Combining GC-MS/MS with a LLE-SPE sample pretreatment step to simultaneously analyse enrofloxacin and ofloxacin residues in chicken tissues and pork.

A widely applicable original gas chromatography-tandem mass spectrometry (GC-MS/MS) method was explored to qualitatively and quantitatively measure enrofloxacin and ofloxacin residues in chicken tissues and pork. The experimental samples were processed based on liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Trimethylsilyl diazomethane (TMSD) was chosen to react derivatively with enrofloxacin and ofloxacin. In total, 78.25% âˆ¼ 90.56% enrofloxacin and 78.43% âˆ¼ 91.86% ofloxacin was recovered from the blank fortified samples. The limits of detection (LODs) were 0.7-1.0 µg/kg and 0.1-0.2 µg/kg, respectively. The limits of quantitation (LOQs) were 1.6-1.9 µg/kg and 0.3-0.4 µg/kg, respectively. It was verified that various experimental data met the requirements of the FAO & WHO (2014) for the detection of veterinary drug residues. Real samples obtained from local markets were analysed using the established method, and no residues of enrofloxacin or ofloxacin were detected in the samples.

Quantitative analysis of erythromycin, its major metabolite and clarithromycin in chicken tissues and eggs via QuEChERS extraction coupled with ultrahigh-performance liquid chromatography-tandem mass spectrometry.

A simple, rapid and novel method involving ultrahigh-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UHPLC-ESI-MS/MS) was developed to simultaneously detect erythromycin, its major metabolite and clarithromycin in chicken tissues (muscle, liver and kidney) and eggs (whole egg, albumen and yolk). Samples were extracted using acetonitrile-water (80:20, v/v), and a Cleanert MAS-Q cartridge was used to perform quick, easy, cheap, effective, rugged, and safe (QuEChERS) purification. The average recoveries were 87.78-104.22 %, and the corresponding intraday and interday relative standard deviations were less than 7.10 %. The decision limits and detection capabilities of the chicken tissues and eggs were 2.15-105.21 µg/kg and 2.26-110.42 µg/kg, respectively. For chicken tissues and eggs, the limits of detection and limits of quantification were 0.5 µg/kg and 2.0 µg/kg, respectively. The proposed method was successfully employed to analyse real samples, demonstrating its applicability.

Transcriptome analysis reveals that gga-miR-2954 inhibits the inflammatory response against Eimeria tenella infection.

Coccidiosis is an important parasitic protozoan disease in poultry farming, causing huge economic losses in the global poultry industry every year. MicroRNAs (miRNAs) are a class of RNA macromolecules that play important roles in the immune response to pathogens. However, the expression profiles and functions of miRNAs during Eimeria tenella (E. tenella) infection in chickens remain mostly uncharacterized. In this study, high-throughput sequencing of cecal tissues of control (JC), resistant (JR), and susceptible (JS) chickens led to the identification of 35 differentially expressed miRNAs among the three groups. Functional enrichment analysis showed that the differentially expressed miRNAs were mainly associated with the TGF-beta, NF-kB, and Jak-STAT signaling pathways. Notably, gga-miR-2954 was found to be significantly upregulated after coccidial infection. Functional analysis showed that gga-miR-2954 inhibited the production of the inflammatory cytokines IL-6, IL-1ß, TNF-α, and IL-8 in sporozoite-stimulated DF-1 cells. Mechanistically, we found that gga-miR-2954 targeted the RORC gene and that RORC promoted the inflammatory response in sporozoite-stimulated DF-1 cells. In conclusion, our study was the first to identify differentially expressed miRNAs in chicken cecal tissue during E. tenella infection and found that gga-miR-2954 regulates the host immune response to coccidial infection in chickens by targeting the RORC gene.

Development of a QuEChERS-HPLC-FLD Procedure for the Simultaneous Detection of Residues of Florfenicol, Its Metabolite Florfenicol Amine, and Three Fluoroquinolones in Eggs.

