RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-ß expression during virus infection, the expression level of IFN-ß in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

Paeoniflorin protects chicken against APEC-induced acute lung injury by affecting the endocannabinoid system and inhibiting the PI3K/AKT and NF-κB signaling pathways.

Avian pathogenic Escherichia coli (APEC) is the causative agent of chicken colibacillosis. Paeoniflorin, a natural ingredient extracted from Paeonia lactiflora, has a variety of pharmacological effects including anti-inflammatory and immunomodulatory. However, its effects and mechanism in APEC-induced acute lung injury (ALI) in chicken is not clear. The aim of this study was to investigate the protective effect of paeoniflorin on APEC-induced ALI and its possible mechanism. Paeoniflorin (25, 50, and 100 mg/kg) was administered by gavage for 5 d starting at 9 d of age and the chicken were infected with APEC by intraperitoneal injection at 12 d of age. The tissues were collected after APEC infection for 36 h for analysis. The results showed that paeoniflorin significantly alleviated the symptoms, increased the survival rate and body weight gain of APEC-infected chicken, and improved the histopathological damages, and reduced APEC loads in lung tissues. In addition, paeoniflorin restored the gene expression of ZO-1, Occludin and Claudin-3 during APEC infection. Moreover, paeoniflorin pretreatment significantly affected the endocannabinoid system (ECs) by increasing DAGL, decreasing MAGL, increasing secretion of 2-AG. Then, paeoniflorin significantly decreased the secretion of IL-1ß, IL-6 and TNF-α in lung tissues, and decreased the mRNA expression of CXCL8, CXCL12, CCL1, CCL5, and CCL17. In addition, paeoniflorin significantly reduced the phosphorylation levels of PI3K, AKT, P65, and IκB. In summary, we found that paeoniflorin inhibited APEC-induced ALI, and its mechanism may be through affecting ECs and inhibiting the activation of PI3K/AKT and NF-κB signaling pathways, which provides a new idea for the prevention and treatment of chicken colibacillosis.

Identification of potential candidate genes for the Huoyan trait in developing Wulong goose embryos by transcriptomic analysis.

1. The Wulong goose is a Chinese breed and a source of high-quality meat and eggs. A characteristic of the Wulong goose is that a proportion of the birds do not have eyelids, known as the Huoyon trait.2. Wulong geese exhibiting the Huoyan trait at embryonic stages of 9 days (E9), 12 days (E12) and 14 days (E14) were selected alongside those with normal eyelids for comprehensive transcriptome sequencing. Differentially expressed gene (DEG) and functional enrichment analyses were performed and finally, eight DEG were chosen to verify the accuracy of qPCR sequencing.3. Overall, 466, 962 and 550 DEG were obtained from the three control groups, D9 vs. N9, D12 vs. N12 and D14 vs. N14, respectively, by differential analysis (p < 0.05). CDKN1C, CRH, CROCC and TYSND1 were significantly expressed in the three groups. Enrichment analysis revealed the enrichment of CROCC and TYSND1 in pathways of cell cycle process, endocytosis, microtubule-based process, microtubule organising centre organisation, protein processing and protein maturation. CDKN1C and CRH were enriched in the cell cycle and cAMP signalling pathway.4. Some collagen family genes were detected among the DEGs, including COL3A1, COL4A5, COL4A2 and COL4A1. FREM1 and FREM2 genes were detected in both Huoyan and normal eyelids. There was a significant difference (p < 0.01) in FREM1 expression between ED9 and ED14 in female embryos, but this difference was not observed in male embryos.

Estrogen promotes gonadotropin-releasing hormone expression by regulating tachykinin 3 and prodynorphin systems in chicken.

The "KNDy neurons" located in the hypothalamic arcuate nucleus (ARC) of mammals are known to co-express kisspeptin, neurokinin B (NKB), and dynorphin (DYN), and have been identified as key mediators of the feedback regulation of steroid hormones on gonadotropin-releasing hormone (GnRH). However, in birds, the genes encoding kisspeptin and its receptor GPR54 are genomic lost, leaving unclear mechanisms for feedback regulation of GnRH by steroid hormones. Here, the genes tachykinin 3 (TAC3) and prodynorphin (PDYN) encoding chicken NKB and DYN neuropeptides were successfully cloned. Temporal expression profiling indicated that TAC3, PDYN and their receptor genes (TACR3, OPRK1) were mainly expressed in the hypothalamus, with significantly higher expression at 30W than at 15W. Furthermore, overexpression or interference of TAC3 and PDYN can regulate the GnRH mRNA expression. In addition, in vivo and in vitro assays showed that estrogen (E2) could promote the mRNA expression of TAC3, PDYN, and GnRH, as well as the secretion of GnRH/LH. Mechanistically, E2 could dimerize the nuclear estrogen receptor 1 (ESR1) to regulate the expression of TAC3 and PDYN, which promoted the mRNA and protein expression of GnRH gene as well as the secretion of GnRH. In conclusion, these results revealed that E2 could regulate the GnRH expression through TAC3 and PDYN systems, providing novel insights for reproductive regulation in chickens.

