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.

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.

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.

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.

Study on the transcriptome for breast muscle of chickens and the function of key gene RAC2 on fibroblasts proliferation.

BACKGROUND: Growth performance is significant in broiler production. In the growth process of broilers, gene expression varies at different growth stages. However, limited research has been conducted on the molecular mechanisms of muscle growth and development in yellow-feathered male chickens. RESULTS: In the study, we used RNA-seq to study the transcriptome of the breast muscle of male Jinghai yellow chickens at 4 (M4F), 8 (M8F) and 12 weeks (M12F) of age. The results showed that 4608 differentially expressed genes (DEGs) were obtained by comparison in pairs of the three groups with Fold Change (FC) ≥ 2 and False Discovery Rate (FDR) ≤ 0.05, and 83, 3445 and 3903 DEGs were obtained separately from M4FvsM8F, M4FvsM12F and M8FvsM12F. Six genes were found as co-differentially expressed in the three age groups, namely SNCG, MYH1A, ARHGDIB, ENSGALG00000031598, ENSGALG00000035660 and ENSGALG00000030559. The GO analysis showed that 0, 304 and 408 biological process (BP) were significantly enriched in M4FvsM8F, M4FvsM12F and M8FvsM12F groups, respectively. KEGG pathway enrichment showed that 1, 2, 4 and 4 pathways were significantly enriched in M4FvsM8F, M4FvsM12F, M8FvsM12F and all DEGs, respectively. They were steroid biosynthesis, carbon metabolism, focal adhesion, cytokine-cytokine receptor interaction, biosynthesis of amino acids and salmonella infection. We constructed short hairpin RNA (shRNA) to interfere the differentially expressed gene RAC2 in DF-1 cells and detected mRNA and protein expression of the downstream genes PAK1 and MAPK8. Results of qPCR showed that RAC2, PAK1 and MAPK8 mRNA expression significantly decreased in the shRAC2-2 group compared with the negative control (NC) group. Western Blot (WB) results showed that the proteins of RAC2, PAK1 and MAPK8 also decreased in the shRAC2-2 group. Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assay both showed that the proliferation of DF-1 cells was significantly inhibited after transfection of shRAC2-2. CONCLUSIONS: The results of RNA-seq revealed genes, BP terms and KEGG pathways related to growth and development of male Jinghai yellow chickens, and they would have important guiding significance to our production practice. Further research suggested that RAC2 might regulate cell proliferation by regulating PAKs/MAPK8 pathway and affect growth of chickens.

Simultaneous Determination of Tetracyclines and Fluoroquinolones in Poultry Eggs by UPLC Integrated with Dual-Channel-Fluorescence Detection Method.

An innovative, rapid and stable method for simultaneous determination of three tetracycline (oxytetracycline, tetracycline and doxycycline) and two fluoroquinolone (ciprofloxacin and enrofloxacin) residues in poultry eggs by ultra-high performance liquid chromatography-fluorescence detection (UPLC-FLD) was established and optimized. The samples were homogenized and extracted with acetonitrile/ultrapure water (90:10, v/v) and then purified by solid-phase extraction (SPE). LC separation was achieved on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm), and the mobile phase was composed of acetonitrile and a 0.1 mol/L malonic acid solution containing 50 mmol/L magnesium chloride (the pH was adjusted to 5.5 with ammonia). When the five target drugs were spiked at the limit of quantification, 0.5 times the maximum residue limit (MRL), 1.0 MRL and 2.0 MRL, the recoveries were above 83.5% and the precision ranged from 1.99% to 6.24%. These figures of merit complied with the parameter validation regulations of the EU and U.S. FDA. The limits of detection and quantifications of the targets were 0.1-13.4 µg/kg and 0.3-40.1 µg/kg, respectively. The proposed method was easily extended to quantitative analyses of target drug residues in 85 egg samples, thus demonstrating its reliability and applicability.

