In ovo green light photostimulation during the late incubation stage affects somatotropic axis activity.

Targeted green light photostimulation during the last stage of broiler incubation increases expression of the somatotropic axis. The purpose of this study was to further shorten the in ovo green light photostimulation and determine the critical age for photostimulation in broilers embryos, as a future strategy for broiler incubation. Fertile broilers eggs (n = 420) were divided into 5 treatment groups. The first group was incubated under standard conditions (in the dark) as the negative control group. The second was incubated under intermittent monochromatic green light using light-emitting diode lamps with an intensity of 0.1 W/m2 at shell level from embryonic day (ED) 0 of incubation until hatch, as a positive control. The third, fourth, and fifth groups were incubated under intermittent monochromatic green light from ED 15, 16, and 18 of incubation, respectively, until hatch. All treatment groups showed elevated somatotropic axis expression compared with the negative control, with the group incubated under monochromatic green light from ED 18 until hatch showing results closest to the positive control. This suggests that broiler embryos can be exposed to in ovo green light photostimulation from a late stage of incubation (when transferring the eggs to the hatchery) and exhibit essentially the same outcome as obtained by photostimulation during the entire incubation period.

Integrated Proteomic and N-Glycoproteomic Analyses of Chicken Egg during Embryonic Development.

To better appreciate the alterations of egg proteins and their modifications during embryonic development, a comparative and quantitative study was performed aimed at chicken egg white and yolk proteome and N-glycoproteome after 12 days of incubation using tandem mass tag (TMT)-labeling technology in conjunction with reversed-phase high-performance liquid chromatography (RP-HPLC). A total of 334 unique N-glycosite-containing peptides from 153 N-glycoproteins were identified, of which 82 N-glycosite-containing peptides showed significant changes after 12 days of incubation. The varied proteome was mainly involved with antibacterial, ionic binding, cell proliferation, and embryonic development, while the different degrading and/or absorbing priorities of egg proteins were proposed. This study provides substantial insight into the effects of N-glycoprotein variations on the utilization of egg proteins by chicken embryo during incubation.

Sex differences in serum steroid hormone levels during embryonic development in hen eggs.

This experiment was conducted to explore the differences of serum steroid hormones with age and sex in hen eggs during incubation periods for identification of males and females. The concentrations of estrone (E1), estradiol (E2), estriol (E3), testosterone (T), androstenedione (A4), and dihydrotestosterone (DHT) in serum were measured by enzyme-linked immunosorbent assays in chicken embryos on 8, 10, 12, 14, and 16 D, respectively. During the development of chicken embryo, egg weight loss was closely related to age but no sex. However, it was found that both age and sex significantly affected hormones and had obviously more effect on androgens levels. Besides E2 and T, other steroids such as E1, A4, and DHT were also significantly correlated with sex (P < 0.05). Notably, the level of T and the ratio of T to E2 were significantly higher in males than females (P < 0.05). The ratio of E1 to E2 displayed different trends in different sexes, which increased in males but decreased in females. The distribution proportions of androgens and estrogens kept stable level during the late hatching periods of 12 to 16 D. The sex differences of steroids were more obvious at the late hatching stage through PCA. These suggested that the serum hormones differences in male and female embryos played a vital role in sexual differentiation. These findings not only provided the theoretical basis for sex determination of fertilized eggs in egg-laying hen strains, but also contributed to develop a non-invasive way to sex determination of fertilized eggs to meet the modern commercial application.

A combination of chicken embryo extract and a nutritional supplement protect a rat model of aging against d-galactose-induced dysfunction of mitochondria and autophagy.

The aging process is usually associated with increased oxidative stress and deficiency of tissues and organs, which causes a decline in the life quality of individuals. Some anti-oxidant factors derived from foods have been implicated in delaying senescence and sustaining health. The present study aimed to assess the anti-aging effects and underlying mechanism of the combined application of chicken embryo (CE) extract and a nutritional mixture (NM) of 52 ingredients on the aging process of d-galactose (d-gal)-induced rats. After 90 days of treatment, we observed that CE plus NM administration significantly improved the body weight, visceral indices and histological damage of aging rats. The use of CE or NM alone exhibited similar effects, but was not as effective as the combined use of CE and NM. In addition, the additional nutrients promoted the mitochondrial function of d-galactose-induced rats. With CE + NM supplementation, cells from senescent rats showed decreased accumulation of reactive oxygen species (ROS), increased levels of mitochondrial membrane potential, elevated numbers of mitochondria, and morphological changes towards normalization compared to the un-treated group. Furthermore, autophagosomes and autophagy-related proteins were up-regulated in aging rats upon exposure to CE + NM. Interestingly, we also found that the protein extracts of CE have similar effects to CE. These results indicate the critical role played by CE + NM in promoting the function of mitochondria and autophagy, therefore regulating the aging process in rats. Our study provides a novel compound design of nutritional support to achieve an optimal nutritional status for anti-oxidation and slowing of aging.

