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MicroRNAs (miRNAs) are a class of RNA macromolecules that play regulatory roles in follicle development by inhibiting protein translation through binding to the 3'UTR of its target genes. Granulosa cell (GC) proliferation, steroidogenesis, and lipid metabolism have indispensable effect during folliculogenesis. In this study, we found that miR-22-3p was highly expressed in the hierarchical follicles of the chickens, which indicated that it may be involved in follicle development. The results obtained suggested that miR-22-3p promoted proliferation, hormone secretion (progesterone and estrogen), and the content of lipid droplets (LDs) in the chicken primary GC. The results from the bioinformatics analysis, luciferase reporter assay, qRT-PCR, and Western blotting, confirmed that PTEN was directly targeted to miR-22-3p. Subsequently, it was revealed that PTEN inhibited proliferation, hormone secretion, and the content of LDs in GC. Therefore, this study showed that miR-22-3p could activate PI3K/Akt/mTOR pathway via targeting PTEN. Taken together, the findings from this study indicated that miR-22-3p was highly expressed in the hierarchical follicles of chickens, which promotes GC proliferation, steroidogenesis, and lipid metabolism by repressing PTEN to activate PI3K/AKT/mTOR pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

In this study, we examined the effects of Heat Shock Protein 90 ( HSP90 ) on adipocyte proliferation and differentiation in chickens. To achieve this, we constructed RNA interference (RNAi) vectors to target HSP90 and transfected the vectors into primary adipocytes. After transfection, oil red O staining was performed to determine the status of triglyceride accumulation in the cells, whereas the CCK-8 cell kit and 5-Ethynyl-2'-Deoxyuridine (EdU) assays were used to determine cell proliferation. Thereafter, the mRNA and protein expression levels of PPARγ , FAS , SREBP-1c, and HSP90 were determined, and the results showed that after the interference of HSP90 , the mRNA and protein expression levels of HSP90 in the chicken adipocytes decreased significantly compared to the control and blank groups ( p < 0.05 ). The decreased mRNA and protein expression of PPARγ , FAS , and SREBP-1c was related to adipocyte differentiation ( p < 0.05 ). However, HSP90 interference had no effect on adipocyte proliferation ( p > 0.05 ). Taken together, the results of this study showed that HSP90 influenced the expression of PPARγ and adipose-differentiation-related genes, thereby regulating triglyceride accumulation and adipocyte differentiation in chickens.

Background: The eight phenotypically distinguishable indigenous chicken breeds in Guizhou province of China are great resources for high-quality development of the poultry industry in China. However, their full value and potential have yet to be understood in depth. To illustrate the genetic diversity, the relationship and population structure, and the genetic variation patterns shaped by selection in Guizhou indigenous chickens, we performed a genome-wide analysis of 240 chickens from 8 phenotypically and geographically representative Guizhou chicken breeds and 60 chickens from 2 commercial chicken breeds (one broiler and one layer), together with 10 red jungle fowls (RJF) genomes available from previous studies., Results: The results obtained in this present study showed that Guizhou chicken breed populations harbored higher genetic diversity as compared to commercial chicken breeds, however unequal polymorphisms were present within Guizhou indigenous chicken breeds. The results from the population structure analysis markedly reflected the breeding history and the geographical distribution of Guizhou indigenous chickens, whereas, some breeds with complex genetic structure were ungrouped into one cluster. In addition, we confirmed mutual introgression within Guizhou indigenous chicken breeds and from commercial chicken breeds. Furthermore, selective sweep analysis revealed candidate genes which were associated with specific and common phenotypic characteristics evolved rapidly after domestication of Guizhou local chicken breeds and economic traits such as egg production performance, growth performance, and body size., Conclusion: Taken together, the results obtained from the comprehensive analysis of the genetic diversity, genetic relationships and population structures in this study showed that Guizhou indigenous chicken breeds harbor great potential for commercial utilization, however effective conservation measures are currently needed. Additionally, the present study drew a genome-wide selection signature draft for eight Guizhou indigenous chicken breeds and two commercial breeds, as well as established a resource that can be exploited in chicken breeding programs to manipulate the genes associated with desired phenotypes. Therefore, this study will provide an essential genetic basis for further research, conservation, and breeding of Guizhou indigenous chickens., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: The Tibetan chicken has adapted well to high altitudes genetically after its long-term habitation in the plateau. In this study, we analyzed the selection signal of Tibetan black chickens (TBCs) and discovered genes associated with the characteristics of germplasm., Methods: Whole-genome sequencing (WGS) was used to identify the single-nucleotide polymorphism (SNP) markers and genetic structures in the genome of Tibetan black chickens. Further, we performed a comparative population genomics analysis between the genomic data obtained in this present study and the genomic data for five wild red jungle fowls (RJFs) accessed from the NCBI database (GenBank accession number PRJNA241474). Thereafter, the Fst and Pi selections were used to identify genes under positive selection in the Tibetan black chicken genome., Results: A total of 9,490,690 SNPs were identified in the Tibetan black chickens. In addition, the results from the gene ontology (GO) analysis showed that 732 genes of TBCs were enriched in a total of 210 GO terms with specific molecular functions such as regulation of cellular catabolic process, the MAPK signaling pathway, regulation of ion transport, growth, morphogenesis and lung alveolus development which may provide a better mechanism to facilitate oxygen transport and utilization in TBCs. Moreover, the results from the KEGG analysis showed that 732 genes of the TBCs were significantly enriched in the calcium signaling pathway, circadian entrainment ( ADCY1 , GNG7 and PER3 ), oxytocin signaling pathway and pathways of multiple neurodegeneration diseases. In addition, the CD86 antigen ( CD86 ) was identified as a gene associated with the immune response in chickens. It was also revealed that genes such as TRIT1 , HPCAL4 , NT5C1A and HEYL were discovered under selection in Tibetan black chickens on chromosome 23. These genes may be related to the local adaptive characteristics of Tibetan black chickens, for instance, NT5C1A and HEYL may be involved in the high-altitude adaption of oxygen delivery in Tibetan black chickens., Conclusions: In summary, we found that selection mainly affects the disease resistance and cold acclimatization of Tibetan black chickens. Hence, these results may provide important genetic information for the evolution and breeding of Tibetan black chickens.

MicroRNAs (miRNAs) are class of 22 nt short RNA sequences which inhibit protein translation through binding to the 3'UTR of its target genes. The continuous ovulatory property of chicken follicle makes it a perfect model for studying granulosa cell (GC) functions. In this study, we found that large number of miRNAs including miR-128-3p, were differentially expressed in the GCs of F1 and F5 follicles of chicken. Subsequently, the results revealed that miR-128-3p inhibited proliferation, the formation of lipid droplets, and hormone secretion in chicken primary GCs through directly targeting YWHAB and PPAR-γ genes. To determine the effects of 14-3-3β (encoded by YWHAB) protein on GCs functions, we overexpressed or inhibited the expression of YWHAB, and the results showed that YWHAB inhibited the function of FoxO proteins. Collectively, we found that miR-128-3p was highly expressed in the chicken F1 follicles compared to the F5 follicles. In addition, the results indicated that miR-128-3p promoted GC apoptosis through 14-3-3β/FoxO pathway via repressing YWHAB, and inhibited lipid synthesis by impeding the PPAR-γ/LPL pathway, as well as reduced the secretion of progesterone and estrogen. Taken together, the results showed that miR-128-3p plays a regulatory role in chicken granulosa cell function via 14-3-3β/FoxO and PPAR-γ/LPL signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The Mountainous Meihua chicken is a unique regional germplasm resource from Tongjiang County, Bazhong City, China, but its genetic structure and evolutionary relationships with other native chicken breeds in the Sichuan region remain unclear. Here, we analyzed a total of 469 sequences, including 199 Mountainous Meihua chicken sequences generated in this study, together with 30 sequences representing 13 clades and 240 sequences from seven different Sichuan local chicken breeds downloaded from NCBI. These sequences were further used to analyze genetic diversity, patterns of population differentiation, and phylogenetic relationships between groups. We show that Mountainous Meihua chicken mtDNA sequences have high haplotypic and nucleotide diversity (0.876 and 0.012, respectively) and with a T bias that is suggestive of good breeding potential. Phylogenetic analysis showed that Mountainous Meihua chickens belong to clades A, B, E, and G and have a low affinity to other chicken breeds, with a moderate degree of differentiation. A non-significant Tajima's D indicates that no demographic expansions occurred in the past. Finally, the four maternal lineages identified in Mountainous Meihua chicken showed unique genetic characteristics.

