LIN28 is essential for the maintenance of chicken primordial germ cells.

Understanding the mechanism of stem cell maintenance underlies the establishment of long-term and mass culture methods for stem cells that are fundamental for clinical and agricultural applications. In this study, we use chicken primordial germ cell (PGC) as a model to elucidate the molecular mechanisms underlying stem cell maintenance. The PGC is a useful experimental model because it is readily gene-manipulatable and easy to test gene function in vivo using transplantation. Previous studies to establish a long-term culture system have shown that secreted factors such as FGF2 are required to maintain the self-renewal capability of PGC. On the other hand, we know little about intracellular regulators responsible for PGC maintenance. Among representative stem cell factors, we focus on RNA-binding factors LIN28A and LIN28B as possible central regulators for the gene regulatory network essential to PGC maintenance. By taking advantage of the CRISPR/Cas9-mediated gene editing and a clonal culture technique, we find that both LIN28A and LIN28B regulate the proliferation of PGC in vitro. We further showed that colonization efficiency of grafted PGC at the genital ridges, rudiments for the gonads, of chicken embryos were significantly decreased by knockout (KO) of LIN28A or LIN28B. Of note, overexpression of human LIN28 in LIN28-KO PGC was sufficient to restore the low colonization rates, suggesting that LIN28 plays a key role in PGC colonization at the gonads. Transcriptomic analyses of LIN28-KO PGC reveal that several genes related to mesenchymal traits are upregulated, including EGR1, a transcription factor that promotes the differentiation of mesodermal tissues. Finally, we show that the forced expression of human EGR1 deteriorates replication activity and colonization efficiency of PGCs. Taken together, this work demonstrates that LIN28 maintains self-renewal of PGC by suppressing the expression of differentiation genes including EGR1.

Developmental Changes in Patterns of Distribution of Fibronectin and Tenascin-C in the Chicken Cornea: Evidence for Distinct and Independent Functions during Corneal Development and Morphogenesis.

The cornea forms the tough and transparent anterior part of the eye and by accurate shaping forms the major refractive element for vision. Its largest component is the stroma, a dense collagenous connective tissue positioned between the epithelium and the endothelium. In chicken embryos, the stroma initially develops as the primary stroma secreted by the epithelium, which is then invaded by migratory neural crest cells. These cells secrete an organised multi-lamellar collagenous extracellular matrix (ECM), becoming keratocytes. Within individual lamellae, collagen fibrils are parallel and orientated approximately orthogonally in adjacent lamellae. In addition to collagens and associated small proteoglycans, the ECM contains the multifunctional adhesive glycoproteins fibronectin and tenascin-C. We show in embryonic chicken corneas that fibronectin is present but is essentially unstructured in the primary stroma before cell migration and develops as strands linking migrating cells as they enter, maintaining their relative positions as they populate the stroma. Fibronectin also becomes prominent in the epithelial basement membrane, from which fibronectin strings penetrate into the stromal lamellar ECM at right angles. These are present throughout embryonic development but are absent in adults. Stromal cells associate with the strings. Since the epithelial basement membrane is the anterior stromal boundary, strings may be used by stromal cells to determine their relative anterior-posterior positions. Tenascin-C is organised differently, initially as an amorphous layer above the endothelium and subsequently extending anteriorly and organising into a 3D mesh when the stromal cells arrive, enclosing them. It continues to shift anteriorly in development, disappearing posteriorly, and finally becoming prominent in Bowman's layer beneath the epithelium. The similarity of tenascin-C and collagen organisation suggests that it may link cells to collagen, allowing cells to control and organise the developing ECM architecture. Fibronectin and tenascin-C have complementary roles in cell migration, with the former being adhesive and the latter being antiadhesive and able to displace cells from their adhesion to fibronectin. Thus, in addition to the potential for associations between cells and the ECM, the two could be involved in controlling migration and adhesion and subsequent keratocyte differentiation. Despite the similarities in structure and binding capabilities of the two glycoproteins and the fact that they occupy similar regions of the developing stroma, there is little colocalisation, demonstrating their distinctive roles.

