Correlation between small-scale methylation changes and gene expression during the development of myopia.

Visually induced changes in the expression of early growth response-1 (EGR1), FBJ osteosarcoma oncogene (FOS), and NGFI-A binding protein-2 (NAB2) appear to form a part of a retinal network fundamental to ocular growth regulation, and thus, the development of myopia (short-sightedness). However, it is unclear how environmental (visual) cues are translated into these molecular changes. One possibility is through epigenetic modifications such as DNA methylation, a known regulator of such processes. By sequencing bisulfite-converted DNA amplicons, this study examined whether changes in DNA methylation occur within specific regulatory and promoter regions of EGR1, FOS, and NAB2 during the periods of increased and decreased ocular growth in chicks. Visually induced changes in ocular growth rates were associated with single-point, but not large-scale, shifts in methylation levels within the investigated regions. Analysis of methylation pattern variability (entropy) demonstrated that the observed methylation changes are occurring within small subpopulations of retinal cells. This concurs with previous observations that EGR1 and FOS are differentially regulated at the peptide level within specific retinal cell types. Together, the findings of this study support a potential role for DNA methylation in the translation of external visual cues into molecular changes critical for ocular growth regulation and myopia development.

Effect of prenatal ambient temperature on the performance physiological parameters, and oxidative metabolism of Japanese quail (Coturnix coturnix japonica) layers exposed to heat stress during growth.

A strategy to mitigate the negative effects of stress on animals is to enhance their ability to beneficially respond to stressful conditions. This study aimed to assess whether prenatal ambient temperature influences the response of Japanese quail (Coturnix coturnix japonica) chicks to environmental challenges during growth. The experiment was conducted in a 2 × 2 factorial arrangement: two temperature conditions for the mothers (thermoneutral and heat stress by continuous exposure to 32 °C) and two offspring ambient temperature conditions (thermoneutral and heat stress by intermittent exposure to 34 °C for 6 h/day from 15 to 35 days of age). Heat stress in mothers led to lower laying rate, egg mass, expression of methionine sulfoxide reductase A (MSRA) gene, and antioxidant capacity as well as higher chick mortality rate (1-15 days of age). Maternal heat stress led to lower weight gain and total antioxidant capacity and higher feed conversion ratio. Maternal temperature × Offspring temperature interaction effects were observed on carbonylated protein content and HSP70, GSS, and MSRA gene expression. It was observed that, for chicks hatched from heat-stressed mothers, exposure to heat stress led to higher carbonylated protein content and HSP70 expression than exposure to thermoneutral conditions. Maternal heat stress was also responsible for increasing GSS expression in chicks grown under thermoneutral conditions. Chicks hatched from non-stressed mothers and subjected to heat stress had higher MSRA expression compared to chicks maintained in a thermoneutral environment. Our results show that, although maternal heat stress had no negative effects on performance or oxidative metabolism of offspring grown under thermoneutral conditions, it was associated with lower performance and higher protein oxidation in offspring exposed to heat stress during growth. These results could be due in part to alterations in the expression of genes related to antioxidant capacity.

Corneal proteome and differentially expressed corneal proteins in highly myopic chicks using a label-free SWATH-MS quantification approach.

Myopia, or short-sightedness, is a highly prevalent refractive disorder in which the eye's focal length is too short for its axial dimension in its relaxed state. High myopia is associated with increased risks of blinding ocular complications and abnormal eye shape. In addition to consistent findings on posterior segment anomalies in high myopia (e.g., scleral remodeling), more recent biometric and biomechanical data in myopic humans and animal models also indicate anterior segment anomalies (e.g., corneal biomechanical properties). Because the cornea is the anterior-most ocular tissue, providing essential refractive power and physiological stability, it is important to understand the biochemical signaling pathway during myopia development. This study first aimed to establish the entire chicken corneal proteome. Then, using the classical form deprivation paradigm to induce high myopia in chicks, state-of-the-art bioinformatics technologies were applied to identify eight differentially expressed proteins in the highly myopic cornea. These results provide strong foundation for future corneal research, especially those using chicken as an animal model for myopia development.

Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens.

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1-35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.

Methionine supplementing effects on intestine, liver and uterus morphology, and on positivity and expression of Calbindin-D28k and TRPV6 epithelial calcium carriers in laying quail in thermoneutral conditions and under thermal stress.

This study aimed to provide the performance, localization and expression of the epithelial calcium transporter channels Calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quail under heat stress (HS), and with methionine supplementation (MS). This study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels in the kidneys, intestine and uterus of 504 laying quails under different MS (100, 110 and 120%) and temperatures (20, 24, 28 and 32°C). The animals under HS (32°C) had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb, and increased positivity in proximal CTs. Deleterious effects of HS were minimized with MS for: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney (P≤0.05). Epithelial calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the MS, increasing egg production in HS and quality in termoneutrality (P≤0.05). MS further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity of Calb, and increase Calb and TRPV6 expression in the kidney (P≤0.001) under thermoneutrality. It was concluded that the use of MS (120%) is justifiable in order to partially reverse the deleterious effects of HS on the production, in the epithelial calcium carriers, and in the digestory and reproductive morphology of laying quail.

Transcriptional control of chicken KLF7 promoter in preadipocytes.

Krüppel-like factor 7 (KLF7) has been reported to inhibit adipogenesis and regulate the development of the nervous system. However, transcription regulation of KLF7 remains poorly understood. In the current study, a 2196-bp-long 5'-flanking sequence of chicken KLF7 (-2286 bp to -91 bp, upstream of the translation start site) was studied for promoter activity, and there was a remarkable promoter activity in this sequence (P<0.05). The 5'-truncated mutation analysis showed that a minimal promoter was on the sequence from -241 bp to -91 bp. In addition, GATA2 overexpression facilitated the promoter activity of pGL3-KLF7(-2286/-91), pGL3-KLF7(-1215/-91), pGL3-KLF7(-521/-91), and pGL3-KLF7(-241/-91), and GATA3 overexpression inhibited the promoter activity of pGL3-KLF7(-1845/-91), pGL3-KLF7(-1215/-91), pGL3-KLF7(-521/-91), and pGL3-KLF7(-241/-91) in chicken preadipocytes (P<0.05). Knockdown of GATA2 expression inhibited the promoter activity of pGL3-KLF7(-1215/-91) and pGL3-KLF7(-241/-91), and knockdown of GATA3 expression facilitated the promoter activity of pGL3-KLF7(-521/-91) and pGL3-KLF7(-241/-91) (P<0.05). Additionally, overexpression and knockdown analyses showed that GATA3 inhibited KLF7 mRNA expression (P<0.05), and both overexpression and knockdown of GATA2 resulted in the downregulation of KLF7 mRNA expression in chicken preadipocytes (P<0.05). Western blot analysis in chicken preadipocytes showed that GATA2 facilitated KLF7 expression and GATA3 inhibited KLF7 expression. Mutation analysis showed that the motif of 'GGATCTATCA' (-107 bp/-98 bp) might be a cis-regulation element, which is involved in the KLF7 expression regulation by GATA3 in chicken preadipocytes. These results provided some details of KLF7 transcription regulation in chicken adipose tissue.

let-7 miRNAs inhibit CHD7 expression and control auditory-sensory progenitor cell behavior in the developing inner ear.