Aged male chickens experience rapid declines in spermatogenesis, antioxidant capacity, immunity, and hormone synthesis. Vitamin E plays a significant role in reproduction, nervous system function, and disease resistance in animals. Quercetin also exerts many biological effects, such as antioxidant ability, immunostimulation, and protection of spermatozoal plasma membranes. This study evaluated the effects of combining dietary quercetin (Q) and vitamin E (VE) on sperm quality, antioxidant capacity, immunity, and expression of genes related to spermatogenesis, immunity, apoptosis, and inflammation in aged male chickens. A total of 120 Tianfu breeder male chickens (65 wk old) were randomly allotted to 4 treatments with 3 replicates (10 birds each). The birds were fed diets containing Q (0.4g/kg), VE (0.2g/kg), Q+VE (0.4g/kg + 0.2g/kg), and a basal diet for 11 wk. At the end of the experimental period, blood, semen, liver, testes, and spleen samples were collected from 2 birds per replicate. Serum hormones, antioxidant parameters, cytokines, and immunoglobulins were evaluated; and the mRNA expression of genes related to spermatogenesis, apoptosis, and inflammation are determined in the testes and liver tissues. The results showed that the combination quercetin and vitamin E significantly promoted the sperm count and motility, as well as elevated the levels of testosterone, follicle-stimulating hormone, and luteinizing hormone, antioxidant enzymes (Superoxide dismutase, Glutathione, and Total antioxidant capacity), and serum immunoglobulins (IgA and IgM) in the aged male chickens; also Q+VE showed protective effects on the liver against injury. In addition, Q+VE significantly increased the expression of genes related to spermatogenesis (AR, pgk2, Cyclin A1, and Cyclin A2), immunity (IFN-γ and IL-2), and anti-inflammatory cytokines (IL-10) (P < 0.05), whereas the expression of proinflammatory cytokines (IL-1β and IL-6) was decreased (P < 0.05). Taken together, these data indicate that the combination of quercetin and vitamin E improved reproductive characteristics such as spermatogenesis, sperm quality, and hormone regulation, as well as promoted antioxidant defense, hepatoprotective capacity, and immune response in aged male chickens without any detrimental effects., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In aged animals, the physiological functions of the gastrointestinal tract (GIT) are reduced. Dietary intervention is necessary to re-activate GIT functions. The objective of this study was to investigate the impacts of dietary combination of quercetin (Q) and vitamin E (VE) on the intestinal structure and barrier integrity in aged breeder chickens. A sum of 400 (65-wks-old) Tianfu breeder hens were randomly allotted into four (4) groups with four (4) replicates, and fed with basal diet; basal diet supplemented with 0.4g/kg of Q; basal diet supplemented with 0.2g/kg of VE; and basal diet supplemented with the combination of Q (0.4 g/kg) and VE (0.2 g/kg) for 14 weeks. At the end of the 14 th week, serum and gut segments were collected from eight hens per group for analyses. The results showed that Q+VE exerted synergistic effects on intestinal morphology by promoting villi height and crypt depth (P < 0.05), as well as mitigated the intestinal inflammatory damage of the aged hens, but decreased the concentration of serum D-lactate and diamine oxidase; and increased the levels of secretory immunoglobulin A (sIgA) and Mucin-2 mRNA (P < 0.05). Furthermore, the mRNA expression of intestinal tight junction proteins including occludin, ZO1 , and claudin-1 was increased by Q+VE (P < 0.05). Moreover, Q+VE decreased the mRNA expression of the pro-inflammatory genes ( TNF-α, IL-6 , and IL-1β ), and increased the expression of anti-inflammatory genes ( IL-10 and IL-4 ) (P < 0.05). These results were consistent with the mRNA expression of Bax and Bcl-2 . In addition, Q+VE protected the small intestinal tract from oxidative damage by increasing the levels of superoxide dismutase, total antioxidant capacity, glutathione peroxidase, catalase (P < 0.05), and the mRNA expression of SOD1 and GPx-2. However, Q+VE decreased malondialdehyde levels in the intestine compared to the control (P < 0.05). These results indicated that dietary Q+VE improved intestinal function in aged breeder hens, by protecting the intestinal structure and integrity. Therefore, Q+VE could act as an anti-aging agent to elevate the physiological functions of the small intestine in chickens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Amevor, Cui, Du, Ning, Deng, Xu, Shu, Wu, Cao, Shuo, Tian, Li, Wang, Zhang, Du, Zhu, Han and Zhao.)

Folliculogenesis is a complex biological process involving a central oocyte and its surrounding somatic cells. Three-dimensional chromatin architecture is an important transcription regulator; however, little is known about its dynamics and role in transcriptional regulation of granulosa cells during chicken folliculogenesis. We investigate the transcriptomic dynamics of chicken granulosa cells over ten follicular stages and assess the chromatin architecture dynamics and how it influences gene expression in granulosa cells at three key stages: the prehierarchical small white follicles, the first largest preovulatory follicles, and the postovulatory follicles. Our results demonstrate the consistency between the global reprogramming of chromatin architecture and the transcriptomic divergence during folliculogenesis, providing ample evidence for compartmentalization rearrangement, variable organization of topologically associating domains, and rewiring of the long-range interaction between promoter and enhancers. These results provide key insights into avian reproductive biology and provide a foundational dataset for the future in-depth functional characterization of granulosa cells., (© 2022. The Author(s).)

Circadian timing system controlled the rhythmic events, for example, ovulation and oviposition in chickens. However, how biological clock mediates eggshell formation remains obscure. Here, A 24-h mRNA transcriptome analysis was carried out in the uterus of 18 chickens with similar oviposition time points to identify the rhythmic genes and to reveal critical genes and biological pathways involved in the eggshell biomineralization. JTK&#95;CYCLE analysis and real-time PCR revealed a total of 1,793 genes from the sequencing database with 23,513 genes (FPKM>1) were rhythmic genes regulating the rhythmic system and the expression of typical clock genes Per2, Cry1, Bmal1, Clock, Per3, and Rev-erbβ were rhythmically expressed, which suggested that endogenous clock in uterus might control the eggshell mineralization. Time of peak expression of the rhythmic genes was analyzed based on their acrophase. The main phases clustered at the periods from Zeitgeber time 0 (ZT0) to ZT4 (6:00-10:00) and from ZT10 to ZT14 (16:00-20:00). The rhythmic genes were annotated to the following Gene Ontology terms rhythmic process, lyase, ATP binding, cell membrane component. KEGG pathway enrichment analysis revealed the top 15 rhythmic genes were involved in vital biological pathways, including syndecan (1, 2, 3)-mediated signaling, post-translational regulation of adheres junction stability and disassembly, FoxO family signaling, TGF-β receptor and transport of small molecular pathways. 166 of total 1,235 genes (13.4%) were defined as rhythmic transfer factors (TFs) and they were investigated expression time distribution of cis-elements of circadian clock system D-box, E-box, B-site, and Y-Box within 24 h. Results indicated that rhythmic TFs at each phase are potential drivers of their circadian transcription activities. Compared with the control, the expression abundances of ion transport elements SCNN1G, CA2, SPP1, and ATP1B1 were significantly decreased after the interference of Bmal1 gene in synchronized uterine tubular gland cells. Clock genes changed their expression along with the eggshell formation, indicating that there is circadian clock in the uterus of chicken and it regulates the expression of eggshell formation genes., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: miRNAs regulate circadian patterns by modulating the biological clocks of animals. In our previous study, we found that the clock gene exhibited a cosine expression pattern in the fallopian tube of chicken uterus. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in the chicken uterus and, if so, how they regulate egg-laying rhythms is unclear. In this study, we selected 18 layer hens with similar ovipositional rhythmicity (each of three birds were sacrificed for study per 4 h throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens., Results: We identified six circadian miRNAs that are mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of the experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in the chicken uterus. Ca 2+ -transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca 2+ transportation during eggshell calcification in the chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in the chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. The expression of miR-449c-5p showed an opposite trend to that of ATP2B4 within a 24 h cycle in the chicken uterus; it inhibited mRNA and protein expression of ATP2B4 in the uterine tubular gland cells. In addition, overexpression of ATP2B4 significantly decreased intracellular Ca 2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca 2+ concentrations. We found similar results after ATP2B4 knockdown by miR-449c-5p. Taken together, these results indicate that ATP2B4 promotes uterine Ca 2+ trans-epithelial transport., Conclusions: Clock-controlled miR-449c-5p regulates Ca 2+ transport in the chicken uterus by targeting ATP2B4 during eggshell calcification., (© 2021. The Author(s).)