A method utilizing high-performance liquid chromatography-fluorescence detection (HPLC-FLD) has been developed and refined for the simultaneous detection of florfenicol (FF) and its metabolite florfenicol amine (FFA) along with three fluoroquinolone (ciprofloxacin (CIP), enrofloxacin (ENR), and sarafloxacin (SAR)) residues in different parts of eggs (whole egg, egg yolk, and egg albumen). The QuEChERS ("Quick, easy, cheap, effective, rugged, and safe") procedure utilized 0.1 M disodium EDTA solution, water, and acetonitrile as extractants; sodium sulfate, sodium chloride, and trisodium citrate as dehydrating salts; and N-propylethylenediamine and C18 as adsorbents. A dual-channel FLD method was utilized to analyze the target compounds using an XBridge BEH C18 chromatographic column (4.6 mm × 150 mm, 5 µm). The mobile phase was employed isocratically using a solution of 0.01 M sodium dihydrogen phosphate, 0.005 M sodium dodecyl sulfate, and 0.1% triethylamine (pH 4.8) in combination with acetonitrile at a ratio of 65:35 (V/V). The limits of detection (LOD) and quantification (LOQ) of the analytes ranged from 0.03 to 1.5 µg/kg and from 0.1 to 5.0 µg/kg, respectively. The recoveries of the analytes in the blank egg samples ranged from 71.9% to 94.8% when reference standard concentrations of the LOQ, half of the maximum residual limit (MRL), MRL, and twice the MRL were added. The parameters of the presented protocol were validated and subsequently applied to the analysis of real samples, demonstrating the applicability and reliability of the method.

Identification of metabolite biomarkers in Salmonella enteritidis-contaminated chickens using UHPLC-QTRAP-MS-based targeted metabolomics.

This study aimed to characterize the metabolic profile of Salmonella enteritidis (S. enteritidis) in chicken matrix and to identify metabolic biomarkers of S. enteritidis in chicken. The UHPLC-QTRAP-MS high-throughput targeted metabolomics approach was employed to analyze the metabolic profiles of contaminated and control group chickens. A total of 348 metabolites were quantified, and the application of deep learning least absolute shrinkage and selection operator (LASSO) modelling analysis obtained eight potential metabolite biomarkers for S. enteritidis. Metabolic abundance change analysis revealed significantly enriched abundances of anthranilic acid, l-pyroglutamic acid, 5-hydroxylysine, n,n-dimethylarginine, 4-hydroxybenzoic acid, and menatetrenone in contaminated chicken samples. The receiver operating characteristic (ROC) curve analysis demonstrated the strong ability of these six metabolites as biomarkers to distinguish S. enteritidis contaminated and fresh chicken samples. The findings presented in this study offer a theoretical foundation for developing an innovative approach to identify and detect foodborne contamination caused by S. enteritidis.

High-fat diet-induced gut microbiota alteration promotes lipogenesis by butyric acid/miR-204/ACSS2 axis in chickens.

The gut microbiota is known to have significant involvement in the regulation of lipogenesis and adipogenesis, yet the mechanisms responsible for this relationship remain poorly understood. The current study aims to provide insight into the potential mechanisms by which the gut microbiota modulates lipogenesis in chickens. Using chickens fed with a normal-fat diet (NFD, n = 5) and high-fat diet (HFD, n = 5), we analyzed the correlation between gut microbiota, cecal metabolomics, and lipogenesis by 16s rRNA sequencing, miRNA and mRNA sequencing as well as targeted metabolomics analysis. The potential metabolite/miRNA/mRNA axis regulated by gut microbiota was identified using chickens treated with antibiotics (ABX, n = 5). The possible mechanism of gut microbiota regulating chicken lipogenesis was confirmed by fecal microbiota transplantation (FMT) from chickens fed with NFD to chickens fed with HFD (n = 5). The results showed that HFD significantly altered gut microbiota composition and enhanced chicken lipogenesis, with a significant correlation between 3. Furthermore, HFD significantly altered the hepatic miRNA expression profiles and reduced the abundance of hepatic butyric acid. Procrustes analysis indicated that the HFD-induced dysbiosis of the gut microbiota might affect the expression profiles of hepatic miRNA. Specifically, HFD-induced gut microbiota dysbiosis may reduce the abundance of butyric acid and downregulate the expression of miR-204 in the liver. Multiomics analysis identified ACSS2 as a target gene of miR-204. Gut microbiota depletion by an antibiotic cocktail (ABX) showed a gut microbiota-dependent manner in the abundance of butyric acid and the expression of miR-204/ACSS2, which have been observed to be significantly correlated. Fecal microbiota transplantation from NFD chickens into HFD chickens effectively attenuated the HFD-induced excessive lipogenesis, elevated the abundance of butyric acid and the relative expression of miR-204, and reduced the expression of ACSS2 in the liver. Mechanistically, our results showed that the gut microbiota plays an antiobesity role by regulating the butyric acid/miR-204/ACSS2 axis in chickens. This work contributed to a better understanding of the functions of gut microbiota in regulating chicken lipogenesis.