Isolation, purification, and structure identification of antioxidant peptides from embryonated eggs.

Antioxidant peptides are increasingly attracting researchers in medicine and foods. In the present study, egg white hydrolysates of embryonated eggs hatched on the sixth day (EWHD) were segmented consecutively by ultrafiltration membranes with small tangential flow ultrafiltration system. Four segments with more than 30, 30 to 10 kDa, 10 to 5 kDa, and less than 5 kDa of molecular weight cut-off (MWCO) values were separated and were labeled as MWCOI, MWCOII, MWCOIII, and MWCOIV, respectively. The antioxidant activities of segments were investigated by performing DPPH•, •OH radical scavenging, ultra oxygen anion (O2-•), total antioxidant capacity, and reducing power experiments. The results indicated that MWCOI has the strongest scavenging activities on DPPH• radical. However, MWCOIV has the strongest scavenging activities on •OH, O2-•, total antioxidant capacity, and reducing power, which revealed that MWCOIV has strong antioxidant activity. MWCOIV was further separated into 14 fractions via semipreparative reverse-phased high-performance liquid chromatography (RP-HPLC), and their antioxidant activity was evaluated by different antioxidant assays in vitro. The fractions 10 and 7 had strong antioxidant activities. The purities of these 2 fractions were determined by analytical RP-HPLC. Moreover, both fractions 10 and 7 displayed high purity levels, and they were identified by quadrupole time-of-flight tandem mass spectrometry. Only fraction 10, with a molecular weight of 204 Da, can be identified to be Ser-Val. EWHD can be considered as a promising source of natural food antioxidants for the development of functional food.

Heavy metals in eggs and chicken and the associated human health risk assessment in the mining areas of Singhbhum copper belt, India.

Metal contamination was studied in locally rearing chicken and eggs in the environs of mining areas of Singhbhum copper belt. Concentrations of metals were below Indian standards except for Cu, Ni and Zn in the case of chicken at some locations. Estimated daily intake (EDI) and target hazard quotient (THQ) suggested that the metals did not pose risk individually. However, considering the geometric mean of the metals, hazard index (HI) was above unity. Cu, Pb and Co were the key components contributing to a potential noncarcinogenic risk. The HI varied from 0.62 to 1.66 among the locations indicating a considerable heath risk to the consumers of locally reared chicken and eggs around the mining areas. Higher HIs were found at the locations in close vicinity to copper mining and processing units compared to other locations.

The relationship between liver-kidney impairment and viral load after nephropathogenic infectious bronchitis virus infection in embryonic chickens.

To examine the relationship of impairments of the liver and kidney with viral load after nephropathogenic infectious bronchitis virus (NIBV) infection in embryonic chickens, 120 specific-pathogen-free Leghorn embryonated chicken eggs were randomly divided into two groups (infected and control), with three replicates per group and 20 eggs in each replicate. The eggs in the infected and control groups were challenged with 0.2 mL of 105.5 ELD50 NIBV and sterile saline solution, respectively. The embryonic chickens' plasma and liver and kidney tissues were collected at 1, 3, and 5 days post-inoculation (dpi), the liver and kidney functional parameters were quantified, and the tissue viral loads were determined with real-time PCR. The results showed that plasma potassium, sodium, chlorine, magnesium, calcium, and phosphorus levels were increased. The infected group exhibited significantly higher plasma uric acid, blood urea nitrogen, and creatinine levels than the control group at 3 dpi. The plasma concentrations of aspartate aminotransferase and alanine aminotransferase were significantly increased in the infected group. The total protein, albumin, and globulin levels in the infected group were significantly lower than those in the control group. The liver-kidney viral load in the infected group peaked at 3 dpi, at which time the kidney viral load was significantly higher than that of the liver. Our results indicated that NIBV infection caused liver and kidney damage in the embryonic chickens, and the results also demonstrated that the liver and kidney damage was strongly related to the tissue viral load following NIBV infection in embryonic chickens.