Background: The microbiota of the female reproductive tract is increasingly recognized as playing fundamental roles in animal reproduction. To explore the relative contribution of reproductive tract microbiomes to egg production in chickens, we investigated the microbiota in multiple reproductive and digestive tract sites from 128 female layer (egg-producing) chickens in comparable environments., Results: We identified substantial differences between the diversity, composition, and predicted function of site-associated microbiota. Differences in reproductive tract microbiota were more strongly associated with egg production than those in the digestive tract. We identified 4 reproductive tract microbial species, Bacteroides fragilis, Bacteroides salanitronis, Bacteroides barnesiae, and Clostridium leptum, that were related to immune function and potentially contribute to enhanced egg production., Conclusions: These findings provide insights into the diverse microbiota characteristics of reproductive and digestive tracts and may help in designing strategies for controlling and manipulating chicken reproductive tract microbiota to improve egg production., (© The Author(s) 2021. Published by Oxford University Press GigaScience.)

The objective of this study was to compare the testicular development, semen quality, and spermatogenic cell apoptosis of roosters reared in colony, single, and large cages. Rohman parental layers (n = 540) were randomly allocated into cages of rearing system groups (135 males and 405 females). The experimental period was 70 to 210 d of age. We compared testicular development and plasma main reproductive hormones (Follicle-stimulating hormone; Luteinizing hormone; Testosterone; Estrogen2;) from d 70 to 210 of roosters among the three systems. In addition, routine semen quality indexes, apoptosis of testicular spermatogenic cells and sperm apoptosis of breeding roosters under three rearing systems on d 175 and d 210 were evaluated. Roosters during the growing period (from d 70 to 140) have rapid testis growth and increasing main reproductive hormones in plasma. At the peak of sexual maturity (d 210), in colony cage, the females have a positive effect and promote the testis development of males. However, the stocking density in colony cage has no effect on testicular development; compared with the single and large cage. Roosters reared in the natural mating system had better semen quality, particularly in semen volume, density, and viability; the hatching % of fertilized eggs and healthy chicks were higher for the colony than single and large cages. Furthermore, the sperm density was higher for colony than single and large cages, which was related to the apoptosis of spermatogonia and spermatocyte, not the apoptosis of mature sperm. This study provided the basic data for the reproductive performance research of chicken reared in the colony cages., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In order to breed new birds with strong disease resistance, it is necessary to first understand the mechanism of avian antiviral response. Interferon regulatory factor 7 (IRF7) is not only a member of type I interferons (IFNs) regulatory factor (IRFs) family, but also a major regulator of the IFN response in mammals. However, whether IRF7 is involved in the host innate immune response remains unclear in poultry, due to the absence of IRF3. Here, we first observed by HE stains that with the increase of the time of ALV-J challenge, the thymus was obviously loose and swollen, the arrangement of liver cell was disordered, and the bursa of fabricius formed vacuolated. Real-time PCR detection showed that the expression level of IRF7 gene and related immune genes in ALV-J group was significantly higher than that in control group (P < 0.05). To further study the role of chicken IRF7 during avian leukosis virus subgroup J (ALV-J) infection, we constructed an induced IRF7 overexpression and interfered chicken embryo fibroblasts (CEFs) cell and performed in vitro infection using low pathogenic ALV-J and virus analog poly(I:C). In ALV-J and poly(I:C) stimulated CEFs cells, the expression level of STAT1, IFN-α, IFN-β, TLR3 and TLR7 were increased after IRF7 overexpressed, while the results were just the opposite after IRF7 interfered, which indicating that IRF7 may be associated with Toll-like receptor signaling pathway and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 is an important regulator of IFN and is involved in chicken anti-ALV-J innate immunity., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The purpose of the study was to evaluate the protective effects of polygonatum sibiricum polysaccharide (PP), an important component of rhizome polygonatum, on cyclophosphamide (CY) induced immunosuppressed chickens. Four hundred and eighty one-day-old Erlang mountainous chickens were randomly allocated into four treatments. The main factors consisted of dietary supplement (PP at 0 or 800 mg/kg of diet) and immunosuppressive challenge (birds challenged with CY or treated with sterile saline). The results showed that PP enhanced chickens' growth performance via elevating daily weight gain (DWG), serum protein production, and decreasing feed conversion ratio (FCR). Moreover, physical measurements revealed that PP accelerated recovery of relative weights of immune organs and maintained their structure and function. Biochemical analysis indicated that PP significantly stimulated immunoglobulin and antioxidant indexes in serum, and improved the proliferation of peripheral blood T lymphocytes. In addition, PP promoted immune organs cells to enter into S and G 2 /M phases as well as inhibited the apoptosis in the spleen, thymus, and bursa of Fabricius. PP up regulated the expression of IL-2, IL-6 and IFN-γ genes. Therefore, PP performs a profile in antagonizing Cy-induced immunosuppression in chickens, and it seems that PP can be used as a potential immunostimulant agent., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ammonia (NH 3 ) at a high concentration has been recognized as a highly poisonous pollutant affecting both air and water quality. NH 3 , as a stimulus, exerts negative impact on broiler growth and production, but the molecular mechanisms are not clear yet. This study was designed to evaluate the effects of dietary supplementation of Bupleurum falcatum L saikosaponins (SP) on the growth and ileum health status in broilers exposed to NH 3 . Day-old Arbor Acers broilers (n = 480) were randomly allocated into 1 of 4 treatments. The main factors were dietary SP supplementation (0 or 80 mg/kg of diet) and NH 3 challenge (with or without 70 ± 5 ppm NH 3 ). The data of growth, intestinal morphology, and mRNA expression related to ileal function were collected from broilers exposed to NH 3 for 7 d. Results showed that NH 3 remarkably suppressed growth performance and intestinal development as well as induced biological injuries in the ileum of broilers, resulting from oxidative stress, mucous barrier damage, and immune dysfunction as well as upregulated apoptosis. These negative effects of NH 3 were alleviated by the SP supplement. In conclusion, dietary supplementation of SP may be helpful in alleviating the detrimental effects of NH 3 on the ileum development in broilers., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian leucosis (AL) is a disease characterized by tumors and is caused by the avian leukosis virus (ALV). Because of the high variability of viruses and complex pathogenic mechanisms, screening and breeding J subgroup of ALV (ALV-J) resistant avian breeds is one of the strategies for prevention and treatment of AL, thus screening of significant immune markers is needed to promote the development of disease-resistant breeds. In this study, data-independent acquisition (DIA) technology was used to detect the DEPs of three breeds of chicken according to different comparison to investigate the potential markers. Results showed special DEPs for spleen development of each breed were detected, such as PCNT, DDB2, and ZNF62. These DEPs were involved in intestinal immune network used in production of IgA signaling pathways and related to immune response which can be used as potential markers for spleen development in different breeds. The DEPs such as RAB44 and TPN involved in viral myocarditis, transcriptional misregulation in cancer, and tuberculosis can be used as potential markers of spleen immune response after ALV-J infection in chickens. Pair-wise analysis was performed for the three breeds after the infection of ALV-J. The proteins such as RFX1, TAF10, and VH1 were differently expressed between three breeds. These DEPs involved in antigen processing and expression, acute myelogenous leukemia, and viral carcinogenesis can be used as potential immune markers after ALV-J infection of different genetic backgrounds. The screening of potential markers at protein level provides a strong theoretical research basis for disease resistance breeding in poultry.

It has been shown that bamboo leaf flavone (BLF) displays biological and pharmacological activities in mammals. However, the effects of BLF on broiler gut microbiota and related immune function have not been investigated. The aim of this study was to test our hypothesis that BLF can improve the health status of broilers by modulating the gut microbiota. A total of 300 one-day-old Arbor Acres (AA) broilers were used to characterize their gut microbiota and immune status after feeding diet supplemented with BLF. The V4 hypervariable region of the 16S rRNA gene from cecal bacteria was sequenced via the Illumina MiSeq platform. The Immune status and related parameters were assessed, including the immune organ index (the spleen, thymus, and bursa), serum concentrations of IL-2 and INF-γ, and spleen IL-2 and INF-γ gene expressions. The results showed the BLF diet had an Immune enhancement effect on broilers. In addition, BFL caused the changes of the gut microbial community structure, resulting in greater proportions of bacterial taxa belonging to Lactobacillus, Clostridiales, Ruminococcus, and Lachnospiraceae. These bacteria have been used as probiotics for producing short chain fatty acids in hosts. These results indicate that BLF supplement improves immune function in chicken via modulation of the gut microbiota.