Comprehensive analysis of gut microbiome and host transcriptome in chickens after Eimeria tenella infection.

Background: Coccidiosis is an intestinal parasitic disease caused by Eimeria protozoa, which endangers the health and growth of animals, and causes huge economic losses to the poultry industry worldwide every year. Studies have shown that poultry gut microbiota plays an important role in preventing the colonization of pathogens and maintaining the health of the host. Coccidia infection also affects host gene expression. However, the underlying potential relationship between gut microbiome and host transcriptome during E. tenella infection in chickens remain unclear. Methods: In this study, metagenomic and transcriptome sequencing were applied to identify microbiota and genes in cecal contents and cecal tissues of infected (JS) and control (JC) chickens on day 4.5 postinfection (pi), respectively. Results: First, microbial sequencing results of cecal contents showed that the abundance of Lactobacillus, Roseburia sp. and Faecalibacterium sp decreased significantly after E. tenella infection (P < 0.05), while the abundance of Alistipes and Prevotella pectinovora increased significantly (P < 0.05). Second, transcriptome sequencing results showed that a total of 434 differentially expressed mRNAs were identified, including 196 up-regulated and 238 down-regulated genes. These differentially expressed genes related to inflammation and immunity, such as GAMA, FABP1, F2RL1 and RSAD2, may play an important role in the process of host resistance to coccidia infection. Functional studies showed that the enriched pathways of differentially expressed genes included the TGF-beta signaling pathway and the ErbB signaling pathways. Finally, the integrated analysis of gut microbiome and host transcriptome suggested that Prevotella pectinovora associated with FABP1, Butyricicoccus porcorum and Colidextribacter sp. associated with RSAD2 were involved in the immune response upon E. tenella infection. Conclusion: In conclusion, this study provides valuable information on the microbiota and key immune genes after chicken E. tenella infection, with the aim of providing reference for the impact of coccidia infection on cecal microbiome and host.

Qualitative and Quantitative Determination of Decoquinate in Chicken Tissues by Gas Chromatography Tandem Mass Spectrometry.

A novel precolumn derivatization-GC-MS/MS method was developed for the determination of decoquinate residues in chicken tissues (muscle, liver, and kidney). The samples were extracted and purified by liquid-liquid extraction combined with solid-phase extraction and derivatized with acetic anhydride and pyridine. The recovery rates for decoquinate were 77.38~89.65%, and the intra-day and inter-day RSDs were 1.63~5.74% and 2.27~8.06%, respectively. The technique parameters meet the necessities for veterinary drug residue detection in China, the US, and the EU. Finally, the method was applied to analyze tissues of 60 chickens bought from a neighborhood supermarket, and solely one sample of chicken muscle contained 15.6 µg/kg decoquinate residue.

Integrated Metabolomic and Transcriptomic Analysis Reveals Potential Gut-Liver Crosstalks in the Lipogenesis of Chicken.