High albumen height by expression of GALNT9 and thin eggshell by decreased Ca2+ transportation caused high hatchability in Huainan partridge chicken.

Hatchability could be quite different among individuals of indigenous chicken breed which might be affected by the egg quality. In this study, hatchability was individually recorded among 800 forty-wk-old Huainan partridge chickens. The chickens were then divided into high and low hatchability groups (HH and LH group) with 50 birds in each group. Egg quality was further determined in the 2 groups. Eight birds from each group were selected for slaughtering and tissue, responsible for egg formation, collection for structure observation by staining and candidate gene expression by transcriptome analysis. The hatchability in HH was 100% and 61.18% in LH. The eggshell thickness and shell strength were significantly lower, while the albumen height and Haugh unit were significantly higher in HH group than those in LH group (P < 0.05). The magnum weight and index, and the expression of polypeptide N-acetylgalactosaminyltransferase 9 (GALNT9), which responsible for thick albumen synthesis, in HH group were also significantly higher than that of LH group (P < 0.05). Compared with the LH group, there were 702 differentially expressed genes (DEGs) in HH group, of which 402 were up-regulated and 300 were down-regulated. Candidate genes of calbindin 1 (CALB1) and solute carrier family 26 member 9 (SLC26A9), which regulate calcium signaling pathway so as to affect Ca2+ transportation, exhibited significant high and low expression, respectively, in HH group compared to those in LH group (P < 0.05). Therefore, indigenous chicken with high expression of GALNT9 in magnum to form thick albumen to provide more protein for embryo, while high CALB1 and low expression of SLC26A9 to decrease Ca2+ transportation so as to form a thinner eggshell and provide better gas exchange during embryo development.

Mechanism of fibroblast growth factor 1 regulating fatty liver disorder in mule ducks.

Mule ducks tend to accumulate abundant fat in their livers via feeding, which leads to the formation of a fatty liver that is several times larger than a normal liver. However, the mechanism underlying fatty liver formation has not yet been elucidated. Fibroblast growth factor 1 (FGF1), a member of the FGF superfamily, is involved in cellular lipid metabolism and mitosis. This study aims to investigate the regulatory effect of FGF1 on lipid metabolism disorders induced by complex fatty acids in primary mule duck liver cells and elucidate the underlying molecular mechanism. Hepatocytes were induced by adding 1,500:750 µmol/L oleic and palmitic acid concentrations for 36 h, which were stimulated with FGF1 concentrations of 0, 10, 100, and 1000 ng/mL for 12 h. The results showed that FGF1 significantly reduced the hepatic lipid droplet deposition and triglyceride content induced by complex fatty acids; it also reduced oxidative stress; decreased reactive oxygen species fluorescence intensity and malondialdehyde content; upregulated the expression of antioxidant factors nuclear factor erythroid 2 related factor 2 (Nrf2), HO-1, and NQO-1; significantly enhanced liver cell activity; promoted cell cycle progression; inhibited cell apoptosis; upregulated cyclin-dependent kinase 1 (CDK1) and BCL-2 mRNA expression; and downregulated Bax and Caspase-3 expression. In addition, FGF1 promoted AMPK phosphorylation, activated the AMPK pathway, upregulated AMPK gene expression, and downregulated the expression of SREBP1 and ACC1 genes, thereby alleviating excessive fat accumulation in liver cells induced by complex fatty acids. In summary, FGF1 may alleviate lipid metabolism disorders induced by complex fatty acids in primary mule duck liver cells by activating the AMPK signaling pathway.

CircRAB11A act as miR-24-5p sponge promotes proliferation and resists apoptosis of chicken granulosa cell via EGFR/ERK1/2 and RAB11A/ PI3K/AKT pathways.

Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that have been implicated in mediating granulosa cell (GC) proliferation and apoptosis. CircRAB11A was found to have a significantly higher expression in normal follicles compared to atrophic follicles. In this study, we determined that the knockdown of circRAB11A resulted in the inhibition of proliferation and promotion of apoptosis in GCs of chicken. Moreover, circRAB11A was found to act as a sponge for miR-24-5p, both member RAS oncogene family (RAB11A) and epidermal growth factor receptor (EGFR) were revealed to be targets of miR-24-5p through a dual-luciferase reporter assay. RAB11A or EGFR promoted proliferation and suppressed apoptosis in GCs through the phosphatidylinositol-kinase (PI3K)/AKT or extracellular signal-regulated kinase (ERK)1/2 pathway. These findings suggest that circRAB11A may function as a competing endogenous RNA (ceRNA) by targeting the miR-24-5p/RAB11A and miR-24-5p/EGFR axes and activating the ERK1/2 and PI3K/AKT pathways, offering a potential avenue for exploring the mechanism of follicle development.

Tea tree oil inhibits hydrogen sulfide-induced oxidative damage in chicken lungs through CYP450s/ROS pathway.

A large amount of hydrogen sulfide (H2S) is produced in the process of chicken breeding, which can cause serious inflammation and oxidative damage to the respiratory system of chickens. Tea tree oil (TTO) has antioxidant and anti-inflammatory properties. No studies have been reported on the use of TTO in H2S-induced lung injury in chickens. Therefore, in this study, 240 one-day-old Roman pink laying hens were randomly and equally divided into 3 groups: control group (CON), H2S exposure group (AVG, containing H2S), and TTO treatment group (TTG, containing H2S and 0.02 mL/L TTO) to establish an experimental model of TTO treatment with H2S exposure for a period of 42 d. Hematoxylin and eosin (H&E) staining was used to detect lung histopathology. Gene expression profiles were analyzed using transcriptomics. The underlying mechanism of the amelioration of lung injury by TTO was further revealed by antioxidant enzyme assays and qRT-PCR. The results showed that H2S exposure induced significant gene expression of CYP450s (CYP1B1 and CYP1C1) (P < 0.05), and caused intense oxidative stress, apoptosis and inflammation compared with CON. TTO could reduce ROS production and enhance antioxidant capacity (SOD, CAT, T-AOC, and GSH-PX) by regulating the CYP450s/ROS pathway (P < 0.05). Compared with the control group, the treatment group showed significantly decreased expression of apoptotic (Caspase-8, Caspase-3, Bid and Fas) (P < 0.05) and inflammatory (IL-4, IL-16, NF-κB, TNF-α and IFN-γ) (P < 0.05) factors in the lung. This study revealed that TTO regulated CYP450s/ROS pathway to alleviate H2S-induced lung injury in chickens. These results enrich the theory of the action mechanism of TTO on H2S-exposed chicken lungs and are of great value for the treatment of H2S-exposed animals.

Characterization and functional analysis of chicken CDK protein.

The family of cell cycle-dependent kinases (CDKs) serves as catalytic subunits within protein kinase complexes, playing a crucial role in cell cycle progression. While the function of CDK proteins in regulating mammalian innate immune responses and virus replication is well-documented, their role in chickens remains unclear. To address this, we cloned several chicken CDKs, specifically CDK6 through CDK10. We observed that CDK6 is widely expressed across various chicken tissues, with localization in the cytoplasm, nucleus, or both in DF-1 cells. In addition, we also found that multiple chicken CDKs negatively regulate IFN-ß signaling induced by chicken MAVS or chicken STING by targeting different steps. Moreover, during infection with infectious bursal disease virus (IBDV), various chicken CDKs, except CDK10, were recruited and co-localized with viral protein VP1. Interestingly, overexpression of CDK6 in chickens significantly enhanced IBDV replication. Conversely, knocking down CDK6 led to a marked increase in IFN-ß production, triggered by chMDA5. Furthermore, targeting endogenous CDK6 with RNA interference substantially reduced IBDV replication. These findings collectively suggest that chicken CDKs, particularly CDK6, act as suppressors of IFN-ß production and play a facilitative role in IBDV replication.

The possibly role of GnIH in stress and gut dysfunction in chicken.