Intra-amniotic administration of l-glutamine promotes intestinal maturation and enteroendocrine stimulation in chick embryos.

Initial nutritional stimulation is a key driving force for small intestinal maturation. In chick embryos, administration of l-glutamine (Gln) into the amniotic fluid stimulates early development of the small intestinal epithelium by promoting enterocyte differentiation. In this study, we evaluated the effects of intra-amniotic administration of Gln on enterocyte morphology and function, and elucidated a potential enteroendocrine pathway through which Gln stimulates small intestinal maturation. Our results show that Gln stimulation at embryonic day 17 significantly increased enterocyte and microvilli dimensions by 10 and 20%, respectively, within 48 h. Post-hatch, enterocytes and microvilli were 20% longer in Gln-treated chicks. Correspondingly, Gln stimulation significantly upregulated mRNA expression of brush border nutrient transporters PepT-1 and SGLT-1 and tight junction proteins TJP-1 and TJP-2, before and after hatch (P < 0.05). Since GLP-2 signaling from intestinal L-cells is associated with enterocyte growth, functionality and integrity, we examined the effects of Gln stimulation on mRNA expression of key hormones and receptors within this enteroendocrine pathway and found significant increases in GLP-2R, IGF-1 and IGF-1R expression before and after hatch (P < 0.05). In conclusion, our findings link primary nutrient stimulation in the developing small intestine with enterocyte morphological and functional maturation and enteroendocrine signaling.

The Chicken Embryo Model: A Novel and Relevant Model for Immune-Based Studies.

Dysregulation of the immune system is associated with many pathologies, including cardiovascular diseases, diabetes, and cancer. To date, the most commonly used models in biomedical research are rodents, and despite the various advantages they offer, their use also raises numerous drawbacks. Recently, another in vivo model, the chicken embryo and its chorioallantoic membrane, has re-emerged for various applications. This model has many benefits compared to other classical models, as it is cost-effective, time-efficient, and easier to use. In this review, we explain how the chicken embryo can be used as a model for immune-based studies, as it gradually develops an embryonic immune system, yet which is functionally similar to humans'. We mainly aim to describe the avian immune system, highlighting the differences and similarities with the human immune system, including the repertoire of lymphoid tissues, immune cells, and other key features. We also describe the general in ovo immune ontogeny. In conclusion, we expect that this review will help future studies better tailor their use of the chicken embryo model for testing specific experimental hypotheses or performing preclinical testing.

Sulfated glycans containing NeuAcα2-3Gal facilitate the propagation of human H1N1 influenza A viruses in eggs.

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galß1-4(SO3--6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galß1-4(SO3--6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos.

New insights into the obligatory nature of cyclooxygenase-2 and PGE2 during early chick embryogenesis.

Temporal expression patterns and activity of two cyclooxygenase (COX-1 and COX-2) isoforms were analysed during early chick embryogenesis to evaluate their roles in development. COX-2 inhibition with etoricoxib resulted in significant structural anomalies such as anophthalmia (born without one or both eyes), phocomelia (underdeveloped or truncated limbs), and gastroschisis (an opening in the abdominal wall), indicating its significance in embryogenesis. Furthermore, the levels of PGE2, PGD2, PGF2α, and TXB2 were assessed using quantitative LC-MS/MS to identify which effector prostanoid (s) had their synthesis initiated by COX-2. COX-2 inhibition was only shown to reduce the level of PGE2 significantly, and hence it could be inferred that the later could be largely under the regulation of activated COX-2 in chick embryos. The compensatory increase in the activity of COX-1 observed in the etoricoxib-treated group helped to maintain the levels of PGD2, PGF2α, and TXB2. Though the roles of these three prostanoids in embryogenesis need to be further clarified, it appears that their contribution to the observed developmental anomalies is minimal. This study has shown that COX-2 is functionally active during chick embryogenesis, and it plays a central role in the structural configuration of several organs and tissues through its downstream effector molecule PGE2.