Growing evidence has shown the involvement of the gut-liver axis in lipogenesis and fat deposition. However, how the gut crosstalk with the liver and the potential role of gut-liver crosstalk in the lipogenesis of chicken remains largely unknown. In this study, to identify gut-liver crosstalks involved in regulating the lipogenesis of chicken, we first established an HFD-induced obese chicken model. Using this model, we detected the changes in the metabolic profiles of the cecum and liver in response to the HFD-induced excessive lipogenesis using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The changes in the gene expression profiles of the liver were examined by RNA sequencing. The potential gut-liver crosstalks were identified by the correlation analysis of key metabolites and genes. The results showed that a total of 113 and 73 differentially abundant metabolites (DAMs) between NFD and HFD groups were identified in the chicken cecum and liver, respectively. Eleven DAMs overlayed between the two comparisons, in which ten DAMs showed consistent abundance trends in the cecum and liver after HFD feeding, suggesting their potential as signaling molecules between the gut and liver. RNA sequencing identified 271 differentially expressed genes (DEGs) in the liver of chickens fed with NFD vs. HFD. Thirty-five DEGs were involved in the lipid metabolic process, which might be candidate genes regulating the lipogenesis of chicken. Correlation analysis indicated that 5-hydroxyisourate, alpha-linolenic acid, bovinic acid, linoleic acid, and trans-2-octenoic acid might be transported from gut to liver, and thereby up-regulate the expression of ACSS2, PCSK9, and CYP2C18 and down-regulate one or more genes of CDS1, ST8SIA6, LOC415787, MOGAT1, PLIN1, LOC423719, and EDN2 in the liver to enhance the lipogenesis of chicken. Moreover, taurocholic acid might be transported from the gut to the liver and contribute to HFD-induced lipogenesis by regulating the expression of ACACA, FASN, AACS, and LPL in the liver. Our findings contribute to a better understanding of gut-liver crosstalks and their potential roles in regulating chicken lipogenesis.

Simultaneous quantitative determination of residues of abamectin, ivermectin, albendazole and its three metabolites in beef and chicken by HPLC-PDA.

Antimicrobial drugs are frequently used in a combination or shuttle way to cope with coinfection of bacteria or parasites and prevent drug resistance, thus the accurate quantification of multiple drug residues in animal-derived foods is crucial to ensure food safety. Here, a simple and efficient high-performance liquid chromatography-photodiode array (HPLC-PDA) method was established for the simultaneous quantitative screening of six common residues of antiparasitic drugs, including abamectin (ABM), ivermectin (IVM), albendazole (ABZ) and the three metabolites of ABZ in beef and chicken. The LODs and LOQs for six target compounds in beef and chicken are determined to be 3.2 to 12.5 µg/kg and 9.0 to 30.0 µg/kg, respectively. The calibration curves show good linearity (R2 ≥ 0.9990) between the peak area and concentration. The recoveries from the fortified blank samples are all above 85.10%. Finally, the applicability of the HPLC-PDA method is successfully demonstrated by the real sample analysis.

Untargeted and targeted metabolomics identify metabolite biomarkers for Salmonella enteritidis in chicken meat.

Salmonella Enteritidis easily contaminate chicken during slaughtering, processing, transportation, and sales, which seriously endangers human health. This study aimed to identify metabolite biomarkers for Salmonella Enteritidis contamination in chicken meat. UPLC-Q-Orbitrap MS untargeted metabolomics analysis identified 441 and 240 confidently metabolites in positive and negative ion mode, respectively. Thirty metabolites were defined as potential biomarkers for Salmonella enteritidis contamination in chicken meat. UPLC-QQQ-MS based targeted metabolomics was used to quantitatively analyze candidate metabolite biomarkers in Salmonella enteritidis contaminated and fresh chicken samples. A total of 10 candidate metabolite biomarkers were confirmed in the validation set, among which acetylcholine, l-Methionine, l-Proline, l-Valine, and l-Norleucine were identified as biomarkers for Salmonella Enteritidis contamination in chicken. The combined receiver operating characteristic curve analysis of the five biomarkers achieved an AUC of 0.956, indicating their high sensitivity and specificity in predicting Salmonella Enteritidis in raw chicken. In conclusion, the present study identified five metabolite biomarkers for Salmonella enteritidis in raw chicken. These results provide a potential theoretical basis for developing Salmonella Enteritidis detection methods in raw chicken.

