Adiponectin and visfatin expression profile in extra-embryonic annexes and role during embryo development in layer and broiler chickens.

Some evidence showed differences between layer and broiler embryo development. We recently showed that two adipokines, adiponectin and visfatin are expressed in the extra embryonic membranes and fluids. However, their role in the embryo development is unknown. Thus, our objectives were 1. to compare the expression of AdipoQ and its receptors AdipoR1 and AdipoR2 and visfatin in extra-embryonic annexes in broiler and layer breeders during the embryo development and 2. to investigate the role of two adipokines in embryo development in both broiler and layer breed after in ovo injection of blocking antibodies against chicken adiponectin or visfatin. We found that adiponectin, AdipoR1, AdipoR2 and visfatin were mainly more expressed in the allantoic that in amniotic membranes. In addition, these expressions increased according the stage of embryo development. We observed a higher expression in layer than in broiler of AdipoQ in allantoic membranes at ED14 and ED18, of AdipoR1 and AdipoR2 in both allantoic and amniotic membranes at ED7 and ED14 and of visfatin only in allantoic membrane from ED7 to ED18. AdipoQ and visfatin were absent in amniotic fluid at ED7 but present at ED14 or ED18 where higher concentrations were detected in layer than in broiler. Interestingly, we showed a strong positive correlation between Adipo and visfatin concentration in amniotic fluid and the body weight of embryo in both breeds. However, after in ovo injection of Adipo antibodies we did not observe any effect on the embryo mortality whereas injection of visfatin antibodies increased in a dose dependent manner the embryo mortality in both breeds. Taken together, Adipo and visfatin are higher expressed in layer than broiler in extra-embryonic membranes and amniotic fluid. Our data suggest also that visfatin could be a main regulator of embryo development.

Expression of chemerin and its receptors in extra-embryonic annexes and role of chemerin and its GPR1 receptor in embryo development in layer and broiler hens.

Intensive genetic selection of broiler breeders and layer hens resulted in differences in the mechanisms of growth and also in cell metabolism during embryogenesis. Previous research has shown that an adipokine named chemerin and one of these receptors, CMKLR1 were potentially involved in broiler embryo development. Here, our objectives were 1) to compare the expression of chemerin and its receptors CMKLR1, GPR1, and CCRL2 and chemerin concentration in extra-embryonic annexes (allantoic and amniotic membranes and fluids and plasma) in broiler and layer fertile eggs during the development (embryonic day (ED) 7, 14, and 18) by RT-qPCR and specific chicken ELISA and 2) to investigate the role of chemerin and one of its receptors GPR1 in embryo development after in ovo injections of neutralizing antibodies against chicken chemerin and GPR1. We found that chemerin expression in amniotic membranes was higher in layer than broiler eggs at ED7 and ED14 whereas the expression of the 3 receptors was higher in layer than broiler in the allantoic membranes at ED14 and ED18. Chemerin concentration was more important in layer than broiler at ED14 and ED18 in amniotic liquid and at all the studied stages in blood plasma. We also showed positive correlation between amniotic chemerin concentration and chemerin amniotic membrane expression, chemerin plasma concentration and embryo body weight in both breeds. Finally, in ovo injection of chicken chemerin and GPR1 neutralizing antibodies increased embryo mortality in both layer and broiler eggs. Taken together, even if chemerin concentration and chemerin system expression in embryonic membranes are mainly higher expressed in layer than in broiler, chemerin potentially through GPR1 could promote embryo development in both breeds.

Chemerin concentration in egg white in layer and broiler hens during the laying period for 2 successive generations.