The evolutionarily conserved lethal-7 (let-7) microRNAs (miRNAs) are well-known activators of proliferative quiescence and terminal differentiation. However, in the murine auditory organ, let-7g overexpression delays the differentiation of mechano-sensory hair cells (HCs). To address whether the role of let-7 in auditory-sensory differentiation is conserved among vertebrates, we manipulated let-7 levels within the chicken auditory organ: the basilar papilla. Using a let-7 sponge construct to sequester let-7 miRNAs, we found that endogenous let-7 miRNAs are essential for limiting the self-renewal of HC progenitor cells. Furthermore, let-7b overexpression experiments revealed that, similar to mice, higher than normal let-7 levels slow/delay HC differentiation. Finally, we identify CHD7, a chromatin remodeler, as a candidate for mediating the repressive function of let-7 in HC differentiation and inner ear morphogenesis. Our analysis uncovered an evolutionarily conserved let-7-5p-binding site within the chicken Chd7 gene and its human and murine homologs, and we show that let-7g overexpression in mice limits CHD7 expression in the developing inner ear, retina and brain. Haploinsufficiency of CHD7 in humans causes CHARGE syndrome and attenuation of let-7 function may be an effective method for treating CHD7 deficiency.

miR-199 family contributes to regulation of sonic hedgehog expression during craniofacial development.

BACKGROUND: The frontonasal ectodermal zone (FEZ) is a signaling center that regulates patterned development of the upper jaw, and Sonic hedgehog (SHH) mediates FEZ activity. Induction of SHH expression in the FEZ results from SHH-dependent signals from the brain and neural crest cells. Given the role of miRNAs in modulating gene expression, we investigated the extent to which miRNAs regulate SHH expression and FEZ signaling. RESULTS: In the FEZ, the miR-199 family appears to be regulated by SHH-dependent signals from the brain; expression of this family increased from HH18 to HH22, and upon activation of SHH signaling in the brain. However, the miR-199 family is more broadly expressed in the mesenchyme of the frontonasal process and adjacent neuroepithelium. Downregulating the miR-199 genes expanded SHH expression in the FEZ, resulting in wider faces, while upregulating miR-199 genes resulted in decreased SHH expression and narrow faces. Hypoxia inducible factor 1 alpha (HIF1A) and mitogen-activated protein kinase kinase kinase 4 (MAP3K4) appear to be potential targets of miR-199b. Reduction of MAP3K4 altered beak development but increased apoptosis, while reducing HIF1A reduced expression of SHH in the FEZ and produced malformations independent of apoptosis. CONCLUSIONS: Our results demonstrate that this miRNA family appears to participate in regulating SHH expression in the FEZ; however, specific molecular mechanisms remain unknown.

Targeted disruption of TORC1 retards young squab growth by inhibiting the synthesis of crop milk protein in breeding pigeon (Columba livia).

This study was conducted to explore the regulatory role of the target of rapamycin complex 1 (TORC1) signaling pathway in crop milk synthesis in breeding pigeons (Columba livia). Three groups of breeding pigeons in the lactation period (n = 30 pairs/group) were respectively injected with rapamycin (RAPA, a specific inhibitor of the target of rapamycin complex) at doses of 0 (vehicle, control), 0.6, or 1.2 mg/kg body weight (BW)/day via the wing vein for 7 days. The average daily feed intake (ADFI) and BW of the breeding pigeons and the BW of young squabs were respectively recorded throughout the experimental period. The breeding pigeons were sacrificed to collect their crop tissues, crop milk, and serum on the eighth day of the experiment. The results showed that neither 0.6 nor 1.2 mg/kg BW RAPA injection affected BW loss or ADFI in breeding pigeons (P > 0.05), while crop thickness and crop relative weight were significantly decreased (P < 0.05) in the 1.2 mg/kg BW rapamycin-injected group. Simultaneously, RAPA (especially at 1.2 mg/kg BW) decreased the crude protein, αs1-casein, αs2-casein, ß-casein, and amino acid contents (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Lys, His, Arg, and Pro) of crop milk (P < 0.05) and the concentrations of albumin, total protein, and uric acid in the serum of breeding pigeons (P < 0.05). Additionally, the expression of TORC1 pathway-related proteins (TORC1, S6K1, S6, 4EBP1, and eIF4E) was downregulated in the crop tissues of breeding pigeons by 0.6 or 1.2 mg/kg BW/day RAPA injection (P < 0.05). Accordingly, the average daily gain (ADG) of young squabs declined, and the mortality rate increased significantly (P < 0.05). Together, the results showed that RAPA reduced protein and amino acid levels in the crop milk of breeding pigeons and retarded young squab growth, suggesting a crucial role of TORC1 in crop milk synthesis in breeding pigeons.