Chicken Interspecies Chimerism Unveils Human Pluripotency.

Human pluripotent stem cells (hPSCs) are commonly kept in a primed state but also able to acquire a more immature naive state under specific conditions in vitro. Acquisition of naive state changes several properties of hPSCs and might affect their contribution to embryonic development in vivo. However, the lack of an appropriate animal test system has made it difficult to assess potential differences for chimera formation between naive and primed hPSCs. Here, we report that the developing chicken embryo is a permissive host for hPSCs, allowing analysis of the pluripotency potential of hPSCs. Transplantation of naive-like and primed hPSCs at matched developmental stages resulted in robust chimerism. Importantly, the ability of naive-like but not of primed hPSCs to form chimera was substantially reduced when injected at non-matched developmental stages. We propose that contribution to chick embryogenesis is an informative and versatile test to identify different pluripotent states of hPSCs.

Maternal conjugated linoleic acid alters hepatic lipid metabolism via the AMPK signaling pathway in chick embryos.

The effects of maternal conjugated linoleic acid (CLA) on embryonic development and hepatic lipid metabolism were investigated in chick embryos. A total of 180 Arbor Acres female broiler breeders (36 wk old) were randomly divided into the following 3 dietary treatment groups: a basic diet (control), a basic diet containing 0.5% CLA (CLA1), and a basic diet containing 1.0% CLA (CLA2). The females were fed for 8 wk, and the eggs from each group were collected and hatched during the last 2 wk. The results showed that the addition of dietary CLA increased the broken egg rate and reduced the fertilization rate and the egg hatchability (P < 0.05). CLA enrichment decreased the polyunsaturated and monounsaturated fatty acids and increased the saturated fatty acids in the yolk sac (P < 0.05). The yolk sac weight, body weight, and body length had a linear decrease with CLA supplementation (P < 0.05). In the developing chick embryo (at E14) and newly hatched chick (D0), the serum triglyceride concentration decreased with maternal CLA supplementation and was accompanied by a reduction in subcutaneous adipose tissue deposition. In addition, maternal CLA supplementation mediated the hepatic lipid metabolism by decreasing the mRNA expression of sterol regulatory element-binding proteins-1c (SREBP-1c), fatty acid synthase and acetyl-CoA carboxylase, and increasing the mRNA expression of adenosine 5'-monophosphate-activated protein kinase α (AMPKα), peroxisome proliferator-activated receptors α (PPARα), liver fatty acid-binding protein, adipose triglyceride lipase and carnitine palmitoyltransferase in embryonic chick livers (P < 0.05). A drop in SREBP-1c protein expression and an increase in the protein expression of p-AMPKα and PPARα were also observed in the liver of chick embryo (P < 0.05). In conclusion, maternal CLA supplementation regulated the fatty acid composition in the yolk sac, and mediated embryonic chick development and hepatic lipometabolism, and these effects may be related to the AMPK pathway. These findings suggest the potential ability of maternal CLA supplementation to reduce fat deposition in chick embryos.

Testosterone Promotes the Proliferation of Chicken Embryonic Myoblasts Via Androgen Receptor Mediated PI3K/Akt Signaling Pathway.

Testosterone (T) is essential for muscle fiber formation and growth. However, the specific mechanism by which T regulates skeletal muscle development in chicken embryos remains unclear. In this study, the role of T in myoblast proliferation both in vivo and in vitro was investigated. Results showed that the T administration significantly increased the ratio of breast muscle and leg muscle. T induced a significant increase in the cross-sectional area (CSA) and density of myofiber and the ratio of PAX7-positive cells in the skeletal muscle. Exogenous T also induced the upregulation of myogenic regulatory factors (MRFs) and cyclin-dependent kinases (CDK2)/Cyclin D1 (CCND1) and protein levels of androgen receptor (AR), p-Akt and PAX7. Furthermore, T treatment significantly promoted myoblasts cultured in vitro entering a new cell cycle and increased PAX7-positive cells. The mRNA and protein expression of AR and PAX7 were upregulated when treated with T compared to that of the control. The addition of T induced proliferation accompanied by increasing AR level as well as PI3K (Phosphoinositide 3-kinase)/Akt activation. However, T-induced proliferation was attenuated by AR, PI3K, and Akt-specific inhibitors. These data indicated that the pro-proliferative effect of T was regulated though AR in response to the activation of PI3K/Akt signalling pathway.