Toxicity of different forms of graphene in a chicken embryo model.

In the present work, the toxicity of three forms of graphene: pristine graphene (pG), graphene oxide (GO), and reduced graphene oxide (rGO) was investigated using a chicken embryo model. Fertilized chicken eggs were divided into the control group and groups administered with pG, GO, and rGO, in concentrations of 50, 500, and 5000 µg/ml. The experimental solutions were injected in ovo into the eggs, and at day 18 of incubation, the embryo survival, body and organ weights, the ultrastructure of liver samples, and the concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the livers were measured. Survival of embryos decreased significantly after treatment with all types of graphene, but not in a dose-dependent manner. The body weights were only slightly affected by the highest doses of graphene, while the organ weights were not different among treatment groups. In all experimental groups, atypical hepatocyte ultrastructure and mitochondrial damage were observed. The concentration of the marker of DNA damage 8-OHdG in the liver significantly decreased after pG and rGO treatments. Further in vivo studies with different animal models are necessary to clarify the level of toxicity of different types of graphene and to estimate the concentrations appropriate to evaluate their biomedical applications and environmental hazard.

Histological and histochemical evaluations on the effects of high incubation temperature on the embryonic development of tibial growth plate in broiler chickens.

The effects of experimentally induced high incubation temperature on the embryonic development of growth plate of the chicken were investigated by means of histological and enzyme histochemical methods. In the experiments, 250 fertile eggs of Ross-308 broiler strain were divided into two groups, the control eggs were maintained under optimal conditions (37.8°C and 65% ± 2% relative humidity, rh) during the whole incubation period. Heat-stress imposed eggs were maintained under normal conditions (37.8°C and 65% ± 2% rh) until the 10th day of incubation, and then, continuously (24 h per day) exposed to high temperature (38.8°C and 65% ± 2% rh). Tissue samples were taken from 10 animals of each group at the 11th, 13th, 15th, 18th, 21st days of incubation. Tissue samples were processed by enzyme histochemical methods in addition to routine histological techniques. The relative tibia weights and tibia length were lower in the heat-stress group compared to the control group. The results of the measurements of the growth plate showed that the proliferative zone was narrowed whereas, the transitional and hypertrophic zone were thickened in the heat stress group. Alkaline phosphatase (ALP) density was significantly decreased in the heat-stress group compared to the control group. In conclusion, bone formation and growth plate formation are crucial for embryo development and 1°C higher from optimum may increase the incidence of skeletal disorders and leg problems in broiler chickens which is one of the major animal welfare concerns for the poultry industry.

Effect of embryonic development on the chicken egg yolk plasma proteome after 12 days of incubation.

To better appreciate the dynamics of yolk proteins during embryonic development, we analyzed the protein quantitative changes occurring in the yolk plasma at the day of lay and after 12 days of incubation, by comparing unfertilized and fertilized chicken eggs. Of the 127 identified proteins, 69 showed relative abundance differences among conditions. Alpha-fetoprotein and two uncharacterized proteins (F1NHB8 and F1NMM2) were identified for the first time in the egg. After 12 days of incubation, five proteins (vitronectin, α-fetoprotein, similar to thrombin, apolipoprotein B, and apovitellenin-1) showed a major increase in relative abundance, whereas 15 proteins showed a significant decrease in the yolks of fertilized eggs. In unfertilized/table eggs, we observed an accumulation of proteins likely to originate from other egg compartments during incubation. This study provides basic knowledge on the utilization of egg yolk proteins by the embryo and gives some insight into how storage can affect egg quality.

The potential of spatially resolved spectroscopy for monitoring angiogenesis in the chorioallantoic membrane.