The genetic selection progress in layers and broilers makes poultry production one of the fastest growing industries. Objectives of the breeding companies are the stability or the increase in the laying rate and the production of viable chicks. New biomarkers are necessary to improve reproductive and egg performances. Chemerin (Chem) produced by oviduct accumulates in egg white (EW). Here, we hypothesized that EW Chem concentration was dependent on the stage of laying and on the breed (layer vs. broiler). In addition, they could be associated to laying performance and fertility parameters. In breeding companies, we collected during 2 successive generations (G0 (mother) and G1 (daughter)) eggs from 100 layers and 100 broilers hens during 5 d at 3 stages: before, after laying peak and at the end of laying period. For each egg, the EW was sampled to measure Chem concentration by ELISA assay. In each generation at the end of laying period, magnums from oviduct, where the EG is formed, were collected in layers and broilers in order to investigate Chem differential expression by RT-qPCR between both breeds. Chem concentration in EW was dependent on the time of laying period and its profile was differently regulated in layers and broilers. Indeed, it increased at the end of laying in layers whereas it decreased after the laying peak in broilers. At the end of laying period, Chem concentration in EW was almost 2-fold higher in layers than in broilers and this was confirmed in both G0 and G1 generations at the Chem mRNA and protein levels in the magnum. For the 2 successive generations, Chem concentration in EW was negatively correlated with the laying rate and the fertility parameter in broiler hens whereas it was negatively correlated with the egg quality (weight of whole egg and weight of albumen) and positively with the fertility rate at some time of laying in layer hens. Taken together, the Chem concentration in EW could be a potential predictive tool for reproductive parameters in genetic selection.

Differential Salmonella Typhimurium intracellular replication and host cell responses in caecal and ileal organoids derived from chicken.

Chicken infection with Salmonella Typhimurium is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the caecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Here, we established intestinal crypt-derived chicken organoids to better decipher the impact of Salmonella intracellular replication on avian intestinal epithelium. Using a 3D organoid model, we observed a significantly higher replication rate of the intracellular bacteria in caecal organoids than in ileal organoids. Our model thus recreates intracellular environment, allowing Salmonella replication of avian epithelium according to the intestinal segment. Moreover, an inhibition of the cellular proliferation was observed in infected ileal and caecal organoids compared to uninfected organoids. This appears with a higher effect in ileal organoids, as well as a higher cytokine and signaling molecule response in infected ileal organoids at 3 h post-infection (hpi) than in caecal organoids that could explain the lower replication rate of Salmonella observed later at 24 hpi. To conclude, this study demonstrates that the 3D organoid is a model allowing to decipher the intracellular impact of Salmonella on the intestinal epithelium cell response and illustrates the importance of the gut segment used to purify stem cells and derive organoids to specifically study epithelial cell -Salmonella interaction.

Chronic dietary exposure to a glyphosate-based herbicide in broiler hens has long-term impacts on the progeny metabolism.

Glyphosate-based herbicides (GBH) are the most commonly used herbicides in agriculture. Several studies reported possible adverse effects on human and animal models after a GBH exposure. However, the effects of a temporary maternal exposure on the progeny have been poorly documented, especially in avian models. We investigated the effects of a hen chronic dietary exposure to a GBH on the progeny, obtained during the period following the withdrawal of GBH from the diet. Hens were exposed to a GBH via their food for 6 wk, after which the GBH was removed from their food. Eggs from these hens were collected 3 wk after the GBH was withdrawn for 1 wk. We monitored the growth performances, metabolic parameters, and behavior from the progeny of the hens (Ex-GBH chicks, n = 186) and compared them with those of unexposed control-hen progeny (CT chicks, n = 213). Ex-GBH chicks were more likely to explore their new environment than CT chicks during the open-field test. In addition, they had an increased fattening and blood triglycerides level, whereas their food consumption was similar to CT chicks. Quantitative PCR on the chemerin system and FASN in chicks livers indicate a transcriptional activity in favor of fatty acid synthesis, and lipidomic analysis on chicks abdominal adipose tissue reveal a global increase in monounsaturated fatty acid and a global decrease in polyunsaturated fatty acids. Seven genes involved in the synthesis of fatty acids were identified with the open access LIPIDMAP software, and their disturbance in Ex-GBH chicks was confirmed via qPCR. Taken together, these results suggest that the progeny of hens temporarily exposed to a GBH are more likely to fatten, even with a balanced diet. The removal of GBH from their contaminated environment would therefore not be sufficient to completely restore their health, has it could induce transgenerational effects.

Adipokines expression in reproductive tract, egg white and embryonic annexes in hen.