Stress is known to disrupt the intestinal barrier and induce intestinal dysfunction. A critical role for gonadotropin inhibitory hormone (GnIH) in stress has emerged. However, whether GnIH mediates stress-induced intestinal dysfunction remains unknown. The present study explored this question through in vivo and in vitro experiments in hens. Our in vivo experiments showed that continuous intraperitoneal injection of GnIH not only significantly increased the concentration of stress hormones in serum, but also significantly elevated the mRNA expression of glucocorticoid receptor (GR) in the duodenum and jejunum. Moreover, morphological and molecular analyses revealed that GnIH disrupted the physical and chemical barriers of the intestine and dramatically increased inflammatory factor levels in the intestine and serum of hens. Interestingly, the microbiomics results showed that GnIH altered the structure and composition of the gut flora in the cecum, revealing an increased abundance of harmful intestinal bacteria such as Desulfovibrionaceae. Similar results were found in in vitro studies in which the GnIH-induced intestinal mucosal barrier was disrupted, and inflammation increased in jejunal explants, although no significant difference was found in the expression of GR between the control and GnIH groups. Our results demonstrated that GnIH not only directly damaged intestinal barriers and elevated intestinal inflammation but also mediated stress and microflora imbalance-induced intestinal function disorder, suggesting that GnIH is a potential therapeutic target for gut dysfunction, stress-induced intestinal function disorder, and inflammatory bowel disease in animals and humans.

The effect of LINC9137 targeting miR-140-3p-NKAIN3 signal axis on the development of goose testis sertoli cells.

Sertoli cells (SC) are a type of important cells in the testes, which can provide transport proteins, regulatory proteins, growth factors, and other cytokines for the spermatogenic process. They participate in the regulation of the maturation and differentiation of spermatogenic cells and play an important supporting role in the migration, proliferation, and differentiation of germ cells at all levels in the testes. Previous studies found differential expression of LINC9137, miR-140-3p, and Sodium/Potassium Transporting ATPase Interacting 3 (NKAIN3) genesin high and low sperm motility goose testicular tissues. This study investigated the effects of the LINC9137-miR-140-3p-NKAIN3 signal axis on the proliferation and apoptosis of goose testicular sertoli cells at the cellular level, respectively. The results showed that through acridine orange staining, oil red O staining, Alkaline phosphatase (AKP) staining, and RT qPCR assay, it was comprehensively identified that the cultured testicular sertoli cells were purified in vitro. Through the dual luciferase activity detection test, it was found that LINC9137 has a targeted binding site with miR-140-3p and NKAIN3. In addition, this study found that overexpression of miR-140-3p significantly inhibited the expression of LINC9137 and NKAIN3 in sertoli cells, and their expression was significantly increased when miR-140-3p was interfered with. By measuring cell proliferation activity and apoptosis related gene expression, it was found that overexpression of LINC9137 decreased cell proliferation activity (P > 0.05), while the expression level of apoptosis factor Bcl2 Associated X Protein (Bax)/B-cell lymphoma-2 (Bcl2) increased (P > 0.05). On the contrary, when interfering with LINC9137, the cell proliferation activity of sertoli cells was significantly increased (P < 0.01), and the expression level of apoptosis factor Bax/Bcl2 was significantly reduced (P < 0.05); The effect of miR-140-3p on the proliferation and apoptosis of sertoli cells is opposite to that of LINC9137. Meanwhile, this study co transfected overexpressed LINC9137 and miR-140-3p plasmids into sertoli cells, and found that the effect of LINC9137 overexpression on supporting cell proliferation was weakened by miR-140-3p. This study elucidates the role and function of the LINC9137 miR-140-3p-NKAIN3 signaling axis in the development of goose testes and spermatogenesis, establishes a regulatory network related to spermatogenesis, and provides a theoretical basis for studying the genetic regulation of goose spermatogenesis.

Chicken gasdermins mediate pyroptosis after the cleavage by caspases.