Quantitative Analysis of Decoquinate Residues in Hen Eggs through Derivatization-Gas Chromatography Tandem Mass Spectrometry.

A novel precolumn derivatization-gas chromatography tandem mass spectrometry (GC-MS/MS) method was developed to detect and confirm the presence of decoquinate residues in eggs (whole egg, albumen and yolk). Liquid-liquid extraction (LLE) and solid phase extraction (SPE) were used to extract and purify samples. The derivatization reagents were pyridine and acetic anhydride, and the derivatives were subjected to GC-MS/MS detection. After the experimental conditions were optimized, satisfactory sensitivity was obtained. The limits of detection (LODs) and limits of quantification (LOQs) for the decoquinate in eggs (whole egg, albumen and yolk) were 1.4-2.4 µg/kg and 2.1-4.9 µg/kg, respectively. At four spiked concentration levels, the average recoveries were 74.3-89.8%, the intraday RSDs ranged from 1.22% to 4.78%, and the inter-day RSDs ranged from 1.61% to 7.54%. The feasibility and practicality of the method were confirmed by testing egg samples from a local supermarket.

Separation and Detection of Abamectin, Ivermectin, Albendazole and Three Metabolites in Eggs Using Reversed-Phase HPLC Coupled with a Photo Diode Array Detector.

An innovative and sensitive approach using high-performance liquid chromatography-photo diode array detection (HPLC-PDAD) was developed and optimized for the simultaneous determination of abamectin (ABM), ivermectin (IVM), albendazole (ABZ) and three metabolites in eggs. The samples were extracted with acetonitrile (MeCN)/water (90:10, v/v), and the extracts containing the targets were cleaned up and concentrated by a series of liquid-liquid extraction (LLE) steps. A reversed-phase C18 column and a mobile phase consisting of 0.1% trifluoroacetic acid (TFA) aqueous solution and methanol (MeOH) were utilized to perform optimal chromatographic separation. The developed method was validated on the basis of international guidelines. The limits of detection (LODs) and quantitation (LOQs) were 2.1-10.5 µg/kg and 7.8-28.4 µg/kg, respectively. Satisfactory linear relationships were observed for the targets in their corresponding concentration ranges. The mean recoveries ranged from 85.7% to 97.21% at 4 addition levels, with intraday and interday relative standard deviations (RSDs) in the ranges of 1.68-4.77% and 1.74-5.31%, respectively. The presented protocol was demonstrated to be applicable and reliable by being applied for the detection of target residues in locally sourced egg samples.

Identification of miRNA-mRNA Networks Associated with Pigeon Skeletal Muscle Development and Growth.

