor in the brains of domestic chicks (Gallus g. domesticus). We compared the neurobehavioural phenotypes of a control group of chicks incubated in an optimal thermal environment (37.8 °C) with those of a group of experimental chicks exposed chronically in ovo to suboptimal temperatures (27.2 °C for 1 hour twice a day). Chronic exposure to a suboptimal temperature delayed hatching and decreased growth rate and experimental chicks had higher neophobic responses than controls in novel food and novel environment tests. In addition, experimental chicks showed higher expression of corticotropin-releasing factor than did controls in nuclei of the amygdala, a structure involved in the regulation of fear-related behaviours. In this study, we report the first evidence of the strong but underappreciated role of incubation microclimate on the development of birds' behaviour and its neurobiological correlates.

Food restriction but not fish oil increases fertility in hens: role of RARRES2?

Overfed hens selected for their rapid growth become fatter and develop reproductive disorders. Herein, we aimed to demonstrate that food restriction leading to a weight reduction and/or a supplementation with fish oil may be effective in preventing reproductive disorders through the regulation of adipokine expression in broiler hens. This study included four groups of food restricted (Rt) or ad libitum hens (Ad, feeding at a rate 1.7 times greater than Rt hens) supplemented or unsupplemented with fish oil (1%). The Rt diet significantly increased plasma chemerin (RARRES2) levels during the laying period, delayed sexual maturity by one week and improved egg quality and fertility. These effects were associated with higher progesterone production in response to IGF1 (or LH) in cultured granulosa cells and in vivo egg yolk, as compared with Ad hens. Fish oil supplementation had similar effects to the Rt diet on progesterone (P < 0.05), but without any effect on fertility. Using RT-PCR, we found that RARRES2 levels were lower in theca cells of Rt hens and NAMPT levels were increased by the fish oil supplementation. A significant positive correlation between RARRES2 expression in granulosa cells and the weight of F1 preovulatory follicle was observed, as well as a negative correlation of plasma RARRES2 levels with hatchability. Thus, food restriction but not fish oil supplementation improved fertility, and this was associated with variations in RARRES2 plasma and ovarian expression in hens.

Yolk hormones influence in ovo chemosensory learning, growth, and feeding behavior in domestic chicks.

In this study, we assessed whether prenatal exposure to elevated yolk steroid hormones can influence in ovo chemosensory learning and the behavior of domestic chicks. We simulated a maternal environmental challenge by experimentally enhancing yolk progesterone, testosterone, and estradiol concentrations in hen eggs prior to incubation. The embryos from these hormones-treated eggs (HO) as well as sham embryos (O) that had received the vehicle-only were exposed to the odor of fish oil (menhaden) between embryonic Days 11 and 20. An additional group of control embryos (C) was not exposed to the odor. All chicks were tested following hatching for their feeding preferences between foods that were or were not odorized with the menhaden odor. In the 3-min choice tests, the behavior of O chicks differed significantly according to the type of food whereas C and HO chicks showed no preference between odorized and non-odorized food. Our result suggests weaker response in HO chicks. In addition, HO chicks showed impaired growth and reduced intake of an unfamiliar food on the 24-h time scale compared to controls. Our data suggest that embryonic exposure to increased yolk hormone levels can alter growth, chemosensory learning, and the development of feeding behaviors.

Moderate heat challenge increased yolk steroid hormones and shaped offspring growth and behavior in chickens.

BACKGROUND: Environmental challenges might affect the maternal organism and indirectly affect the later ontogeny of the progeny. We investigated the cross-generation impact of a moderate heat challenge in chickens. We hypothesized that a warm temperature--within the thermotolerance range--would affect the hormonal environment provided to embryos by mothers, and in turn, affect the morphology and behavioral phenotype of offspring. METHODOLOGY/PRINCIPAL FINDINGS: Laying hens were raised under a standard thermal condition at 21°C (controls) or 30°C (experimental) for 5 consecutive weeks. A significant increase was observed in the internal temperature of hens exposed to the warm treatment; however plasma corticosterone levels remained unaffected. The laying rate was not affected, but experimental hens laid lighter eggs than the controls during the treatment. As expected, the maternal thermal environment affected yolk hormone contents. Eggs laid by the experimental hens showed significantly higher concentrations of yolk progesterone, testosterone, and estradiol. All chicks were raised under standard thermal conditions. The quality of hatchlings, growth, feeding behavior and emotional reactivity of chicks were analyzed. Offspring of experimental hens (C30 chicks) were lighter but obtained better morphological quality scores at hatching than the controls (C21 chicks). C30 chicks expressed lesser distress calls when exposed to a novel food. Unlike C21 chicks, C30 chicks expressed no preference for energetic food. CONCLUSION/SIGNIFICANCE: Our findings suggest that moderate heat challenge triggers maternal effects and modulate the developmental trajectory of offspring in a way that may be adaptive. This suggests that the impact of heat challenges on captive or wild populations might have a cross-generation effect.