Gasdermin (GSDM) proteins are executioners of pyroptosis in many species. Gasdermin proteins can be cleaved at their linker region between the amino domain (NT) and carboxyl domain (CT) by enzymes. The released GSDM-NTs bind cell membrane and form pores, thereby leading to the release of cellular components and lytic cell death. GSDM-mediated pyroptosis is considered to play important role in immune responses. However, little is known about the GSDM proteins and GSDM-mediated pyroptosis in birds. In the current study, genes encoding chicken gasdermin A (chGSDMA) and chGSDME were cloned. The cleavage of chGSDMA and chGSDME by chicken caspase-1 (chCASP1), chCASP3 and chCASP7 and the cleavage sites were determined. The chGSDMA-NT obtained form chCASP1-mediated cleavage and chGSDME-NT obtained from chCASP3/chCASP7-mediated cleavage could bind and damage cell membrane and lead to cell death of HEK293 cells. chGSDMA-NT also strongly localized to and formed puncta in nucleus. Besides, both chGSDMA-NT and chGSDME-NT showed growth inhibition and bactericidal activity to bacteria. In chickens challenged with Pasteurella multocida and Salmonella typhimurium, the expression of chGSDMA and chGSDME was upregulated and the activation of chCASP3 and the cleavage of chGSDME were observed. The work provides essential information for expanding our knowledge on pyroptosis in birds.

The ciliary protein C2cd3 is required for mandibular musculoskeletal tissue patterning.

The mandible is composed of several musculoskeletal tissues including bone, cartilage, and tendon that require precise patterning to ensure structural and functional integrity. Interestingly, most of these tissues are derived from one multipotent cell population called cranial neural crest cells (CNCCs). How CNCCs are properly instructed to differentiate into various tissue types remains nebulous. To better understand the mechanisms necessary for the patterning of mandibular musculoskeletal tissues we utilized the avian mutant talpid2 (ta2) which presents with several malformations of the facial skeleton including dysplastic tendons, mispatterned musculature, and bilateral ectopic cartilaginous processes extending off Meckel's cartilage. We found an ectopic epithelial BMP signaling domain in the ta2 mandibular prominence (MNP) that correlated with the subsequent expansion of SOX9+ cartilage precursors. These findings were validated with conditional murine models suggesting an evolutionarily conserved mechanism for CNCC-derived musculoskeletal patterning. Collectively, these data support a model in which cilia are required to define epithelial signal centers essential for proper musculoskeletal patterning of CNCC-derived mesenchyme.

Divergent Regulatory Roles of Transcriptional Variants of the Chicken LDB3 Gene in Muscle Shaping.

LIM domain binding 3 (LDB3) serves as a striated muscle-specific Z-band alternatively spliced protein that plays an important role in mammalian skeletal muscle development, but its regulatory role and molecular mechanism in avian muscle development are still unclear. In this study, we reanalyzed RNA sequencing data sets of 1415 samples from 21 chicken tissues published in the NCBI GEO database. First, three variants (LDB3-X, LDB3-XN1, and LDB3-XN2) generated by alternative splicing of the LDB3 gene were identified in chicken skeletal muscle, among which LDB3-XN1 and LDB3-XN2 are novel variants. LDB3-X and LDB3-XN1 are derived from exon skipping in chicken skeletal muscle at the E18-D7 stage and share three LIM domains, but LDB3-XN2 lacks a LIM domain. Our results preliminarily suggest that the formation of three variants of LDB3 is regulated by RBM20. The three splice isomers have divergent functions in skeletal muscle according to in vitro and in vivo assays. Finally, we identified the mechanism by which different variants play different roles through interactions with IGF2BP1 and MYHC, which promote the proliferation and differentiation of chicken myoblasts, in turn regulating chicken myogenesis. In conclusion, this study revealed the divergent roles of three LDB3 variants in chicken myogenesis and muscle remodeling and demonstrated their regulatory mechanism through protein-protein interactions.

E2F transcription factor 5, a new regulator in adipogenesis to mediate the role of Krüppel-like factor 7 in chicken preadipocyte differentiation and proliferation.

E2F transcription factor 5 (E2F5) gene is a transcription factor, plays an important role in the development of a variety of cells. E2F5 is expressed in human and mouse adipocytes, but its specific function in adipogenesis is unclear. Krüppel-like factor 7 (KLF7) facilitates proliferation and inhibits differentiation in chicken preadipocytes. Our previous KLF7 chromatin immunoprecipitation-sequencing analysis revealed a KLF7-binding peak in the 3' flanking region of the E2F5, indicating a regulatory role of KLF7 in this region. In the present study, we investigated E2F5 potential role, the overexpression and knockdown analyses revealed that E2F5 inhibited the differentiation and promoted the proliferation of chicken preadipocytes. Moreover, we identified enhancer activity in the 3' flanking region (nucleotides +22661/+22900) of E2F5 and found that KLF7 overexpression increased E2F5 expression and luciferase activity in this region. Deleting the putative KLF7-binding site eliminated the promoting effect of KLF7 overexpression on E2F5 expression. Further, E2F5 reversed the KLF7-induced decrease in preadipocyte differentiation and increase in preadipocyte proliferation. Taken together, our findings demonstrate that KLF7 inhibits differentiation and promotes proliferation in preadipocytes by enhancing E2F5 transcription.