Inhibin α ( INHA ) is a candidate gene controlling ovulation in poultry. As the functional center of inhibin, INHA is a molecular marker associated with egg-laying performance. The objective of the current study was to analyze the expression differences of INHA in reproductive system and single nucleotide polymorphisms (SNPs) associations with reproductive traits in chickens. A total of 260 LuHua chickens (barred-feather chicken) were adopted. Twelve SNPs were detected in INHA gene. Among the exonic SNPs, three (g. 22177991A>G, g. 22178249G>C, and g. 22178414G>A) were missense mutations, resulting in the amino acid substitutions Val→Ala, Ala→Gly, and Ala→Gly, respectively. Four SNPs in the 3＇ untranslated region of INHA were predicted to either disturb or create microRNA-target interactions. Five SNPs (g. 22176870T>C, g. 22177100T>C, g. 22177149T>C, g. 22177991A>G, and g. 22178975G>A) were significantly associated with the number of eggs at 300 d of age (EN) ( P < 0.05). Birds carrying GA genotype exhibited more EN than those with AA genotype ( P < 0.01). In addition, quantitative real-time PCR revealed that INHA is mainly expressed in follicles on d 300 in chickens. Firstly, INHA expression increased and then decreased. The highest INHA mRNA abundance was found in the fifth largest preovulatory follicle (F5) ( P < 0.01). In the prehierarchical follicles, INHA mRNA expression increased dramatically in small yellow follicles (SYF) ( P < 0.01). Western blotting analysis showed that the INHA protein expression profile in the follicle was similar to its mRNA counterpart with greater expression in F5 and SYF follicles and lowest expression in F1 follicles ( P < 0.05). These results suggest that INHA is a potential candidate gene improving reproductive traits in chickens., Competing Interests: The authors declared no conflicts of interest.

Background: There are hundreds of phenotypically distinguishable domestic chicken breeds or lines with highly specialized traits worldwide, which provide a unique opportunity to illustrate how selection shapes patterns of genetic variation. There are many local chicken breeds in China., Results: Here, we provide a population genome landscape of genetic variations in 86 domestic chickens representing 10 phenotypically diverse breeds. Genome-wide analysis indicated that sex chromosomes have less genetic diversity and are under stronger selection than autosomes during domestication and local adaptation. We found an evidence of admixture between Tibetan chickens and other domestic population. We further identified strong signatures of selection affecting genomic regions that harbor genes underlying economic traits (typically related to feathers, skin color, growth, reproduction and aggressiveness) and local adaptation (to high altitude). By comparing the genomes of the Tibetan and lowland fowls, we identified genes associated with high-altitude adaptation in Tibetan chickens were mainly involved in energy metabolism, body size maintenance and available food sources., Conclusions: The work provides crucial insights into the distinct evolutionary scenarios occurring under artificial selection for agricultural production and under natural selection for success at high altitudes in chicken. Several genes were identified as candidates for chicken economic traits and other phenotypic traits.

The aim of the study was to investigate GDF9 gene polymorphisms and their association with reproductive traits in chicken using DNA sequencing. A total of 279 Dongxiang blue-shelled (DX) chickens and 232 Luhua (LH) chickens were used for validation. We detected 15 single nucleotide polymorphisms (SNPs): nine SNPs were previously unreported in chicken, two were missense mutations, and only three exhibited significant associations with reproductive traits. G.17156387C>T was significantly associated with age at first egg (AFE) and weight of first egg (WFE) in both breeds. Birds carrying the CC genotype exhibited higher AFE and WFE values than those with the TT genotype. The SNP g.17156427A>G exhibited an association with egg weight at 300 days of age (EWTA) in DX but not in LH chickens. The SNP g.17156703A>C affected the AFE and EN (total number of eggs at 300 days of age) in DX chickens. In addition, certain diplotypes significantly affected AFE, BWTA (body weight at 300 days of age), and EN in both breeds. RT-PCR results showed that the GDF9 gene was highly expressed in stroma with cortical follicles (STR) and prehierarchal follicles. These results provided further evidence that the GDF9 gene is involved in determining reproductive traits in chicken.

Peroxisome proliferator activated receptor-alpha (PPARα) and Egl nine homolog 3 (EGLN3) play critical roles in facilitating the adaptation to a hypoxic environment. However, the relationship between EGLN3 and PPARα variants and hypoxic adaptation remains poorly understood in Tibetan chickens. To better understand the effects of genetic variation, we sequenced exons of PPARα and EGLN3 in 138 Lowland chickens (LC) from 7 breeds that were located in Emei, Miyi, Shimian, Wanyuan, Pengxian, and Muchuan in the Sichuan province, and Wenchang in the Hainan province (altitudes for these locations are below 1800 meters). Total 166 Tibetan chickens (TC) from 7 subpopulations that were located in Shigatse, Lhoka, Lhasa, Garze, Aba, Diqing and Yushu in the Tibetan area were also sequenced (altitudes greater than 2700 meters). One single-nucleotide polymorphism (rs316017491, C > T) was identified in EGLN3 and was shared by TC and LC with no significant difference for allele frequencies between them (P > 0.05). Six single-nucleotide polymorphisms (SNP1, A29410G; SNP2, rs13886097; SNP3, T29467C; SNP4, rs735915170; SNP5, rs736599044; and SNP6, rs740077421) including one non-synonymous mutation (SNP2, T > C) were identified in PPARα. This is the first report of SNP1 and SNP3. There was a difference between TC and LC for allele frequencies (P <0.01), except for SNP1, SNP4, and SNP5) The fix index statistic test indicated that there was population differentiation between TC and LC for SNP2, SNP3, and SNP6 in PPARα (P < 0.05). Phylogenetic analysis showed that the genetic distance among chickens, finch and great tit were close for both EGLN3 and PPARα. Bioinformatics analysis of PPARα showed that SNP2 leads to an amino acid substitution of Ile for Met, which results in the protein being more likely to be hydrolyzed. Thus, genetic variation in PPARα may play a role in the ability of TC to adapt to a high altitude environment; however we were unable to identify a relationship between polymorphisms in EGLN3 and environmental adaptability.

GPR133 (G protein-coupled receptor 133) plays significant roles in various physiological processes. Alternatively splicing (AS) variants of GPR133 in many species have been predicted in multiple databases, but there is no available information about the AS events of chicken GPR133 (cGPR133). In the present study, two chicken GPR133 variants, cGPR133-va and cGPR133-vb, were identified by a combination of reverse transcription PCR (RT-PCR) and rapid amplification of cDNA 5'-ends (5' RACE). Sequence analysis shows that cGPR133-va and cGPR133-vb are resulting from different AS modules and their sequences are predicted to encode two distinct putative proteins, respectively. Quantitative real-time PCR (qRT-PCR) analysis revealed that cGPR133-va and cGPR133-vb are widely expressed in different tissues, while exhibiting specific expression profile. Altogether, our results first demonstrate the existence of novel cGPR133 variants and illustrate its transcriptional diversity and their widespread distribution, which provides a foundation for the further research of GPR133.

The circadian clock is reported to play a role in the ovaries in a variety of vertebrate species, including the domestic hen. However, the ovary is an organ that changes daily, and the laying hen maintains a strict follicular hierarchy. The aim of this study was to examine the spatial-temporal expression of several known canonical clock genes in the granulosa and theca layers of six hierarchy follicles. We demonstrated that the granulosa cells (GCs) of the F1-F3 follicles harbored intrinsic oscillatory mechanisms in vivo. In addition, cultured granulosa cells (GCs) from F1 follicles exposed to luteinizing hormone (LH) synchronization displayed Per2 mRNA oscillations, whereas, the less mature GCs (F5 plus F6) displayed no circadian change in Per2 mRNA levels. Cultures containing follicle-stimulating hormone (FSH) combined with LH expressed levels of Per2 mRNA that were 2.5-fold higher than those in cultures with LH or FSH alone. These results show that there is spatial specificity in the localization of clock cells in hen preovulatory follicles. In addition, our results support the hypothesis that gonadotropins provide a cue for the development of the functional cellular clock in immature GCs.

Background: Since the domestication of the red jungle fowls ( Gallus gallus ; dating back to ∼10 000 B.P.) in Asia, domestic chickens ( Gallus gallus domesticus ) have been subjected to the combined effects of natural selection and human-driven artificial selection; this has resulted in marked phenotypic diversity in a number of traits, including behavior, body composition, egg production, and skin color. Population genomic variations through diversifying selection have not been fully investigated. The whole genomes of 78 domestic chickens were sequenced to an average of 18-fold coverage for each bird. By combining this data with publicly available genomes of five wild red jungle fowls and eight Xishuangbanna game fowls, we conducted a comprehensive comparative genomics analysis of 91 chickens from 17 populations. After aligning ∼21.30 gigabases (Gb) of high-quality data from each individual to the reference chicken genome, we identified ∼6.44 million (M) single nucleotide polymorphisms (SNPs) for each population. These SNPs included 1.10 M novel SNPs in 17 populations that were absent in the current chicken dbSNP (Build 145) entries. The current data is important for population genetics and further studies in chickens and will serve as a valuable resource for investigating diversifying selection and candidate genes for selective breeding in chickens., (© The Authors 2017. Published by Oxford University Press.)