The growth and development of skeletal muscle determine the productivity of pigeon meat production, and miRNA plays an important role in the growth and development of this type of muscle. However, there are few reports regarding miRNA regulating the growth and development of skeletal muscle in pigeons. To explore the function of miRNA in regulating the growth and development of pigeon skeletal muscle, we used RNA sequencing technology to study the transcriptome of pigeons at two embryonic stages (E8 and E13) and two growth stages (D1 and D10). A total of 32,527 mRNAs were identified in pigeon skeletal muscles, including 14,378 novel mRNAs and 18,149 known mRNAs. A total of 2362 miRNAs were identified, including 1758 known miRNAs and 624 novel miRNAs. In total, 839 differentially expressed miRNAs (DEmiRNAs) and 11,311 differentially expressed mRNAs (DEGs) were identified. STEM clustering analysis assigned DEmiRNAs to 20 profiles, of which 7 were significantly enriched (p-value < 0.05). These seven significantly enriched profiles can be classified into two categories. The first category represents DEmiRNAs continuously downregulated from the developmental stage to the growth stage of pigeon skeletal muscle, and the second category represents DEmiRNAs with low expression at the development and early growth stage, and significant upregulation at the high growth stage. We then constructed an miRNA−mRNA network based on target relationships between DEmiRNAs and DEGs belonging to the seven significantly enriched profiles. Based on the connectivity degree, 20 hub miRNAs responsible for pigeon skeletal muscle development and growth were identified, including cli-miR-20b-5p, miR-130-y, cli-miR-106-5p, cli-miR-181b-5p, miR-1-z, cli-miR-1a-3p, miR-23-y, cli-miR-30d-5p, miR-1-y, etc. The hub miRNAs involved in the miRNA−mRNA regulatory networks and their expression patterns during the development and growth of pigeon skeletal muscle were visualized. GO and KEGG enrichment analysis found potential biological processes and pathways related to muscle growth and development. Our findings expand the knowledge of miRNA expression in pigeons and provide a database for further investigation of the miRNA−mRNA regulatory mechanism underlying pigeon skeletal muscle development and growth.

Concurrent Determination of Tigecycline, Tetracyclines and Their 4-Epimer Derivatives in Chicken Muscle Isolated from a Reversed-Phase Chromatography System Using Tandem Mass Spectrometry.

A quantitative and qualitative method using a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) detection approach was developed and validated for the analysis of tigecycline, four tetracyclines and their three 4-epimer derivatives in chicken muscle. Samples were extracted repeatedly with 0.1 mol/L Na2EDTA-McIlvaine buffer solution. After vortexing, centrifugation, solid-phase extraction, evaporation and reconstitution, the aliquots were separated using a C8 reversed-phase column (50 mm × 2.1 mm, 5 µm) with a binary solvent system consisting of methanol and 0.01 mol/L trichloroacetic acid aqueous solution. The typical validation parameters were evaluated in accordance with the acceptance criteria detailed in the guidelines of the EU Commission Decision 2002/657/EC and the U.S. Food and Drug Administration Bioanalytical Method Validation 05/24/18. The matrix-matched calibration curve was linear over the concentration range from the limit of quantitation (LOQ) to 400 µg/kg for doxycycline, and the calibration graphs for tetracycline, chlortetracycline, oxytetracycline, their 4-epimer derivatives and tigecycline showed a good linear relationship within the concentration range from the LOQ to 200 µg/kg. The limits of detection (LODs) for the eight targets were in the range of 0.06 to 0.09 µg/kg, and the recoveries from the fortified blank samples were in the range of 89% to 98%. The within-run precision and between-run precision, which were expressed as the relative standard deviations, were less than 5.0% and 6.9%, respectively. The applicability was successfully demonstrated through the determination of residues in 72 commercial chicken samples purchased from different sources. This approach provides a novel option for the detection of residues in animal-derived food safety monitoring.

Effects of dietary Acremonium terricola culture supplementation on the quality, conventional characteristics, and flavor substances of Hortobágy goose meat.

This study aimed to determine the effect of dietary supplementation with Acremonium terricola culture (ATC) on the quality, conventional characteristics, and flavor substances of Hortobágy goose meat. A total of 720 one-day-old goslings were divided into four dietary treatments, each consisting of six cages of 30 goslings. The dietary conditions consisted of the control group and three treatment groups supplemented with 3, 5, or 7 g/kg ATC. In male geese, supplementation with 3 g/kg ATC elevated the crude ash (CA) content of the thigh muscle compared to the control group, and the CA content of the pectoralis major was significantly elevated when geese were supplemented with 5 g/kg ATC (p < 0.05). In females, compared with the control group, supplementation with 7 g/kg ATC enhanced the crude protein (CP) content of the pectoralis major. Supplementation with 7 g/kg ATC also increased the crude fat (CF) content of the pectoralis major in females as well as in both sexes; moreover, this supplementation dose increased the inosinic acid content of the thigh muscle in males and in both sexes. In contrast, supplementation with 5 g/kg ATC decreased the pH of the thigh muscle at 12 h postmortem (p < 0.01). No significant changes in meat color, water loss rate, shear force, moisture content or amino acid (AA) levels were observed after ATC supplementation (p > 0.05). Levels of saturated fatty acids (SFAs) and polyunsaturated FAs (PUFAs) in the pectoralis major and levels of SFAs, monounsaturated FAs (MUFAs), and PUFAs in the thigh muscle were not affected by the supplementation. Overall, ATC supplementation had positive effects on the pH, and CA, CP, CF, inosinic acid contents as well as on the FA composition of gosling meat. The optimal level of ATC supplementation was 7 g/kg in goslings from 1 to 70 days of age.