Rapid Generation of Chicken Immune Libraries for Yeast Surface Display.

Fluorescence-activated cell sorting (FACS) in combination with yeast surface display has emerged as a vital tool for the isolation and engineering of antibodies and antibody-derived fragments from synthetic, naïve, and immune libraries. However, the generation of antibodies against certain human antigens from immunized animals, e.g., mice, can remain challenging due to the homology to the murine counterpart. Due to the phylogenetic distance from humans, avian immunization can be a powerful technique for the generation of antibodies with high specificity against human antigens. Additionally, the peculiar Ig gene diversification in chickens enables the amplification of heavy and light chain genes utilizing single primer pairs, resulting in a convenient library generation. Herein, we describe the protocol for the construction of a single chain fragment variable (scFv) library derived from chickens after immunization with epidermal growth factor receptor (EGFR) for subsequent yeast surface display as well as the screening process utilizing FACS for the isolation of high-affinity antibodies.

Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFß pathway in endodermal epithelial cells of Japanese quail.

Nutrients are utilized and re-constructed by endodermal epithelial cells (EECs) of yolk sac membrane (YSM) in avian species during embryonic development. Sterol O-acyltransferase 1 (SOAT1) is the key enzyme to convert cholesterol to cholesteryl ester for delivery to growing embryos. During embryonic development, yolk absorption is concomitant with significant changes of SOAT1 mRNA concentration and enzyme activity in YSM. Presence of microRNAs (miRNAs) are observed in the embryonic liver and muscle during avian embryogenesis. However, the expression of miRNAs in YSM during embryogenesis and the involvement of miRNAs in lipid utilization are not known. Using a miRNA sequencing technique, we found several miRNA candidates and confirmed their expression patterns individually by real time PCR. MiRNA candidates were selected based on the expression pattern and their possible roles in inhibiting transforming growth factor beta receptor type 1 (TGFBR1) that would regulate the function of SOAT1. Similar to SOAT1 mRNA, the gga-miR-181a-5p expression was gradually elevated during embryonic development. However, the expression of gga-miR-429-3p in YSM was gradually decreased during embryonic development. The inhibitory effects of gga-miR-181a-5p or gga-miR-429-3p on the potential targets (SOAT1 and TGFBR1) were demonstrated by transient miRNA transfections in EECs. We also found that mutated TGFBR1 3'UTR prevented the direct pairings of gga-miR-181a-5p and gga-miR-429-3p. Treatment of TGFBR1 inhibitor, LY364947, further decreased SOAT1 transcription. Similar results were also observed by the miRNA transfection studies. The results showed the vital participations of gga-miR-181a-5p and gga-miR-429-3p in regulating TGFß pathway, and affecting downstream SOAT1 expression and function in the YSM. This is indicative of possible regulation of avian yolk lipid utilization by changing YSM miRNA expressions.

In vitro effects of PNP and PNMC on apoptosis and proliferation in the hen ovarian stroma and prehierarchal follicles.

This study aimed to examine the mRNA expression, activity, and immunolocalisation of apoptosis/proliferation regulating factors following in vitro exposure of the stroma, white (WFs), and yellowish (YFs) follicles of the chicken ovary to 4-nitrophenol (PNP) or 3-methyl-4-nitrophenol (PNMC). PNMC increased the mRNA expression of caspase-3, -8, Apaf-1, and cytochrome c in the ovarian stroma. The activity of caspase-3, -8, and -9 decreased in WFs in both nitrophenol-treated groups. PNP reduced the number of caspase-3-positive cells in the stromal connective tissue (CT) and the theca interna and externa layers of WFs. In the stroma, the proliferating index decreased in the wall of primary follicles in both nitrophenol-treated groups, however, in the CT, the effect of PNMC was opposite. In the theca interna of WFs, PNP diminished the proliferating index. These results suggest that nitrophenols might impact the development of chicken ovarian follicles by affecting cell death and proliferation.