The study was conducted to optimize lighting schedule for pre-pubertal (12 to 22 weeks) Chinese native breed Pengxian yellow pullet. A total of 414 healthy pullets (10 weeks), with similar body weight were randomly distributed into three groups (n = 138) and housed in individual cages for up to 12 weeks of age in light controlled rooms and provided normal lighting schedule (10L:14D). At 12 to 18 weeks of age, pullets were housed in three rooms, having varying lighting schedule viz. G1 (8L: 16D), G2 (10L:14D), or G3 (12L:12D). From 19th week onwards lighting schedule was gradually increased every week in incremental manner till all groups started receiving 16L:8D lighting schedule. The age at first egg, weight of first egg laid, percent peak hen day egg production, concentration of plasma luteinizing and follicle-stimulating hormones and expression of genes regulating synthesis or/and secretion of hypothalamic gonadotropin-releasing hormone-I (GnRH-I), and pituitary LH-β and FSH-β were studied during experimental period (12 to 43 weeks of age) of this study. The result indicated that pullets of long day length (G3) group had higher plasma levels of FSH and LH and also better mRNA expression that regulates synthesis or/and secretion of GnRH-I, FSH-β, and LH-β before egg laying. The age at first egg (151.3 days) in pullets of G3 group receiving longer lighting hours (12L:12D) was 8.8 days less (P<0.05) compared to pullets of G1 group, while it was 6.9 days less (P>0.05) compared to G2. However, significantly higher (P<0.05) plasma levels of LH and FSH in pullets of G1 as compared to pullets belonging to G3 group corresponded with the higher (P<0.05) cumulative egg production during the experimental period, while these attributes in G2 group didn't differ from either G1 or G3 groups. Pullets of G1 group had significantly higher levels (P<0.05) of GnRH-I, FSH-β, and LH-β mRNA abundances at 43 weeks of age than other two groups and this corresponded with the percent (hen day) peak egg production (75.38%) in pullets in this G1 group that was attained at 32 weeks of age, while the peak production of 71.24% was attained at 30 weeks of age in G3 group. There was no effect of lighting schedule on body weight of pullets, recorded during experimental period, at all occasions; belonging to three groups (G1,G2 and G3) and receiving varying hours of photo-stimulation (P>0.05). It was inferred that the optimum lighting schedule for Chinese native breed Pengxian yellow pullets during 10 weeks of pre-pubertal growth period is short hours of photo-stimulation (i.e 8L:16D).

TRAF family member-associated NF-κB activator (TANK)-binding kinase1 (TBK1) is a serine-threonine kinase at the crossroads of multiple interferon (IFN)-inducing signaling pathways in innate immunity. The importance of TBK1 in antiviral immunity is well established in mammal models, but in chicken, the molecular characterization and potential function of TBK1 remain unclear. In the present study, the open-reading frame (ORF) of chicken TBK1 (chTBK1) was cloned and characterized. The sequencing results revealed that the chTBK1 ORF consists of 2190 base pairs (bp) encoding a deduced protein of 729 amino acid residues. Multiple sequence alignment analysis demonstrated chTBK1 similarity to other birds and mammals, which indicates that it is evolutionarily conserved. Quantitative real-time PCR (qRT-PCR) results showed that chTBK1 was ubiquitously expressed in chicken tissues and expression was especially high in immune tissues. In addition, the expression of chTBK1 was significantly up-regulated by infection with avian leukosis virus subgroup J (ALV-J) both in vivo and in chicken embryo fibroblasts (CEFs) challenged with ALV-J or stimulated with poly I:C in vitro. Consistent with the activation of chTBK1, the interferon regulatory factor 3 (IRF3) and IFNβ gene in CEFs were also up-regulated after challenge with ALV-J or polyI:C. In contrast, the expression of IRF3 and IFNβ in CEFs was significantly reduced by siRNA targeting the chTBK1 gene compared with a negative control (NC) during ALV-J infection or polyI:C transfection. In conclusion, our results demonstrated that chTBK1 may be an important immunoregulator for IRF3 and IFNβ induction in response to viral stimulation in chicken.

Avian leukosis virus (ALV) is detrimental to poultry health and causes substantial economic losses from mortality and decreased performance. Because tumorigenesis is a complex mechanism, the regulatory architecture of the immune system is likely to include the added dimensions of modulation by miRNAs and long-noncoding RNA (lncRNA). To characterize the response to ALV challenge, we developed a novel methodology that combines four datasets: mRNA expression and the associated regulatory factors of miRNA and lncRNA, and ALV gene expression. Specific Pathogen-Free (SPF) layer chickens were infected with ALV-J or maintained as non-injected controls. Spleen samples were collected at 40 days post injection (dpi), and sequenced. There were 864 genes, 7 miRNAs and 17 lncRNAs differentially expressed between infected and non-infected birds. The combined analysis of the 4 RNA expression datasets revealed that ALV infection is detected by pattern-recognition receptors (TLR9 and TLR3) leading to a type-I IFN mediated innate immune response that is modulated by IRF7 and IRF1. Co-expression network analysis of mRNA with miRNA, lncRNA and virus genes identified key elements within the complex networks utilized during ALV response. The integration of information from the host transcriptomic, epigenetic and virus response also has the potential to provide deeper insights into other host-pathogen interactions.

Chicken is the most common poultry species and is important to human societies. Tibetan chicken (Gallus gallus domesticus) is a breed endemic to China that is distributed mainly on the Qinghai-Tibet Plateau. However, its origin has not been well characterized. In the present study, we sequenced partial mitochondrial DNA (mtDNA) control region of 239 and 283 samples from Tibetan and Sichuan indigenous chickens, respectively. Incorporating 1091 published sequences, we constructed the matrilineal genealogy of Tibetan chickens to further document their domestication history. We found that the genetic structure of the mtDNA haplotypes of Tibetan chickens are dominated by seven major haplogroups (A-G). In addition, phylogenetic and network analyses showed that Tibetan chickens are not distinguishable from the indigenous chickens in surrounding areas. Furthermore, some clades of Tibetan chickens may have originated from game fowls. In summary, our results collectively indicated that Tibetan chickens may have diverged from indigenous chickens in the adjacent regions and hybridized with various chickens.

The hypoxia adaptation to high altitudes is of considerable interest in the biological sciences. As a breed with adaptability to highland environments, the Tibetan chicken (Gallus gallus domestics), provides a biological model to search for genetic differences between high and lowland chickens. To address mechanisms of hypoxia adaptability at high altitudes for the Tibetan chicken, we focused on the Endothelial PAS domain protein 1 (EPAS1), a key regulatory factor in hypoxia responses. Detected were polymorphisms of EPAS1 exons in 157 Tibetan chickens from 8 populations and 139 lowland chickens from 7 breeds. We then designed 15 pairs of primers to amplify exon sequences by Sanger sequencing methods. Six single nucleotide polymorphisms (SNPs) were detected, including 2 missense mutations (SNP3 rs316126786 and SNP5 rs740389732) and 4 synonymous mutations (SNP1 rs315040213, SNP4 rs739281102, SNP6 rs739010166, and SNP2 rs14330062). There were negative correlations between altitude and mutant allele frequencies for both SNP6 (rs739010166, r = 0.758, p<0.001) and SNP3 (rs316126786, r = 0.844, P<0.001). We also aligned the EPAS1 protein with ortholog proteins from diverse vertebrates and focused that SNP3 (Y333C) was a conserved site among species. Also, SNP3 (Y333C) occurred in a well-defined protein domain Per-AhR-Arnt-Sim (PAS domain). These results imply that SNP3 (Y333C) is the most likely casual mutation for the high-altitude adaption in Tibetan chicken. These variations of EPAS1 provide new insights into the gene's function.

GPR1 is a G protein-coupled receptor that plays critical roles in eukaryotic cells: typically, response to glucose stimulation, lipid accumulation, and transmitting nutrition signals to cAMP pathway. However, the alternative splicing of the GPR1 gene and its expression pattern in chicken tissues and ovarian follicles were unknown. In our current study, we used RACE-PCR to identify three GPR1 variants, including the full-length variant ( GPR1-va1 ) and two alternatively spliced variants ( GPR1-va2 , GPR1-vb ). Quantitative real-time PCR examined the expression pattern of GPR1 mRNA in chicken tissues and ovarian follicles. The result reveals that the coding sequence of the three variants cDNA is 1053, 1053, and 627 bp in length, encoding 350, 350, and 208 amino acids, respectively. The three variants of GPR1 show similar tissue distributions; GPR1 expression was abundant in the abdominal fat, lung, and heart. With the follicular development, the expression of GPR1 gene gradually increased, and GPR1-va1 and GPR1-va2 spliced variants expression in F2 were significantly higher than in F5, F4, and prehierarchical follicles ( P < 0.05). Taken together, we found three novel variants of GPR1 , and the results of GPR1 expression profiling in adipose tissues and ovarian follicles suggest that GPR1 may play a significant role in the lipid accumulation and progression of follicular development., Competing Interests: The authors declare that they have no competing interests.