Enriching ISA brown and Shaver white breeder diets with sources of n-3 polyunsaturated fatty acids increased embryonic utilization of docosahexaenoic acid.

There is limited information on feeding egg-type chick breeders n-3 polyunsaturated fatty acids (PUFA) and its impact on hatching egg quality and embryonic fatty acid (FA) utilization. We investigated the effects of feeding brown and white egg-type chick breeders diets containing sources of n-3 PUFA on egg composition, apparent embryonic FA utilization, and intestinal FA transporter in hatchlings. Twenty-six-week-old ISA brown and Shaver white breeders were fed either 1) control (CON); 2) CON + 1% of microalgae (DMA, Aurantiochytrium limacinum) fermentation product, as a source of docosahexaenoic acid (DHA); or 3) CON + 2.60% of coextruded full-fat flaxseed and pulse mixture (FFF, 1:1 wt/wt) as a source of α-linolenic acid (ALA). Test diets had similar total n-3 and n-6:n-3 ratio. Eggs were hatched, and residual yolk (RY) samples taken for FA analyses. Apparent embryonic FA utilization was calculated by subtracting concentration of FA in RY from concentration of FA in yolk before incubation. There was an interaction between strains and diets (P < 0.05) on DHA in phospholipid and triglyceride fractions of yolk. Both n-3 PUFA sources increased DHA to a greater extent in Shaver white than in ISA brown. The interactive effect of strains and diets (P = 0.019) on embryonic utilization of ALA was such that DMA and FFF reduced ALA utilization, and this pattern was more prevalent in Shaver white birds than in ISA brown birds. There was no interaction between strains and diets on DHA utilization (P > 0.05). Embryos from hens fed n-3 PUFA sources used less total FA in phospholipid fraction (P < 0.001), and they preferentially used more DHA than CON embryos. Shaver white embryos used more (P < 0.05) ALA and DHA than ISA brown embryos. Although data suggested Shaver white had higher propensity of depositing DHA than ISA brown, irrespective of strain, feeding n-3 PUFA modified embryonic pattern of FA utilization toward utilization of DHA.

Longitudinal developmental analysis of prethalamic eminence derivatives in the chick by mapping of Tbr1 in situ expression.

The prethalamic eminence (PThE) is the most dorsal subdomain of the prethalamus, which corresponds to prosomere 3 (p3) in the prosomeric model for vertebrate forebrain development. In mammalian and avian embryos, the PThE can be delimited from other prethalamic areas by its lack of Dlx gene expression, as well as by its expression of glutamatergic-related genes such as Pax6, Tbr2 and Tbr1. Several studies in mouse embryos postulate the PThE as a source of migratory neurons that populate given telencephalic centers. Concerning the avian PThE, it is visible at early embryonic stages as a compact primordium, but its morphology becomes cryptic at perinatal stages, so that its developmental course and fate are largely unknown. In this report, we characterize in detail the ontogeny of the chicken PThE from 5 to 15 days of development, according to morphological criteria, and using Tbr1 as a molecular marker for this structure and its migratory cells. We show that initially the PThE contacts rostrally the medial pallium, the pallial amygdala and the paraventricular hypothalamic alar domain. Approximately from embryonic day 6 onwards, the PThE becomes progressively reduced in size and cell content due to massive tangential migration of many of its neuronal derivatives towards nearby subpallial and hypothalamic regions. Our analysis supports that these migratory neurons from the avian PThE target telencephalic centers such as the commissural septal nuclei, as previously described in mammals, but also the diagonal band and preoptic areas, and hypothalamic structures in the paraventricular hypothalamic area.