Corticosterone-mediated physiological stress modulates hepatic lipid metabolism, metabolite profiles, and systemic responses in chickens.

The impact of physiological stress on lipid metabolism, the metabolome, and systemic responses was examined in chickens. To incite a stress response, birds were continuously administered corticosterone (CORT) in their drinking water at three doses (0 mg/L, 10 mg/L, and 30 mg/L), and they were sampled 1, 5, and 12 days after commencement of CORT administration. Corticosterone administration to birds differentially regulated lipogenesis genes (i.e. FAS, ACC, ME, and SREBF1), and histopathological examination indicated lipid deposition in hepatocytes. In addition, CORT affected water-soluble metabolite profiles in the liver, as well as in kidney tissue and breast muscle; thirteen unique metabolites were distinguished in CORT-treated birds and this was consistent with the dysregulation of lipid metabolism due to physiological stress. Acute phase responses (APRs) were also altered by CORT, and in particular, expression of SAA1 was decreased and expression of CP was increased. Furthermore, CORT administration caused lymphoid depletion in the bursa of Fabricius and elevated IL6 and TGFß2 mRNA expression after 5 and 12 days of CORT administration. Collectively, incitement of physiological stress via administration of CORT in chickens modulated host metabolism and systemic responses, which indicated that energy potentials are diverted from muscle anabolism during periods of stress.

Effect of Bitter Compounds on the Expression of Bitter Taste Receptor T2R7 Downstream Signaling Effectors in cT2R7/pDisplay-Gα16/gust44/pcDNA3.1 (+) Cells.

Bitterness is an important taste sensation for chickens, which provides useful sensory information for acquisition and selection of diet, and warns them against ingestion of potentially harmful and noxious substances in nature. Bitter taste receptors (T2Rs) mediate the recognition of bitter compounds belonging to a family of proteins known as G-protein coupled receptors. The aim of this study was to identify and evaluate the expression of T2R7 in chicken tongue tissue and construct cT2R7-1 and cT2R7-2-expressing HEK-293T cells to access the expression of PLCß2 and ITPR3 after exposure with different concentrations of the bitter compounds. Using real-time PCR, we show that the relative expression level of T2R7 mRNA in 5, 1, 0.1, and 10-3 mM of camphor and erythromycin solutions and 5 mM of chlorpheniramine maleate solutions was significantly higher than that in 50 mM KCL solutions. We confirmed that the bitter taste receptor T2R7 and downstream signaling effectors are sensitive to different concentrations of bitter compounds. Moreover, T2R7-1 (corresponding to the unique haplotype of the Tibetan chicken) had higher sensitivity to bitter compounds compared with that of T2R7-2 (corresponding to the unique haplotype of the Jiuyuan black-chicken). These results provide great significance of taste response on dietary intake to improve chicken feeding efficiency in poultry production and have certain reference value for future taste research in other bird species.

Light at night disrupts diel patterns of cytokine gene expression and endocrine profiles in zebra finch (Taeniopygia guttata).

Increased exposure to light pollution perturbs physiological processes through misalignment of daily rhythms at the cellular and tissue levels. Effects of artificial light-at-night (ALAN) on diel properties of immunity are currently unknown. We therefore tested the effects of ALAN on diel patterns of cytokine gene expression, as well as key hormones involved with the regulation of immunity, in zebra finches (Taeniopygia guttata). Circulating melatonin and corticosterone, and mRNA expression levels of pro- (IL-1ß, IL-6) and anti-inflammatory (IL-10) cytokines were measured at six time points across 24-h day in brain (nidopallium, hippocampus, and hypothalamus) and peripheral tissues (liver, spleen, and fat) of zebra finches exposed to 12 h light:12 h darkness (LD), dim light-at-night (DLAN) or constant bright light (LLbright). Melatonin and corticosterone concentrations were significantly rhythmic under LD, but not under LLbright and DLAN. Genes coding for cytokines showed tissue-specific diurnal rhythms under LD and were lost with exposure to LLbright, except IL-6 in hypothalamus and liver. In comparison to LLbright, effects of DLAN were less adverse with persistence of some diurnal rhythms, albeit with significant waveform alterations. These results underscore the circadian regulation of biosynthesis of immune effectors and imply the susceptibility of daily immune and endocrine patterns to ALAN.