Myozenin3 ( Myoz3 ) has been reported to bind multiple Z-disc proteins and hence play a key role in signal transduction and muscle fiber type differentiation. The purpose of current study is to better understand the basic characteristics of Myoz3 . Firstly, we cloned the ORF (open reading frame) of the Myoz3 gene. AA (amino acid) sequence analysis revealed that the Myoz3 gene encodes a 26 kDa protein which have 97% identities with that of turkey. Expression profiling showed that Myoz3 mRNA is mainly expressed in leg muscle and breast muscle. Furthermore, we investigated Myoz3 gene polymorphisms in two broiler breeds, the Yellow Bantam (YB) and the Avian. Five SNPs (single nucleotide polymorphisms) were identified in the YB breed and 3 were identified in the Avian breed. Genotypes and haplotype were constructed and their associations with carcass traits were analyzed. In the YB breed, c.516 C>T had a strong effect on both shank bone length and the [Formula: see text] value of breast muscle, and the H1H3 diplotype had the highest FC compared to other diplotypes. The markers identified in this study may serve as useful targets for the marker-assisted selection (MAS) of growth and meat quality traits in chickens.

The problem of hypoxia adaptation in high altitudes is an unsolved brainteaser in the field of life sciences. As one of the best chicken breeds with adaptability to highland environment, the Tibetan chicken, is genetically different from lowland chicken breeds. In order to gain a better understanding of the mechanism of hypoxic adaptability in high altitude, in the present study, we focused on the MT-COI together with ATP-6 gene to explore the regulatory mechanisms for hypoxia adaptability in Tibet chicken. Here, we sequenced MT-COI of 29 Tibetan chickens and 30 Chinese domestic chickens and ATP-6 gene of 28 Tibetan chickens and 29 Chinese domestic chickens. In MT-COI gene, 9 single nucleotide polymorphisms (SNPs) were detected though none of these was a missense mutation, confirming the fact that MT-COI gene is a largely conservative sequence. In ATP-6 gene, 6 single nucleotide polymorphisms (SNPs) were detected and we found a missense mutation (m.9441G > A) in the ATP-6 gene of Tibetan chicken resulting in an amino acid substitution. Due to the critical role of ATP-6 gene in the proton translocation and energy metabolism, we speculated the possibility of this mutation playing an important role in easier energy conversion and metabolism in Tibetan chickens than Chinese domestic chickens so as to better adapt to the harsh environment of the high-altitude areas. The Median-joining profile also suggested that haplotype Ha2 has the ancestral position to the other haplotypes and has significant relationship with high-altitude adaptation in ATP-6 gene. Therefore, we considered that the polymorphism (m.9441G > A) in the ATP-6 gene may affect the specific functions of ATP-6 enzyme relating to high-altitude adaptation of Tibetan chicken and MT-COI gene is a largely conservative sequence.

Chickens selected for low (LWS) and high (HWS) juvenile body weight (BW) for 55 generations differ in BW by 10-fold at selection age. High (HWR) and low (LWR) body weight-relaxed lines have been random-bred since the 46th generation. Our objective was to evaluate the developmental and nutritional regulation of pancreatic mRNA abundance of pancreatic and duodenal homeobox 1 (PDX1), preproinsulin (PPI), preproglucagon (PPG), and glucose transporter 2 (GLUT2). At day of hatch (DOH) and days 1, 3, 7, and 15 (D1, 3, 7 and 15, respectively), pancreas was collected and real time PCR was performed in Experiment 1. In Experiment 2, HWS and LWS were fed or delayed access to food for 72 h post-hatch, and pancreas collected at D15. There was an interaction of line and age for GLUT2 (P=0.001), PPI (P<0.0001), PPG (P=0.034), and PDX1 (P<0.0001). Expression was greater in chicks from LWR and LWS than HWR and HWS. There was an interaction of line and nutrition on PPG (P<0.0001) and GLUT2 (P=0.001) mRNA, where expression was similar among chicks that were fed but greater in LWS than HWS when chicks were delayed access to food. Thus, the first two weeks is important for maturation of pancreatic endocrine function. Long-term selection for BW is associated with differences in pancreas development, and delaying access to food at hatch may have persisting effects on glucose regulatory function., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Housing systems used in the production of poultry meat vary worldwide dependent on climate, land availability, and other resources essential for production. Reported here are comparisons between pen and cage rearing (the housing system, denoted HS: ), line crosses LC: ), two native Chinese lines (EM males were mated to Y1 and Y2 and their offspring denoted as EMY1 and EMY2), and sex in determining broiler traits. At hatch, 320 males and 320 females from each LC (giving a total of 1,280 chicks) were randomly assigned within each subgroup to 16 battery pens. There were 4 replicates for each combination of LC by sex. On d 28, half of the chicks were transferred to indoor floor pens, and the others were raised in single cages from d 29 to 91. Weekly body weights, livability, and feed conversion ratios ( FCR: ) were obtained to d 91, the age at which the broilers were slaughtered for carcass measurements. The caged males and females were heavier (P < 0.05) than their penned counterparts (2,292 vs 2,219 g). Except for females from line EMY1 (94.9%), the livability for each unit from 1 to 28 d, and 29 to 91 d was greater than 95%. Penned EMY2 broilers had the highest FCR (3.02), whereas penned EMY1 broilers had the lowest FCR (2.96) among the housing systems by LC combinations (P < 0.05). Caged chickens had thicker subcutaneous fat (7.24 mm), a higher percentages of abdominal fat (5.01%) and liver mass (3.13%) , but lower eviscerated carcass (60.63%) and breast muscle weights (pectoralis major and minor, 17.10%). Males were heavier and had higher percentages of leg muscle (boneless drum plus thigh, 24.22%) and heart muscle (1.08%) than the females (P < 0.05). However, the females had thicker subcutaneous fat (7.19 mm) and higher percentages of carcass weight (87.28%), breast muscle (18.11%), abdominal fat (6.54%), and liver mass (3.15%) than males. Penned females had the highest percentage of breast muscle (18.94%), and caged females had the highest percentage of liver mass (3.72%). Females of EMY1 had the highest percentage of breast muscle (18.40%). Generally, the housing system employed and the sex of the broilers greatly affect the carcass traits., (© 2015 Poultry Science Association Inc.)

The MyoD1, MyoG, Myf5, and Mrf4 proteins belong to the family of muscle regulatory factors (MRFs) and play important roles in skeletal muscle hyperplasia and hypertrophy. We hypothesized that exercise would affect MRF mRNA and protein abundance in postnatal chicken skeletal muscle driving molecular changes that could ultimately lead to increased muscle fiber diameter. At day (d) 43, twelve hundred chickens with similar body weight were randomly assigned to cage, pen, and free-range groups. The MRF mRNA abundance was measured in the pectoralis major and thigh muscle at d56, d70, and d84, and the protein levels of MRFs were determined from the thigh muscle at d84. The results showed no significant difference in mRNA of the MRFs among the three groups at d56 (P>0.05). At d84, chicken in the pen and free-range group showed higher MyoD1, MyoG, Myf5, and Mrf4 mRNA abundance compared to the caged chickens (P<0.05). Free-range chickens had higher Mrf4 and MyoG expression than those in penned ones (P<0.05). Protein abundances of all four factors were lowest in the caged group, and Mrf4 and MyoG protein quantities were greatest in free-range chickens (P<0.05), but Myf5 and MyoD1 protein abundance did not differ between penned and caged groups. The results suggested that exercise up-regulated MRF expression in the postnatal skeletal muscles, which led to an increase in muscle fiber diameter, and eventually affected the meat quality of the skeletal muscles in adult chickens., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this study, we quantified the expression of CRBP1 and CRBP3 in Roman layer (R) and Erlang mountainous chickens (SD02 and SD03), to discern the tissue, breed and age-related expression patterns in order to discover potential involvement in egg production and other related reproduction traits. Real-time quantitative PCR assays were developed for accurate measurement of CRBP1 and CRBP3 mRNA levels in different tissues from chickens at four ages (12, 20, 32 and 45 weeks). We found that the CRBP1 and CRBP3 were expressed in all six tissues examined in all three breeds of chicken at 32 weeks. CRBP1 mRNA levels in SD02 kidneys were slightly higher than those in SD03 and R at 12 weeks, whereas, at the other three time points, the expression levels of CRBP1 in SD03 were higher than those in SD02 and R. In addition, there was higher hepatic expression of CRBP3 mRNA in layers (R) compared to broilers (SD02 and SD03) at 20 and 32 weeks. An age-related expression pattern of CRBP1 gene was evident in liver (P < 0.01), but not in pituitary (P > 0.05). Overall, the expression level of CRBP1 gene in kidney, ovary and oviduct at the different ages had a "decline-rise-decline" trend in all three breeds. In contrast, in pituitary, hypothalamus, liver and kidney CRBP3 mRNA expression levels were significantly different at various ages (P < 0.05) and exhibited a "rise-decline-rise" pattern in all three breeds. Our results show that the expression of CRBP1 and CRBP3 in chicken tissues exhibit specific developmental changes and age-related patterns.