Hypoxic hypometabolism in chicken embryos: conformism and downregulation.

Postnatally, during hypoxia the decrease in oxygen consumption ( [Formula: see text] ) can exceed what expected from the limitation in O2 availability, meaning that [Formula: see text] -downregulation exceeds O2-conformism. We questioned whether a similar phenomenon could occur prenatally, in chicken embryos at mid- (E11, out of 20.5 days) or near end- (E18) incubation. [Formula: see text] was measured with an open-flow system in the sequence of normoxia-normothermia (21% O2, 37 °C, 30 min), hypoxia in normothermia (Hx-NT, either 18, 15, 12 or 9% O2, 37 °C, 1 hour), hypoxia in hyperthermia (Hx-HT, up to 43 °C, 1 hour) and return to normoxia-normothermia (30 min). During Hx-NT [Formula: see text] invariably decreased in a [O2]-dependent fashion. The hypoxic drop in [Formula: see text] did not require a post-hypoxic payment of the O2-debt, implying that the decrease in [Formula: see text] reflected hypometabolism. [Formula: see text] did not differ significantly between Hx-HT and Hx-NT for [O2] = 15% or less, as expected by O2-conformism. Differently, with milder hypoxia (18% O2), [Formula: see text] during Hx-HT significantly exceeded that in Hx-NT, meaning that the value of [Formula: see text] in Hx-NT was not limited by O2 supply. We conclude that a phenomenon of hypoxic [Formula: see text] downregulation like that observed in postnatal mammals can occur also prenatally, in the chicken embryos. The mechanisms at the basis of the downregulation remain unresolved and could combine physiological and cellular processes.

GSK-3 signaling is involved in proliferation of chicken primordial germ cells.

Primordial germ cells (PGCs) are precursors of sperms and oocytes and responsible for passing the genetic information from one generation to the next. Chicken PGCs segregate from somatic cells in early embryo and could be isolated and cultured in vitro, making it a useful tool to produce genetically modified animals. However, the number of PGCs isolated from embryo is limited and these cells are not efficient to proliferation in vitro. GSK-3 plays an important role in multiple intracellular signaling pathways and inhibition of GSK-3-mediated ß-catenin phosphorylation is known to reduce apoptosis and promote proliferation in T cells and embryo stem cells (ESC). In this study, we investigate the effect of GSK-3 inhibitor on the proliferation of PGCs in vitro and found significant increases of cell proliferation in the culture supplemented with CHIR. We further found that CHIR regulates PGC cell cycle by activating Wnt signaling and antagonizing the apoptosis of PGCs by inhibition of the expression of caspase-3 and Beclin-1. PGCs treated with CHIR expressed the germ cell-related markers and retain the capability to colonize the embryonic gonad after re-introduction to vasculature of HH stage-15 embryos. These results suggest that GSK-3 is involved in cell renewal and apoptosis in chicken PGCs.

In ovo metabolism of estradiol to estrone sulfate in chicken eggs: Implications for how yolk estradiol influences embryonic development.

The steroid 17ß-estradiol (herein "estradiol") is a potent regulator of sexual differentiation that exerts wide-ranging effects on the developing brain and other tissues. The developing gonads are an important source of estradiol but most, if not all, vertebrate embryos are also exposed to maternally derived estradiol during development. In birds, this maternally derived estradiol is present in the egg at the time of oviposition but very little is known about how this source of estradiol influences development. A critical aspect of understanding yolk estradiol effects is deciphering how steroid metabolism may regulate embryonic exposure to yolk estradiol. In this study, we examine the metabolic fate of estradiol during the first five days of incubation in chicken (Gallus gallus) eggs. Using tritiated estradiol to trace the movement and metabolism of estradiol, we demonstrate that estradiol is metabolized to estrone, which is subsequently conjugated to estrone sulfate as the primary metabolite. Estrone sulfate then accumulates in the albumen by day five of incubation. Overall, these findings have important implications for how yolk estradiol may influence development and alter offspring phenotype. Mechanisms through which estradiol, as well as estrone sulfate, might elicit effects are discussed.