Developmental conditions modulate DNA methylation at the glucocorticoid receptor gene with cascading effects on expression and corticosterone levels in zebra finches.

Developmental conditions can impact the adult phenotype via epigenetic changes that modulate gene expression. In mammals, methylation of the glucocorticoid receptor gene Nr3c1 has been implicated as mediator of long-term effects of developmental conditions, but this evidence is limited to humans and rodents, and few studies have simultaneously tested for associations between DNA methylation, gene expression and phenotype. Adverse environmental conditions during early life (large natal brood size) or adulthood (high foraging costs) exert multiple long-term phenotypic effects in zebra finches, and we here test for effects of these manipulations on DNA methylation and expression of the Nr3c1 gene in blood. Having been reared in a large brood induced higher DNA methylation of the Nr3c1 regulatory region in adulthood, and this effect persisted over years. Nr3c1 expression was negatively correlated with methylation at 2 out of 8 CpG sites, and was lower in hard foraging conditions, despite foraging conditions having no effect on Nr3c1 methylation at our target region. Nr3c1 expression also correlated with glucocorticoid traits: higher expression level was associated with lower plasma baseline corticosterone concentrations and enhanced corticosterone reactivity. Our results suggest that methylation of the Nr3c1 regulatory region can contribute to the mechanisms underlying the emergence of long-term effects of developmental conditions in birds, but in our system current adversity dominated over early life experiences with respect to receptor expression.

Tissue-specific expression profiles and positive selection analysis in the tree swallow (Tachycineta bicolor) using a de novo transcriptome assembly.

Tree swallows (Tachycineta bicolor) are one of the most commonly studied wild birds in North America. They have advanced numerous research areas, including life history, physiology, and organismal responses to global change; however, transcriptomic resources are scarce. To further advance the utility of this system for biologists across disciplines, we generated a transcriptome for the tree swallow using six tissues (brain, blood, ovary, spleen, liver, and muscle) collected from breeding females. We de novo assembled 207,739 transcripts, which we aligned to 14,717 high confidence protein-coding genes. We then characterized each tissue with regard to its unique genes and processes and applied this transcriptome to two fundamental questions in evolutionary biology and endocrinology. First, we analyzed 3,015 single-copy orthologs and identified 46 genes under positive selection in the tree swallow lineage, including those with putative links to adaptations in this species. Second, we analyzed tissue-specific expression patterns of genes involved in sex steroidogenesis and processing. Enzymes capable of synthesizing these behaviorally relevant hormones were largely limited to the ovary, whereas steroid binding genes were found in nearly all other tissues, highlighting the potential for local regulation of sex steroid-mediated traits. These analyses provide new insights into potential sources of phenotypic variation in a free-living female bird and advance our understanding of fundamental questions in evolutionary and organismal biology.

Arih2 gene influences immune response and tissue development in chicken.

Ariadne homolog 2 (ARIH2), as an E3 ubiquitin ligase, is one of the important factors involved in regulating biological functions, such as inflammation and skeletal muscle degeneration. In the present study, the full-length coding sequence of Arih2 gene was cloned from Hy-Line Brown chicken. The tissue transcriptional profiles of Arih2 gene at different developmental stages were detected using quantitative real-time PCR (qRT-PCR), and the Arih2 functional characteristics in immune response were analyzed. The results showed that the full-length coding sequence of Arih2 gene was 1473 bp, encoding 490 amino acids, and conservative between different species. The Arih2 gene was transcribed in various tissues at different developmental stages, and its transcriptional activities varied significantly between multiple tissues. With the development of chicken, Arih2 gene was basically up-regulated in heart, liver, kidney, skeletal muscle and glandular stomach, but fluctuated significantly in large intestine. In immune response, the transcriptional activities of Arih2 gene exhibited significant changes in the bursa, thymus and blood (P<0.05). The results showed that Arih2 might be a multifunctional gene involved in tissue development and immune response in chicken, and have a potential possible application as diagnostic marker for identifying immune response.