In mammals, molecules mainly secreted by white adipose tissue named adipokines are also synthetized locally in the reproductive tract and are able to influence reproductive functions. In avian species, previous studies indicated that the adipokine chemerin is highly abundant in the albumen, compared to the yolk and this was associated to high chemerin expression in the magnum. In addition, the authors observed that chemerin and its receptors are expressed by allantoic and amniotic membranes and chemerin is present in fluids during the embryo development. Here, we studied other adipokines, including adiponectin, visfatin, apelin, and adipolin in egg white and their known receptors in the active (egg-laying hen) and regressed (hen not laying) oviduct and embryonic annexes during embryo development. By using Western blot, RT-qPCR analysis and immunohistochemistry, we demonstrated the expression of different adipokines in the egg albumen (visfatin) and the reproductive tract (adiponectin, visfatin, apelin, adipolin, and their cognate receptors) according the position of egg in the oviduct. We showed that the expression of adipokines and adipokines receptors was strongly reduced in the regressed oviducts (arrested laying hen). Results indicated that visfatin and adiponectin appeared at ED11 to 14 and increased until ED18 in amniotic fluid whereas it was found from ED7 and was unchanged during embryo development in allantoic fluid. Taken together, adipokines and their receptors are expressed in the egg white, the reproductive tract and the embryonic annexes. Data obtained suggest important functions of theses metabolic hormones during the chicken embryo development. Thus, adipokines could be potential biomarkers to improve the embryo development.

The influence of selection in wild pheasant (Phasianus colchicus) breeding on reproduction and the involvement of the chemerin system.

Chemerin is a hormone produced mainly by adipose tissue and liver. We have recently shown that it is locally produced in the reproductive tract in hens, particularly at the magnum level, leading to its accumulation in the egg albumen. We have also determined that chemerin is necessary for egg fertilization, embryo development, and angiogenesis within the chorio-allantoic membrane in chicken species. We, therefore, hypothesize that chemerin, widely present in various gallinacean species, could be a marker of egg fertility in this animal order. To demonstrate this, we used a model close to the hen: the pheasant. By RT-qPCR, we have shown that chemerin and its three receptors CMKLR1, GPR1, and CCRL2 are expressed in the reproductive tract of females. In addition, chemerin is also produced predominantly in the magnum and accumulates in the egg albumen as determined by immunoblot. We then compared two lines of pheasants with different reproductive characteristics: the F11 and F22 breeds. F22 lays more eggs than F11, but have significantly lower fertility and hatchability rates. In addition, F22 exhibit a significantly lower amount of chemerin protein in their magnum (P < 0.01) and in the egg albumen (P < 0.0001) compared to F11. Finally, we observed a positive correlation between the chemerin amount in the albumen of F11 eggs and the hatching rate of the eggs (r = 0.5; P = 0.04) as well as a negative correlation between the chemerin quantity in the albumen of F22 eggs and the rate of unfertilized eggs (r = -0.37; P = 0.04). Finally, chemerin system (ligand and receptors) is also expressed within embryo annexes (chorioallantoic and amniotic membranes) during incubation. These data demonstrate an interspecies conservation of chemerin production in the magnum, its accumulation in the egg albumen and its possible use as a marker for determining the quality of eggs in term of fertility and embryo development.

Chicken white egg chemerin as a tool for genetic selection for egg weight and hen fertility.

Embryo mortality rate, which can reach up to 40% in avian species, is a major issue for breeding. It is therefore important to identify new embryo development biomarkers for genetic selection to improve reproductive performances. We have recently shown that chemerin is expressed in the oviductal hen magnum, accumulates in egg white, is correlated with embryo survival and could thus be used as a molecular marker of embryo development. Eggs from seven hen breeds (n = 70) were collected during five successive days at the end of the laying period. After weighing eggs, yolk and albumen, an egg white sample from each egg was collected and a blood sample was taken from each hen. Chemerin concentrations in albumen and blood samples were measured by a specific home made ELISA assay. Hen's plasma and egg's albumen chemerin levels were found to be correlated with reproductive parameters such as fecundity, fertility, embryo mortality, hatchability and laying rates. The inter-hen chemerin level variability in albumen was higher than intra-hen except for one breed (R+). We observed significantly different levels of chemerin in egg white between breeds. However, chemerin concentrations in egg white were not significantly associated to variations of hen plasma chemerin levels. Interestingly, we observed negative correlations between albumen chemerin concentrations and egg weight (r = -0.43, p = 0.001), between albumen weight (r = -0.40, p = 0.002), and between yolk weight (r = -0.28, p = 0.03). We also showed negative correlations between egg white chemerin concentrations and fecundity (r = -0.32, p = 0.011) and fertility (r = -0.27, p = 0.04) whereas no significant correlation was observed with the laying rate. Taken together, these results suggest that egg white chemerin concentration might be a good biomarker for genetic selection for egg weight and fertility in hens, provided these data are confirmed on a larger scale.