Over the last decade, the poultry sector has sought to develop ways to monitor chicken embryonic development as to optimize the incubation conditions. One of the parameters of development which may change under different incubation conditions is the angiogenesis in the chorioallantoic membrane (CAM). To be able to quantify these changes in the angiogenesis and detect long-term effects on health, a non-destructive technique is necessary. In this article, the first steps toward such a non-destructive technique are successfully taken. A spatially resolved spectroscopy set-up is built and tested for its potential to measure changes in angiogenesis with incubation time, and differences between a normal and hypercapnic incubation. In this first study, reflectance measurements are performed directly on the CAM as the eggshell considerably complicates the analysis. This issue should be addressed in future research to come to a really non-destructive technique. An experiment was conducted in which one group was incubated under normal conditions, and another under early prenatal hypercapnic conditions (i.e., increased CO(2) concentrations). The angiogenesis in the CAM was measured at embryonic day (ED) 10, 13, and 16. The measurements showed a clear blood spectrum with an increasing amount of blood in time, and significant differences in the reflectance as function of the source-detector distances. However, no significant differences between the hypercapnia and the control group could be detected.

Proteome profile of the pipping muscle in broiler embryos.

This study is the first proteomics analysis of the muscularis complexus (pipping muscle) in chicken (Gallus gallus) broiler embryos. We used differential detergent fractionation and nano-HPLC-MS/MS analysis to identify 676 proteins from all cellular components. The identified proteins were functionally classified in accordance with their involvement in various cellular activities.

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification.

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.

Production of chick embryo extract for the cultivation of murine neural crest stem cells.

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest(1). Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest(2,3,4,5). These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone(6,7,8,9). To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required(10). The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

Transcriptional profiling of Toll-like receptors in chicken embryos and in the ovary during sexual maturation and in response to Salmonella enteritidis infection.

One of the key members of the innate immune system in many vertebrate species is the family of Toll-like receptors (TLRs). These molecules, which initiate the innate immune response and mount an anti-microbial response in both vertebrates and invertebrates, have recently been identified in the chicken genome. The recent findings of chicken TLRs (cTLRs) expression in ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS) infection are very important for reproductive physiology due to the transovarian transmission of Salmonella enteritidis (SE) in laying hens. The aim of this study was to investigate the expression of the ten cTLRs identified to date, in the chicken ovary in vivo and embryos during early embryonic development, to investigate whether sexual maturation affects their ovarian mRNA abundance and to investigate the transcriptional changes of TLRs in the chicken ovary in response to SE infection. RNA was extracted from embryos from day 3 to day 10 of embryonic development as well as from the ovaries of healthy prepubertal, sexually mature and aged birds, and from sexually mature and aged SE infected birds. RT-PCR analysis revealed that all TLRs apart from TLRs 1-1 and 2-2 were expressed in the ovary of sexually mature chickens, while all TLRs apart from TLR1-1 were expressed in the chicken embryos during embryonic development. Quantitative real-time PCR analysis revealed that the ovarian mRNA abundance of TLRs differ with respect to sexual maturation. SE infection resulted in a significant induction of TLR4, and 15 in the ovary of sexual mature birds, and in a significant induction of TLR15 in the ovary of aged birds, while a significant down-regulation was observed for TLR3 in the ovary of aged birds. These findings suggest that a TLR-mediated immune response mechanism exists in the chicken ovary.

Immunohistochemical localization of oestrogen receptor alpha in the various cell categories of chick embryo ovary.

The immunohistochemical (IHC) localization of oestrogen receptor alpha (ERα) was studied in the developing left ovary of 14.5-day-old chick embryos. The study was focused in particular on distinguishing in cortex and medulla the different cell categories that proved positive to the reaction, in order to gain further understanding of gonadal cell interactions during ovarian development. Immunostained cells were observed in both the cortex and medulla, but the reactivity for ERα was discontinuous, probably due to variable cell requirements. In the cortex, positivity was observed in cells of the ovarian surface epithelium, in germ cells and in prefollicular cells. In the medulla, positivity was found in the following cell categories: interstitial cells, poorly differentiated somatic cord cells, including those delimiting lacunae, germ cells and their accompanying cells of epithelial origin. Furthermore, the IHC results showed that the intracellular localization of the antigen was cytoplasmic, nuclear, or both. The significance of ERα presence and intracellular localization was discussed in relation and as supplementary to previous research by various Authors. In particular, as regards the unusual cytoplasmic immunoreactivity, a gradual shift of ERα localization from cytoplasmic to nuclear during the embryonic period is suggested.

Whole mount in situ hybridization detection of mRNAs using short LNA containing DNA oligonucleotide probes.