Differential regulation of protein synthesis by skeletal muscle type in chickens.

In mammalian skeletal muscles, protein synthesis rates vary according to fiber types. We herein demonstrated differences in the regulatory mechanism underlying the protein synthesis in the pectoralis major (a glycolytic twitch muscle), adductor superficialis (an oxidative twitch muscle), and adductor profound (a tonic muscle) muscles of 14-day-old chickens. Under ad libitum feeding conditions, protein synthesis is significantly higher in the adductor superficialis muscle than in the pectoralis major muscle, suggesting that protein synthesis is upregulated in oxidative muscles in chickens, similar to that in mammals. In the pectoralis major muscle, fasting significantly inhibited the Akt/S6 pathway and protein synthesis with a corresponding decrease in plasma insulin concentration. Conversely, the insulin like growth factor-1 (IGF-1) mRNA levels significantly increased. These findings suggest that the insulin/Akt/S6 pathway plays an important role in the regulation of protein synthesis in the pectoralis major muscle. Interestingly, protein synthesis in the adductor superficialis muscle appears to be regulated in an Akt-independent manner, because fasting significantly decreased S6 phosphorylation and protein synthesis without affecting Akt phosphorylation. In the adductor profound muscle, IGF-1 expression, phosphorylation of Akt and S6, and protein synthesis were decreased by fasting, suggesting that insulin and/or skeletal IGF-1 appear contribute to protein synthesis via the Akt/S6 pathway. These findings revealed the differential regulation of protein synthesis depending on skeletal muscle types in chickens.

Leucine promotes the growth of squabs by increasing crop milk protein synthesis through the TOR signaling pathway in the domestic pigeon (Columba livia).

Leucine (Leu) plays a critical regulatory role in protein synthesis, however, the effects and molecular mechanisms of Leu on crop milk protein in the domestic pigeons (Columba livia) are still unknown. Therefore, the study aimed to investigate the effects of dietary Leu supplementation on crop milk protein synthesis and the growth performance of squabs and the possible underlying mechanism. A total of 240 pairs of breeding pigeons (1102.3 ± 9.5 g/pair) were randomly assigned to 1 of 5 treatments, including a positive control (PC) diet that had adequate crude protein (crude protein, CP = 18%; Leu = 1.30%), a negative control (NC) diet that was low in CP (CP = 16%, Leu = 1.30%), and NC diets supplemented with Leu at 0.15%, 0.45%, or 1.05%. Compared with the NC diet, 0.15 to 0.45% Leu supplementation decreased BW loss and increased relative crop weight, crop thickness, and protein levels in the crop tissue and milk of breeding pigeons. However, dietary supplementation with 1.05% Leu inhibited ADFI in breeding pigeons. Dietary supplementation with 0.15 to 0.45% Leu decreased the mortality rate and increased the BW, eviscerated yield, and breast muscle yield of young squabs. The protein expression levels of the target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1), ribosomal protein S6 kinase (S6), eukaryotic initiation factor 4E binding protein 1 (4EBP1), and eukaryotic translation initiation factor 4E (eIF4E) were upregulated in the crop tissue of breeding pigeons in PC, 0.15% and 0.45% Leu-supplemented groups. Collectively, these results indicated that 0.15 to 0.45% Leu supplementation could decrease BW loss, increase milk protein synthesis in the crop of breeding pigeons, and enhance the survival rate and growth performance of young squabs through the TOR signaling pathway.