miRNA-seq analysis in skeletal muscle of chicken and function exploration of miR-24-3p.

The regulation of skeletal muscle growth and development in chicken is complex. MicroRNAs (miRNAs) have been found to play an important role in the process, and more research is needed to further understand the regulatory mechanism of miRNAs. In this study, leg muscles of Jinghai yellow chickens at 300 d with low body weight (slow-growing group) and high body weight (fast-growing group) were collected for miRNA sequencing (miRNA-seq) and Bioinformatics analysis revealed 12 differentially expressed miRNAs (DEMs) between the two groups. We predicted 150 target genes for the DEMs, and GO and KEGG pathway analysis showed the target genes of miR-24-3p and novel_miR_133 were most enriched in the terms related to growth and development. Moreover, networks of DEMs and target genes showed that miR-24-3p and novel_miR_133 were the 2 core miRNAs. Hence, miR-24-3p was selected for further functional exploration in chicken primary myoblasts (CPMs) with molecular biology technologies including qPCR, cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) and immunofluorescence. When proliferating CPMs were transfected with miR-24-3p mimic, the expression of cyclin dependent kinase inhibitor 1A (P21) was up-regulated and both CCK-8 and EdU assays showed that the proliferation of CPMs was inhibited. However, when the inhibitor was transfected into the proliferating CPMs, the opposite results were found. In differentiated CPMs, transfection with miR-24-3p mimic resulted in up regulation of MYOD, MYOG and MYHC after 48 h. Myotube areas also increased significantly compared to the mimic negative control (NC) group. When treated with inhibitor, differentiation CPMs produced the opposite effects. Overall, we revealed 2 miRNAs (novel_miR_133 and miR-24-3p) significantly related with growth and development and further proved that miR-24-3p could suppress the proliferation and promote differentiation of CPMs. The results would facilitate understanding the effects of miRNAs on the growth and development of chickens at the post-transcriptional level and could also have an important guiding role in yellow-feathered chicken breeding.

miRNA-10a-5p Targeting the BCL6 Gene Regulates Proliferation, Differentiation and Apoptosis of Chicken Myoblasts.

Proliferation, differentiation, and apoptosis are three essential stages in cell development, and miRNAs can achieve extensive regulation of cellular developmental processes by repressing the expression of target genes. According to our previous RNA-seq results, miRNA-10a-5p was differentially expressed at different periods in chicken myoblasts, revealing a possible association with muscle development. In this study, we concluded that miRNA-10a-5p inhibited chicken myoblasts' proliferation and differentiation and promoted chicken myoblasts' apoptosis by directly targeting BCL6, a critical transcription factor involved in muscle development and regeneration. Overexpression of BCL6 significantly facilitated myoblasts' proliferation and differentiation and suppressed myoblasts' apoptosis. On the contrary, knockdown of BCL6 significantly repressed myoblasts' proliferation and differentiation and induced myoblasts' apoptosis. The results above suggest that miRNA-10a-5p plays a potential role in skeletal muscle growth, development and autophagy by targeting the BCL6 gene. We first revealed the functions of miRNA-10a-5p and BCL6 in the proliferation, differentiation, and apoptosis of chicken myoblasts.