Androgen and mineralocorticoid receptors are present on the germinal disc region in laying hens: Potential mediators of sex ratio adjustment in birds?

Female birds skew offspring sex ratios based on environmental and social stimuli; however, the mechanism mediating this phenomenon remains unknown. Growing evidence suggests that testosterone and corticosterone may influence meiosis, as they skew sex ratios when given immediately before chromosomal segregation. It is unclear if these hormones act on the germinal disc (GD) or through a downstream mediator. It is also unknown whether the GD contains receptors for these hormones. If testosterone and/or corticosterone act on the GD to skew sex ratios, then the GD should have receptors for them and that receptor levels should be higher in the GD regions compared to other follicular regions. Furthermore, fluctuations of receptor levels should occur near meiotic segregation. We collected ovarian follicles at 5 h pre-ovulation (just before meiotic segregation) and 20 h pre-ovulation (when sex chromosomes are arrested), and measured androgen receptor (AR) and mineralocorticoid receptor (MR) protein levels via Western blot. ARs and MRs were on the follicle in the GD and non-GD regions, and at 5 h and 20 h pre-ovulation. Both AR and MR protein levels were higher in the GD region than the non-GD region at both time points, but did not differ between time points. These results suggest that hen ovarian follicles have receptors for testosterone and corticosterone, and that the ability for testosterone to respond may be specifically higher in the GD-region, providing further support for the role of testosterone in the alteration of meiotic segregation.

Label-free proteomic analysis reveals the differentiation between unfertilized and fertilized Beijing-You chicken eggs.

Egg fertilization is a dynamic process, including varieties of biochemical changes. To better understand the molecular mechanisms during the egg embryo development, the objective of this study was to quantify protein expression changes between fertilized and unfertilized Beijing-You chicken eggs using label-free liquid chromatography-tandem mass spectrometry method. The results showed that a total of 1241 proteins were identified from fertilized and unfertilized eggs, 229 proteins were observed difference in fertilized eggs (p < 0.05) compared with that in unfertilized eggs. The expressions of 86 proteins were up-regulated and 48 proteins were down-regulated in fertilized eggs. STRING database analysis and Gene Ontology analysis results showed that these differentially expressed proteins significantly interacted and were involved in lipid transport and inflammatory response biological processes. The mRNA and protein expression levels of most differentially expressed proteins Apolipoprotein B, Fibrinogen alpha chain, Transferrin receptor protein 1, Phospholipid transfer protein and Vimentin were validated by RT-PCR and western blot. These results could provide possible novel insights for the molecular mechanism of egg fertilization.

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.

Glycerol in ovo feeding as an energy substrate improves performance of broilers from young breeders.

Glycerol is one of the substrates used for glycogen production by the chicken embryo, which is the predominant energy source during the last days of incubation and during hatching. The objective of the present study was to evaluate the in ovo feeding (IOF) of glycerol in the light and heavy broiler eggs derived from breeders of two different ages. Two experiments, with 672 eggs each, were carried out. The only difference between the experiments was breeder age: 32 weeks old in Exp. I and 60 weeks old in Exp. II. A completely randomized experimental design in a 3 × 2 factorial arrangement was applied. Treatments consisted of three glycerol IOF doses (0, 6, or 12 mg/ml) and two egg weights (light or heavy). Incubation parameters, glycogen reserves and live performance parameters (1-7 days of age) were evaluated. Hatch of fertile eggs, embryo mortality after IOF and the number of early-hatching chicks were not affected by the treatments in both experiments. Hatchlings from heavy eggs (68.03 ± 0.64 g) laid by young breeders and receiving 6 mg glycerol/ml showed higher liver glycogen levels than those injected with 0 or 12 mg/ml. Glycerol IOF of embryos from young breeders increased feed intake and weight gain at 7 days of age, independently of egg weight. However, different glycerol dosages had no effect on the performance of the progeny of 60-week-old breeders. These results show that glycerol may be used as an IOF ingredient without affecting incubation parameters. The chickens from young breeders had greater glycogen deposition with inoculation of 6 mg/ml of glycerol and better performance with glycerol administration. However, glycerol IOF did not improve the performance of the progeny of 60-week-old breeders. Therefore, glycogen IOF may be recommended for eggs laid by young breeders.