The relationship of MITF gene expression and promoter methylation with plumage colour in quail.

1. This study focused on the relationship between MITF mRNA expression and plumage colour in quail and the effect of promoter methylation on the expression of MITF mRNA.2. The CDS region of MITF mRNA was cloned by RT-PCR, followed by DNA sequencing. The RT-qPCR method was used to analyse the expression levels of MITF mRNA in dorsal skin tissue in Korean quail and Beijing white quail. The promoter region of the MITF gene was cloned, and the CpG island was predicted by the CpGplot program. The methylation levels of the CpG island were analysed using BS-PCR technology.3. Quail MITF mRNA contains a 1,476 bp complete ORF, which encodes a 492 amino acid residue protein. The MITF protein has no signal peptide or transmembrane region. The expression of MITF mRNA in dorsal tissue of Korean quail was significantly higher than that in Beijing white quail (p < 0.01). Abundant cis-elements and a 346 bp CpG island were found in the promoter region of the MITF gene. The average methylation level of the CpG island was 22 (22%) in Korean quail, and 46 (30%) in Beijing white quail (p < 0.05).4. The hypermethylation of the MITF gene promoter region in Beijing white quail resulted in a decrease in expression level, which was related to white feather colour.

Research Note: Chicken breast muscle satellite cell function: effect of expression of CNN1 and PHRF1.

The Wooden Breast myopathy results in the necrosis and fibrosis of breast muscle fibers in fast-growing heavy weight meat-type broiler chickens. Myogenic satellite cells are required to repair and regenerate the damaged muscle fibers. Using Genome Wide Association, candidate genes affected with Wooden Breast have been previously reported. The effect of these genes on satellite cell proliferation, differentiation, and the synthesis of lipids by satellite cells is unknown. Satellite cells isolated from the pectoralis major muscle from commercial Ross 708 broilers and a Randombred chicken (RBch) line were used. Expression of calponin 1 (CNN1) and PHD and ring fingers domains 1 (PHRF1) were knocked down by silent interfering RNA to determine their effect on satellite cell-mediated proliferation, differentiation, and lipid accumulation. CNN1 and PHRF1 affected satellite cell activity and lipid accumulation in both lines. Proliferation was reduced in the Ross 708 and RBch lines by knocking down the expression of both genes, and differentiation was affected with a line and treatment interaction when gene expression was reduced at the beginning of proliferation. During differentiation lipid accumulation was decreased with knocking down the expression of CNN1 and PHRF1. Both CNN1 and PHRF1 have not been reported previously in skeletal muscle and further research is required to determine their effect on satellite cell-mediated growth and regeneration of the pectoralis major (breast) muscle.

Proteomic analyses on chicken breast meat with white striping myopathy.

White striping (WS) is an emerging myopathy that results in significant economic losses as high as $1 billion (combined with losses derived from other breast myopathies including woody breast and spaghetti meat) to the global poultry industry. White striping is detected as the occurrence of white lines on raw poultry meat. The exact etiologies for WS are still unclear. Proteomic analyses of co-expressed WS and woody breast phenotypes previously demonstrated dysfunctions in carbohydrate metabolism, protein synthesis, and calcium buffering capabilities in muscle cells. In this study, we conducted shotgun proteomics on chicken breast fillets exhibiting only WS that were collected at approximately 6 h postmortem. After determining WS severity, protein extractions were conducted from severe WS meat with no woody breast (WB) condition (n = 5) and normal non-affected (no WS) control meat (n = 5). Shotgun proteomics was conducted by Orbitrap Lumos, tandem mass tag (TMT) analysis. As results, 148 differentially abundant proteins (|fold change|>1.4; p-value < 0.05) were identified in the WS meats compared with controls. The significant canonical pathways included BAG2 signaling pathway, glycogen degradation II, isoleucine degradation I, aldosterone signaling in epithelial cells, and valine degradation I. The potential upstream regulators include LIPE, UCP1, ATP5IF1, and DMD. The results of this study provide additional insights into the cellular mechanisms on the WS myopathy and meat quality.