This experiment used 2 lines of chickens that have been selected 54 generations for either low (LWS) or high (HWS) 8-wk BW from the same founder population, sublines (HWR and LWR) in which selection was relaxed in generation 43 in the selected lines, and crosses (HL and LH) made from generation 54 of HWS and LWS. For 8-wk BW, the difference between lines LWS and HWS in generation 54 was approximately 10-fold, whereas for the relaxed contemporary lines they were approximately 7-fold. Three trials were designed to measure developmental, nutritional, and genetic aspects of blood glucose homeostasis during the first 2 wk posthatch. In trial 1, we measured BW, whole blood glucose (BG), and weights (relative to BW) of liver, pancreas, and yolk sac of chicks fed from day of hatch to d 15. In trial 2, we compared those traits in chicks feed-delayed 72 h posthatch and in chicks without feed delay. In trial 3, we evaluated the effect of a 16-h fast on BW and BG on d 3, 8, and 15. There were higher levels of BG in HWS than LWS, and males than females in the fed state. Delayed access to feed for 72 h after hatch was associated with a dramatic reduction in BG. Feeding triggered a compensatory response whereby LWS displayed greater BG but smaller pancreases (% BW; d 15), compared with the controls. There were maternal effects for BW in both fed and fasted states and the reciprocal crosses exhibited heterosis for BG in the fasted state. These results show that chickens selected for high or low BW differ in BG regulation during the early posthatch period.

The major histocompatibility complex (MHC) among chickens has been well established as being associated with disease resistance and pathogens infection, but the genetic differences in MHC between chickens susceptible to certain infections and those chickens that remain uninfected have not been sufficiently determined. In this study, we sought the genetic basis that may underlie differences in susceptibility to infection among chickens by challenging four groups of broilers with Marek's disease virus (MDV). Over the course of the experiment, lesions began to appear between 21 and 35 days post challenge (dpc), and commercial broilers were not necessarily better than indigenous chickens in terms of disease resistance. The four groups showed neutral resistance to MDV infection validated by challenge results and evolutionary analysis of exons 2 and 3 of the MHC class I region. Several variable sites in exon 2 and exon 3 were exclusively appeared in infected chickens. Exon 3 was likely more crucial than exon 2 in disease resistance. Our observations offered a support for a potential association between promiscuous pathogens and conspicuous genetic diversity in the MHC class I region., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Adipose differentiation-related protein gene (ADP) plays an important role in controlling lipid accumulation in mammals. It may also affect lipid deposition in birds. However, the molecular mechanism of its actions in birds remains unknown. In the present study, the coding sequence of ADP cDNA for Chinese native breed Sichuan Mountainous Black-bone chicken (MB) was first cloned from abdominal fat using reverse transcription-PCR (RT-PCR). This putative MB ADP cDNA (1,881 bp) encodes an open reading frame of 438 amino acids (AA) and shares high AA sequence identity with that of red jungle fowl (99%), duck (92%), house mouse (70%), human (70%), chimpanzee (70%), pig (70%), domestic cow (69%) and domestic sheep (68%). Further analysis using bioinformatics shows the deduced MB ADP protein has the typical characters of PAT (Perilipin, Adipophilin and Tip47) family. Quantitative real-time PCR (qRT-PCR) analysis revealed that ADP expresses in chicken leg muscle, whole brain, heart, liver, pectoralis muscle, abdominal fat and subcutaneous fat. Ontogenetic expression studies shows ADP expression levels in abdominal fat, subcutaneous fat and pectoralis muscle were prior to that in leg muscles at posthatch day (P) 84. But, its levels in abdominal fat and subcutaneous fat were less than that in leg muscles at P28, 42, 56 and 70, respectively. The ADP expression levels in subcutaneous fat and abdominal fat were stable from P28 to P70 and both were less than their counterparts at P84. However, it changed greatly in pectoralis muscle, liver, brain, heart and leg muscle at all points. Patterns of ADP expression suggest that ADP plays an important role in fat development, but further study is needed to reconfirm its function in a large population and in other breeds with different genetic backgrounds.

Background: miRNAs regulate circadian patterns by modulating the biological clocks of animals. In our previous study, we found that the clock gene exhibited a cosine expression pattern in the fallopian tube of chicken uterus. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in the chicken uterus and, if so, how they regulate egg-laying rhythms is unclear. In this study, we selected 18 layer hens with similar ovipositional rhythmicity (each of three birds were sacrificed for study per 4 h throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens. Results: We identified six circadian miRNAs that are mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of the experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in the chicken uterus. Ca2+-transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca2+ transportation during eggshell calcification in the chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in the chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. The expression of miR-449c-5p showed an opposite trend to that of ATP2B4 within a 24 h cycle in the chicken uterus; it inhibited mRNA and protein expression of ATP2B4 in the uterine tubular gland cells. In addition, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca2+ concentrations. We found similar results after ATP2B4 knockdown by miR-449c-5p. Taken together, these results indicate that ATP2B4 promotes uterine Ca2+ trans-epithelial transport. Conclusions: Clock-controlled miR-449c-5p regulates Ca2+ transport in the chicken uterus by targeting ATP2B4 during eggshell calcification. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) is known to be an important regulator of muscle growth and development. The regulation of microRNA on the skeletal muscle phenotype of animals is mainly achieved by regulating the proliferation and differentiation of myoblasts. In this study, we sequenced a total of 60 samples from 15 developing stages of the pectoral muscle and five other tissues at 300 days of Tibetan chicken. We characterized the expression patterns of miRNAs across muscle developmental stages, and found that the chicken growth and development stage was divided into early-embryonic and late-embryonic as well as postnatal stages. We identified 81 and 21 DE-miRNAs by comparing the miRNA profiles of pectoral muscle of three broad periods and different tissues, respectively; and 271 miRNAs showed time-course patterns. Their potential targets were predicted and used for functional enrichment to understand their regulatory functions. Significantly, GgmiRNA-454 is a time-dependent and tissue-differential expression miRNA. In order to elucidate the role of gga-miRNA-454 in the differentiation of myoblasts, we cultured chicken myoblasts in vitro. The results show that although gga-miRNA-454-3p initiates increase and thereafter decrease during the chicken myoblasts differentiation, it had no effect on primary myoblasts proliferation. Furthermore, we confirm that gga-miRNA-454 inhibits myoblast differentiation by targeting the myotube-associated protein SBF2. [ABSTRACT FROM AUTHOR]

Previous studies have indicated that bone morphogenetic protein (BMP) 6 may play an important role in skeletal system development and progression. However, the mechanism underlying the effects of BMP6 in cartilage cell proliferation and differentiation remains unknown. In this study, cartilage cells were isolated from shanks of chicken embryos and treated with different concentrations of Growth Hormone. Cell proliferation potential was assessed using real-time polymerase chain reaction (RT-PCR), western blotting and CCK-8 assays in vitro. The results showed that at 48 h, the Collagen II and BMP6 expression levels in 50 ng/μl GH-treated cartilage cells were significantly higher than in groups treated with 100 ng/μl or 200 ng/μl GH. We further observed that knockdown of BMP6 in cartilage cells led to significantly decreased expression mRNAs and proteins of Collagen II and Collagen X. Moreover, the suppression of BMP6 expression by a specific siRNA led to significantly decreased expression mRNA levels of IGF1R, JAK2, PKC, PTH, IHH and PTHrP and decreased protein levels of PKC, IHH and PTHrP. Taken together, our data suggest that BMP6 may play a critical role in chicken cartilage cell proliferation and differentiation through the regulation of IGF1, JAK2, PKC, PTH, and IHH-PTHrP signaling pathways. [ABSTRACT FROM AUTHOR]

This study evaluated the effects of housing system (cage versus pen), sex and line cross (EMY1 and EMY2) on meat quality in meat-type chickens. Chickens (n=640) from each line cross (males: females=1:1) were housed in batteries from d 1 to 28. Then, half of them were transferred to indoor floor pens, and the others were raised in single cages. Meat quality traits of breast fillets were measured at 91 d of age. Percent lipid and histidine were higher, whereas % total protein and myofibre density (MDS) were lower in caged than penned chickens. Cross EMY1 had higher MDS, but lower lipid % and myofibre diameters (MDM) than EMY2 (p<.05). Males had redder and brighter muscles and higher MDM and contents of glycine and proline than the females (p<.05). Penned females had smaller MDM and higher MDS than their caged counterparts (p<.05). Generally, housing systems alone, or interacting with sex and genetic line, affected yellowness, myofibre characteristics, % protein, % lipid, myofibre density, and % His of breast muscle. [ABSTRACT FROM AUTHOR]