Long noncoding RNA profiling reveals that LncRNA BTN3A2 inhibits the host inflammatory response to Eimeria tenella infection in chickens.

Coccidiosis is a widespread parasitic disease that causes serious economic losses to the poultry industry every year. Long noncoding RNAs (lncRNAs) play important roles in transcriptional regulation and are involved in a variety of diseases and immune responses. However, the lncRNAs associated with Eimeria tenella (E. tenella) resistance have not been identified in chickens. In addition, the expression profiles and functions of lncRNAs during E. tenella infection remain unclear. In the present study, high-throughput sequencing was applied to identify lncRNAs in chicken cecal tissues from control (JC), resistant (JR), and susceptible (JS) groups on day 4.5 post-infection (pi), and functional tests were performed. A total of 564 lncRNAs were differentially expressed, including 263 lncRNAs between the JS and JC groups, 192 between the JR and JS groups, and 109 between the JR and JC groups. Functional analyses indicated that these differentially expressed lncRNAs were involved in pathways related to E. tenella infection, including the NF-kappa B signaling, B cell receptor signaling and natural killer cell-mediated cytotoxicity pathways. Moreover, through cis regulation network analysis of the differentially expressed lncRNAs, we found that a novel lncRNA termed lncRNA BTN3A2 was significantly increased in both cecum tissue and DF-1 cells after coccidia infection or sporozoite stimulation. Functional test data showed that the overexpression of lncRNA BTN3A2 reduced the production of inflammatory cytokines, including IL-6, IL-1ß, TNF-α and IL-8, while lncRNA BTN3A2 knockdown promoted the production of these inflammatory cytokines. Taken together, this study identify the differentially expressed lncRNAs during E. tenella infection in chickens for the first time and provide the direct evidence that lncRNA BTN3A2 regulates the host immune response to coccidia infection.

Transcriptome Integration Analysis at Different Embryonic Ages Reveals Key lncRNAs and mRNAs for Chicken Skeletal Muscle.

The growth and development of skeletal muscle at embryonic stages are vital and it directly affects the growth performance of chickens. Long non-coding RNA (lncRNA) plays an important role in this process. In the experiment, we collected the leg muscles of fast- and slow-growing Bian chickens both at 14- and 20-day embryo ages (14E and 20E) for RNA-seq. Finally, 292 and 347 differentially expressed (DE) lncRNAs were identified in F14vsF20 and S14vsS20, and 1,295 and 1,560 DE mRNAs were also screened, respectively. Then we constructed lncRNA-mRNA networks for the two groups, respectively, and found that 6 of the top 10 lncRNAs ranked with degree are same. GO analysis showed that 12 of the top 20 terms were same in the two comparison groups and most of them were related to energy metabolisms, such as cellular respiration and aerobic respiration. KEGG enrichment revealed that up to 16 pathways of the top 20 in F14vsF20 were same as that of S14vsS20 and most of them were related to growth, including citrate cycle (TCA cycle) and oxidative phosphorylation. Further analysis showed that there were 602 and 102 same DE mRNAs and DE lncRNAs between the two comparison groups. We then identified 442 lncRNA-mRNA pairs, including 201 mRNAs and 32 lncRNAs. Protein-Protein Interactions (PPI) network was predicted for the 201 mRNAs and three core networks were obtained using the plug-in MCODE of Cytoscape. Then the function of genes in the three core networks was further analyzed with ClueGo and they were mainly enriched in six groups of biological processes. On this basis, combined with KEGG pathways and lncRNA-mRNA networks, we identified several candidate lncRNAs and mRNAs. Among them, lncRNAs mainly include TCONS_00061389, TCONS_00025495, TCONS_00017622, TCONS_00216258 and TCONS_00084223, and mRNAs include PLK1, BUB1, TTK, NDUFS7 NDUFAB1, PDHA1, CDK1, SDHA, ACO2 and MDH1. The results would provide a foundation for further experiments on the role of lncRNAs in the regulation of muscle development. And it could also contribute to further clarify the regulatory mechanism of chicken skeletal muscle.