(-)-Hydroxycitric Acid Influenced Fat Metabolism via Modulating of Glucose-6-phosphate Isomerase Expression in Chicken Embryos.

The current research aimed to explore the impact of (-)-hydroxycitric acid (HCA) on fat metabolism and investigate whether this action of (-)-HCA was associated with modulation of glucose-6-phosphote isomerase (GPI) expression in chicken embryos. We constructed a recombinant plasmid (sh2-GPI) to inhibit GPI expression, and then embryos were treated with (-)-HCA. Results showed that (-)-HCA reduced lipid droplet accumulation, triglyceride content, and lipogenesis factors mRNA level and increased lipolysis factors mRNA expression, while this effect caused by (-)-HCA was markedly reversed when the chicken embryos were pretreated with sh2-GPI. (-)-HCA increased phospho (p)-acetyl-CoA carboxylase, enoyl-CoA hydratase short chain-1, carnitine palmitoyl transferase 1A, p-AMP-activated protein kinase, and peroxisome proliferators-activated receptor α protein expression, and this action of (-)-HCA also dispelled when the chicken embryos were pretreated with sh2-GPI. These data demonstrated that (-)-HCA decreased fat deposition via activation of the AMPK pathway, and the fat-reduction action of (-)-HCA was due to the increasing of GPI expression in chicken embryos.

Potential role of ALDH3A2 on the lipid and glucose metabolism regulated by (-)-hydroxycitric acid in chicken embryos.

This study aimed to investigate the effect of (-)-hydroxycitric acid ((-)-HCA) on lipid and glucose metabolism, and further analyzed these actions whether associated with modulation of aldehyde dehydrogenase 3 family member A2 (ALDH3A2) expression in chicken embryos. Results showed that (-)-HCA decreased triglyceride content and lipid droplet counts, while these effects induced by (-)-HCA were reversed in chicken embryos pre-transfected with sh4-ALDH3A2. (-)-HCA decreased malic enzyme, acetyl-CoA carboxylase, fatty acid synthase, and sterol regulatory element binding protein-1c mRNA level, while increased carnitine palmitoyl transferase 1A (CPT1A) and peroxisome proliferators-activated receptor α (PPARα) mRNA level; and the action of (-)-HCA on lipid metabolism factors had completely eliminated in embryos pre-transfected with sh4-ALDH3A2. Chicken embryos pre-transfected with sh4-ALDH3A2 had eliminated the increasing of serum glucose and hepatic glycogen content induced by (-)-HCA. (-)-HCA decreased phosphofructokinase-1 and increased G6P, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate carboxylase mRNA level in chicken embryos. Similarly, the effect of (-)-HCA on these key enzyme mRNA level was reversed in embryos pre-transfected with sh4-ALDH3A2. Furthermore, (-)-HCA increased PPAR-γ-coactivator-1α (PGC-1α), PPARα, hepatic nuclear factor-4A, PEPCK, and CPT1A protein level, and these actions of (-)-HCA disappeared in embryos pre-transfected with sh4-ALDH3A2. These results indicated that (-)-HCA reduced fat accumulation and accelerated gluconeogenesis via activation of PGC-1α signaling pathway, and these effects of (-)-HCA might associate with the increasing of ALDH3A2 expression level in chicken embryos.