In situ hybridization is widely used to visualize transcribed sequences in embryos, tissues, and cells. For whole mount detection of mRNAs in embryos, hybridization with an antisense RNA probe is followed by visual or fluorescence detection of target mRNAs. A limitation of this approach is that a cDNA template of the target RNA must be obtained in order to generate the antisense RNA probe. Here we investigate the use of short (12-24 nucleotides) locked nucleic acid (LNA) containing DNA probes for whole mount in situ hybridization detection of mRNAs. Following extensive protocol optimization, we show that LNA probes can be used to localize several mRNAs of varying abundances in chicken embryos. LNA probes also detected alternatively spliced exons that are processed in a tissue specific manner. The use of LNA probes for whole mount in situ detection of mRNAs will enable in silico design and chemical synthesis and will expand the general use of in situ hybridization for studies of transcriptional regulation and alternative splicing.

Gluconeogenesis differs in developing chick embryos derived from small compared with typical size broiler breeder eggs.

We hypothesized that, as the supply of preformed glucose diminishes during development, the embryo would transition to a greater rate of gluconeogenesis (GNG) and that GNG would be greater in embryos from small vs. typical size eggs. Gluconeogenesis by embryos from small (51.1 +/- 3.46 g) and typical size (65 +/- 4.35 g) broiler breeder eggs was measured by dosing [(13)C(6)]glucose (15 mgxegg(-1)) into the chorio-allantoic fluid for 3 consecutive days to achieve isotopic steady-state before blood collection on embryonic day (e) 12, e14, e16, and e18 (4 to 5 eggsxsize(-1)xd(-1)). The (13)C-Mass isotopomer enrichment of blood glucose was determined by gas chromatography-mass spectrometry. On e14, e16, and e18, but not on e12, embryos from small eggs weighed less (P < 0.05) than typical size eggs. For both sizes of eggs, blood glucose concentration, glucose entry rate (g.d(-1)), and Cori cycling and glucose (13)C-recycling (% of entry rate) increased (P < 0.05) with development. On e12 and e14, rates of glucose entry and Cori cycle flux were greater (P < 0.05) for embryos from small eggs. When standardized to BW (g.100 g of BW(-1)xd(-1)), glucose entry and Cori and non-Cori cycle fluxes were greater for embryos from small eggs. From e12 through e18, blood concentrations of gluconeogenic AA (threonine, glutamine, arginine, proline, isoleucine, and valine) were 25 to 48% less (P < 0.01) in embryos from small eggs. In conclusion, embryos from small eggs exhibit greater rates of GNG earlier in development compared with typical size eggs and, perhaps as a consequence, their reduced embryonic growth may result from diverting greater supplies of AA toward GNG.

Assessment of embryonic growth in chicken eggs by means of visible transmission spectroscopy.

During this work, it was investigated whether spectral measurements can be used to monitor embryonic growth. An experiment was conducted in which both the transmission spectra and embryonic weight were determined on 240 eggs (Cobb, 37 weeks) between Day 5 and Day 10 of incubation. The spectral data were linked to embryonic weight by means of a partial least squares analysis. Different preprocessing procedures were compared during this work, that is, smoothing, multiplicative scatter correction (MSC), and first- and second-order derivative. Compared to the remainder of the preprocessing procedures, MSC leads to a considerable improvement of the prediction capability of the embryonic weight. The ratio of performance to deviation obtained for the MSC spectra equaled 4.5 indicating that a very accurate prediction of embryonic weight is feasible based on the VIS/NIR transmission measurements. Important regions for the prediction are situated around 685-740 nm. It is suggested that the spectral changes in these spectral regions result from the displacement of carotenoids from the yolk into the blood circuitry.

New methods for chicken embryo manipulations.

The capacity to image a growing embryo while simultaneously studying the developmental function of specific molecules provides invaluable information on embryogenesis. However, until recently, this approach was accomplished with difficulty both because of the advanced technology needed and because an easy method of minimizing damage to the embryo was unavailable. Here, we present a novel way of adapting the well-known EC culture of whole chick embryos to time-lapse imaging and to functional molecular studies using blocking agents. The novelty of our method stems from the ability to apply blocking agents ex ovo as well as in ovo. We were able to study the function of a set of molecules by culturing developing embryos ex ovo in tissue culture media containing these molecules or by injecting them underneath the live embryo in ovo. The in ovo preparation is particularly valuable, because it extends the period of time during which the developmental function of the molecule can be studied and it provides an easy, reproducible method for screening a batch of molecules. These new techniques will prove very helpful in visualizing and understanding the role of specific molecules during embryonic morphogenesis, including blood vessel formation.