Embryonic muscle development and fibre type differentiation has always been a topic of great importance due to its impact on both human health and farm animal financial values. Myozenin3 (Myoz3) is an important candidate gene that may regulate these processes. In the current study, we knocked down and overexpressed Myoz3 in chicken embryonic fibroblasts (CEFs) and chicken myoblasts, then utilized RNA-seq technology to screen genes, pathways and biological processes associated with Myoz3. Multiple differentially expressed genes were identified, including MYH10, MYLK2, NFAM1, MYL4, MYL9, PDZLIM1; those can in turn regulate each other and influence the development of muscle fibres. Gene ontology (GO) terms including some involved in positive regulation of cell proliferation were enriched. We further validated our results by testing the activity of cells by cell counting kit-8(CCK-8) and confirmed that under the condition of Myoz3 overexpression, the proliferation rate of CEFs and myoblasts was significantly upregulated, in addition, expression level of fast muscle specific gene was also significantly upregulated in myoblasts. Pathway enrichment analysis revealed that the PPAR (Peroxisome Proliferator-Activated Receptor) pathway was enriched, suggesting the possibility that Myoz3 regulates muscle fibre development and differentiation through the PPAR pathway. Our results provide valuable evidence regarding the regulatory functions of Myoz3 in embryonic cells by screening multiple candidate genes, biological processes and pathways associated with Myoz3. [ABSTRACT FROM AUTHOR]

Thiram pollution is one of the main causes of tibial dyschondroplasia (TD) induced by feed sources. Several studies have speculated that miRNA, circRNA and lncRNA may have significant impact on the development of TD, however, the specific mRNAs and noncoding RNAs and their respective regulatory mechanisms and functions in the development of TD have not been explored. Therefore, in this present study, we screened the differentially expressed mRNA, miRNA, circRNA and lncRNA by whole-transcriptome sequencing (RNA-seq) and differentially expressed genes (DEGs) enrichment, as well as constructed the interaction network among the mRNA-miRNA, mRNA-lncRNA and mRNA-miRNA-circRNA. The sequencing results were verified by fluorescence real-time quantitative PCR (RT-qPCR). The results obtained in this study, revealed that the cells were atrophied and disordered in the TD group, and the expression of BMP6, TGF-β and VEGF were significantly reduced. A total of 141 mRNAs, 10 miRNAs, 23 lncRNAs and 35 circRNAs of DEGs were obtained (p <0.05) Theses DEGs were enriched in the adhere junction and insulin signaling pathways. In addition, the mRNA-miRNA-circRNA network suggested that several pivotal ceRNA showed a regulatory relationship between the transcripts with miRNA, circRNA or lncRNA. Taken together, the results in the present study, represent an insight for further functional research on the ceRNA regulatory mechanism of TD in broilers. • Tibial dyschondroplasia (TD) is a crucial factor restricting the development of modern poultry industry. • This study defined and annotated differentially expressed non-coding RNAs (ncRNAs) and mRNAs in healthy broilers and thiram-induced TD broilers. • The ceRNA network of lncRNA and circRNA displays a complex interaction between ncRNAs and TD-related mRNA. • This study provide an insight for further functional research on the ceRNA regulatory mechanism of TD in broilers. [ABSTRACT FROM AUTHOR]

Immunosuppression caused by avian leukemia virus J subgroup (ALV-J) infection includes atrophy or regeneration disorders of the lymphoid organs, decreased immune response, and termination of B lymphocyte maturation process and inhibition of T-lymphocyte development. The regulatory mechanism of the related resistance genes and protein expression is not clear. While searching for a molecular marker for the immune response to ALV-J infection, we detected differentially expressed proteins (DEPs) of spleens from chicken infected by ALV-J at 15th day and 30th day by the data-independent acquisition technique. Approximately 220 DEPs from the spleens of chickens infected by ALV-J were detected. To find a relatively stable biomarker molecule, we summarized the DEPs at two timepoints and selected activating signal cointegrator 1 complex subunit 3 (ASCC3), TBC1 domain family member 2 (TBC1D2), MHC class II beta chain 1 (BLB2), ensconsin (MAP7), complement component 1 Q subcomponent B chain (C1QB), and Follistatin-like 1 (FSTL1) from both comparisons for protein interaction network analysis. ASCC3, BLB2, C1QB, and FSTL1 were potential biomarkers for the complex infection mechanism of ALV-J and the dynamic immune mechanism of the body. [ABSTRACT FROM AUTHOR]

Low oxygen levels and extremely cold weather in high-altitude environments requires more energy intake to maintain body temperature in animals. However, little is known about the characteristics of cecal and ileac microbiota in Tibetan chicken and how the high and low altitude environments affect the gut microbiota communities in Tibetan chicken. In the present study, In the present study, Tibetan chickens (Group HA, 3572 m, 578.5 Pa) and their introduced flatland counterparts (Group LA, 580 m, 894.6 Pa) in the cecum and ileum to identify the possible bacterial species that are helpful for their host in environmental adaption. High-throughput sequencing was used to sequence the V3 to V4 hypervariable regions of the bacterial 16S rRNA gene. By comparing the gut microbial diversity of HA chicken with that of LA, the results indicated that the microbial diversity of the cecum and ileum in group HA was significantly lower (P < 0.05) than those in group LA. The cecum microbiome maintained higher population diversity and richness than the ileum (P < 0.05). Four phyla Firmicutes, Bacterioidetes, Actinobacteria , and Proteobacteria were dominant in two groups. Interestingly, there were significant differences in abundance ratio among the four groups (P < 0.05). The predominant bacteria in HA and LA ileum belong to Proteobacteria and Firmicutes , whereas in cecum, Bacterioidetes and Actinobacteria were predominant in both groups (P < 0.05). Correlation analysis showed that Sporosarcina, Enterococcus , and Lactococcus were strongly related to air pressure, and Peptoclostridium and Ruminococcaceae_UCG-014 are related to altitude and gut microbiota of LA group was influenced by altitude, while HA group affected by air pressure. Meanwhile, the Ruminococcus-torques- group was negatively correlated with the relative abundance of Paenibacillus , and positive correlated with those of other microorganisms. Furthermore, HA has higher abundance of microbiota involved in energy and glycan biosynthesis metabolism pathway, while LA has higher abundance of microbiota involved in membrane transport, signal transduction, and xenobiotics biodegradation and metabolism. Generally, our results suggested that the composition and diversity of gut microbes changed after Tibetan chickens were introduced to the plain. Tibetan chicken may adapt to new environment via reshaping the gut microbiota. Gut microbes may contribute to the host adaption to high altitude environments by increasing host energy and glycan biosynthesis. [ABSTRACT FROM AUTHOR]

PGC-1β-shRNA vector were transfected into chicken preadipocytes, and translocated into nucleus. PGC-1β expression abundance was decreased and its coactivations with Sterol Regulatory Element Binding Protein 1c (SREBP-1c) and Peroxisome Proliferator-activated Receptor-gamma (PPARγ) were suppressed and further impeded the activation of Fatty Acid Synthetase (FAS), which finally blocked the fatty acid synthesis in cytoplasm of adipocytes. • PGC-1β-shRNA decreased the mRNA and protein abundances of PGC-1β in adipocytes. • Interference decreased preadipocytes differentiation, not proliferation. • Interference impeded TAG deposition and PPARγ, SREBP-1c, and FAS expression. • PGC-1β modulated the differentiation and TAG accumulation of chicken adipocytes. To study the influence of the PGC-1β gene on chicken adipocyte proliferation and differentiation, we constructed RNA interference (RNAi) vectors that target the PGC-1β gene and transfected these vectors into adipocytes. Oil Red O staining and a CCK-8 cell kit were used to determine cell triglyceride accumulation status and cell proliferation after transfection, respectively. The mRNA abundances of PGC-1β and adipocyte-differentiation-related genes (PPARγ , C/EBPα , SREBP-1c , FAS, and A-FABP) were detected by real-time PCR. The results showed that the mRNA and protein abundances of PGC-1β in PGC-1β-shRNA transfected adipocytes were significantly lower than those in the control. Interference decreased cell differentiation, but did not depress the cell proliferation. PGC-1β interference impeded the triglyceride accumulation, the mRNA expression levels of nuclear receptors PPARγ and SREBP-1c, and fatty acid synthetase (FAS) , and both proteins PPARγ and SREBP-1c, and the fatty acids transporting protein A-FABP. Generally, PGC-1β modulated the cell differentiation and triglyceride accumulation in chicken adipocytes. [ABSTRACT FROM AUTHOR]