Zinc alleviates thermal stress-induced damage to the integrity and barrier function of cultured chicken embryonic primary jejunal epithelial cells via the MAPK and PI3K/AKT/mTOR signaling pathways.

Zinc (Zn) could alleviate the adverse effect of high temperature (HT) on intestinal integrity and barrier function of broilers, but the underlying mechanisms remain unclear. We aimed to investigate the possible protective mechanisms of Zn on primary cultured broiler jejunal epithelial cells exposed to thermal stress (TS). In Exp.1, jejunal epithelial cells were exposed to 40℃ (normal temperature, NT) and 44℃ (HT) for 1, 2, 4, 6, or 8 h. Cells incubated for 8 h had the lowest transepithelial resistance (TEER) and the highest phenol red permeability under HT. In Exp.2, the cells were preincubated with different Zn sources (Zn sulfate as iZn and Zn proteinate with the moderate chelation strength as oZn) and Zn supplemental levels (50 and 100 µmol/L) under NT for 24 h, and then continuously incubated under HT for another 8 h. TS increased phenol red permeability, lactate dehydrogenase (LDH) activity and p-PKC/PKC level, and decreased TEER, cell proliferation, mRNA levels of claudin-1, occludin, zona occludens-1 (ZO-1), PI3K, AKT and mTOR, protein levels of claudin-1, ZO-1 and junctional adhesion molecule-A (JAM-A), and the levels of p-ERK/ERK, p-PI3K/PI3K and p-AKT/AKT. Under HT, oZn was more effective than iZn in increasing TEER, occludin, ZO-1, PI3K, and AKT mRNA levels, ZO-1 protein level, and p-AKT/AKT level; supplementation with 50 µmol Zn/L was more effective than 100 µmol Zn/L in increasing cell proliferation, JAM-A, PI3K, AKT, and PKC mRNA levels, JAM-A protein level, and the levels of p-ERK/ERK and p-PI3K/PI3K; furthermore, supplementation with 50 µmol Zn/L as oZn had the lowest LDH activity, and the highest ERK, JNK-1, and mTOR mRNA levels. Therefore, supplemental Zn, especially 50 µmol Zn/L as oZn, could alleviate the TS-induced integrity and barrier function damage of broiler jejunal epithelial cells possibly by promoting cell proliferation and tight junction protein expression via the MAPK and PI3K/AKT/mTOR signaling pathways.

Transcriptome analysis of pigeon pituitary gland: expression changes of genes encoding protein and peptide hormones at different breeding stages.

Unlike other poultry, parent pigeons produce "pigeon milk" in their crops to nurture their squabs, which is mainly controlled by prolactin (PRL). Exception for PRL, the pituitary gland may also release various other peptide and protein hormones. However, whether these hormones change during pigeon crop lactation and their potential physiological functions remain unclear. Here, to identify potential peptide or protein hormone genes that regulate crop lactation, we conducted transcriptome analysis of pigeon pituitary glands at 3 different breeding stages (the ceased stage-nonincubation and non-nurturing stage, the 11th d of the incubation, and the 1st d of the nurturing stage) using RNA sequencing (RNA-Seq). Our analysis identified a total of 15,191 mRNAs and screened out 297 differentially expressed genes (DEG), including PRL, VIP, etc. The expression abundance of PRL mRNA on the 1st d of the nurturing stage was respectively 4.93 and 3.62 folds higher when compared to the ceased stage and the 11th d of the incubation stage. Additionally, the expression abundance of VIP is higher in the 1st d of the nurturing stage than in the ceased stage. Protein-protein interaction (PPI) network and Molecular Complex Detection (MCODE) analysis identified several vital DEGs (e.g., GHRHR, VIP, etc.), being closely linked with hormone and enriched in neuropeptide signaling pathway and response to the hormone. Expression pattern analysis revealed that these DEGs exhibited 4 distinct expression patterns (profile 10, 16, 18, 19). Genes in profile 10 and 19 presented a trend with the highest expression level on 1st d of the nurturing stage, and functional enrichment analysis indicated that these genes are involved in neuropeptide hormone activity, receptor-ligand activity, and the extracellular matrix, etc. Taken together, being consistent with PRL, some genes encoding peptide and protein hormones (e.g., VIP) presented differentially expressed in different breeding stages. It suggests that these hormones may be involved in regulation of the crop lactation process or corresponding behavior in domestic pigeons. The results of this study help to gain new insights into the role of pituitary gland in regulating pigeon lactation.