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.

Adipokines in metabolic and reproductive functions in birds: An overview of current knowns and unknowns.

Adipose tissue is now recognized as an active endocrine organ, which synthesizes and secretes numerous peptides factors called adipokines. In mammals, they exert pleiotropic effects affecting energy metabolism but also fertility. In mammals, secretion of adipokines is altered in adipose tissue dysfunctions and may participate to obesity-associated disorders. Thus, adipokines are promising candidates both for novel pharmacological treatment strategies and as diagnostic tools. As compared to mammals, birds exhibit several unique physiological features, which make them an interesting model for comparative studies on endocrine control of metabolism and adiposity and reproductive functions. Some adipokines such as leptin and visfatin may have different roles in avian species as compared to mammals. In addition, some of them found in mammals such as CCL2 (chemokine ligand 2), resistin, omentin and FGF21 (Fibroblast Growth factor 21) have not yet been mapped to the chicken genome model and among its annotated gene models. This brief review aims to summarize data (structure, metabolic and reproductive roles and molecular mechanisms involved) related to main avian adipokines (leptin, adiponectin, visfatin, and chemerin) and we will briefly discuss the adipokines that are still lacking in avian species.

Oviduct Fluid Extracellular Vesicles Change the Phospholipid Composition of Bovine Embryos Developed In Vitro.

Oviduct fluid extracellular vesicles (oEVs) have been proposed as bringing key molecules to the early developing embryo. In order to evaluate the changes induced by oEVs on embryo phospholipids, fresh bovine blastocysts developed in vitro in the presence or absence of oEVs were analyzed by intact cell MALDI-TOF (Matrix assisted laser desorption ionization-Time of flight) mass spectrometry (ICM-MS). The development rates, cryotolerance, and total cell number of blastocysts were also evaluated. The exposure to oEVs did not affect blastocyst yield or cryotolerance but modified the phospholipid content of blastocysts with specific changes before and after blastocoel expansion. The annotation of differential peaks due to oEV exposure evidenced a shift of embryo phospholipids toward more abundant phosphatidylcholines (PC), phosphatidylethanolamines (PE), and sphingomyelins (SM) with long-chain fatty acids. The lipidomic profiling of oEVs showed that 100% and 33% of the overabundant masses in blastocysts and expanded blastocysts, respectively, were also present in oEVs. In conclusion, this study provides the first analysis of the embryo lipidome regulated by oEVs. Exposure to oEVs induced significant changes in the phospholipid composition of resulting embryos, probably mediated by the incorporation of oEV-phospholipids into embryo membranes and by the modulation of the embryonic lipid metabolism by oEV molecular cargos.

Metabolomic Profile of Oviductal Extracellular Vesicles across the Estrous Cycle in Cattle.

Oviductal extracellular vesicles (oEVs) have been proposed as key modulators of gamete/embryo maternal interactions. The aim of this study was to examine the metabolite content of oEVs and its regulation across the estrous cycle in cattle. Oviductal EVs were isolated from bovine oviducts ipsilateral and contralateral to ovulation at four stages of the estrous cycle (post-ovulatory stage, early and late luteal phases, and pre-ovulatory stage). The metabolomic profiling of EVs was performed by proton nuclear magnetic resonance spectroscopy (NMR). NMR identified 22 metabolites in oEVs, among which 15 were quantified. Lactate, myoinositol, and glycine were the most abundant metabolites throughout the estrous cycle. The side relative to ovulation had no effect on the oEVs' metabolite concentrations. However, levels of glucose-1-phosphate and maltose were greatly affected by the cycle stage, showing up to 100-fold higher levels at the luteal phase than at the peri-ovulatory phases. In contrast, levels of methionine were significantly higher at peri-ovulatory phases than at the late-luteal phase. Quantitative enrichment analyses of oEV-metabolites across the cycle evidenced several significantly regulated metabolic pathways related to sucrose, glucose, and lactose metabolism. This study provides the first metabolomic characterization of oEVs, increasing our understanding of the potential role of oEVs in promoting fertilization and early embryo development.