Determination of Eight Coccidiostats in Eggs by Liquid-Liquid Extraction-Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

A method for the simultaneous determination of robenidine, halofuginone, lasalocid, monensin, nigericin, salinomycin, narasin, and maduramicin residues in eggs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The sample preparation method used a combination of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) technology to extract and purify these target compounds from eggs. The target compounds were separated by gradient elution using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC). Tandem mass spectrometry was used to quantitatively and qualitatively analyze the target compounds via electrospray ionization (ESI+) and multiple reaction monitoring mode. The HPLC-MS/MS and UPLC-MS/MS methods were validated according to the requirements defined by the European Union and the Food and Drug Administration. The limits of detection and limits of quantification of the eight coccidiostats in eggs were 0.23-0.52 µg/kg and 0.82-1.73 µg/kg for HPLC-MS/MS, and 0.16-0.42 µg/kg and 0.81-1.25 µg/kg for UPLC-MS/MS, respectively. The eggs were spiked with four concentrations of the eight coccidiostats, and the HPLC-MS/MS and UPLC-MS/MS average recoveries were all higher than 71.69% and 72.26%, respectively. Compared with the HPLC-MS/MS method, utilizing UPLC-MS/MS had the advantages of low reagent consumption, a short detection time, and high recovery and precision. Finally, the HPLC-MS/MS and UPLC-MS/MS methods were successfully applied to detect eight coccidiostats in 40 eggs.

Analysis of gene co-expression networks and function modules at different developmental stages of chicken breast muscle.

The development of poultry muscle fibers after hatching is closely related to meat quality and production efficiency. It is necessary to identify functional modules (groups of functionally related genes) related to muscle development at different developmental stages, and to investigate their relationships based on the weighted gene co-expression network analysis (WGCNA) methods. Accordingly, we investigated the co-expression associations between genes related to chicken breast muscle at four different developmental stages (between 2 and 14 weeks of age), and systematically analyzed the network topology in Jinmao Hua chicken. As a result, 2341 differentially expressed genes were identified and subjected to co-expression analysis. Four modules were identified to be related to a particular growth stage for the development of breast muscle. A series of genes with the highest connectivity were identified in the pink (2 weeks), yellow (6 weeks), green (10 weeks) and black modules (14 weeks), respectively, and visualized by Cytoscape. These hub genes (FGF, MAPKAPK5, NRG1, SCD, ACSL1, PPAR etc.) were mainly enriched in 15 pathways, such as MAPK signaling pathway, NRG/ErbB signaling pathway, and insulin signaling pathway. They shared biological functions related to development of breast muscle and adipogenesis. This is the first study of gene network with different stages of muscle development in Jinmao Hua chicken to observe co-expression patterns. It may contribute to the underlied molecular mechanisms of chicken breast muscle development.

Optimization of ASE and SPE conditions for the HPLC-FLD detection of piperazine in chicken tissues and pork.

Accelerated solvent extraction (ASE) and solid-phase extraction (SPE) conditions were optimized by a high-performance liquid chromatography-fluorescence detector (HPLC-FLD) method for the detection of piperazine in chicken tissues and pork. Piperazine residues were determined by precolumn derivatization with trimethylamine and dansyl chloride. Samples were extracted with 2% formic acid in acetonitrile using an ASE apparatus and purified using a Strata-X-C SPE column. The monosubstituted product of the reaction of piperazine with dansyl chloride was 1-dansyl piperazine (1-DNS-piperazine). Chromatographic separations were performed on an Athena C18 column (250 × 4.6 mm, id: 5 µm) with gradient elution using ultrapure water and acetonitrile (5:95, V/V) as the mobile phase. The calibration curves showed good linearity over a concentration range of LOQ-200.0 µg/kg with a coefficient of determination (R2 ) ≥ .9992. The recoveries and relative standard deviations (RSD values) ranged from 78.49% to 97.56% and 1.19% to 5.32%, respectively, across the limit of quantification (LOQ) and 0.5, 1, and 2.0 times the maximum residue limit (MRL; µg/kg). The limits of detection (LODs) and LOQs were 0.96 to 1.85 µg/kg and 3.20 to 5.50 µg/kg, respectively. The method was successfully applied for the validation of animal products in the laboratory.

Development and Validation of an HPLC-ESI/MS/MS Method for the Determination of Amoxicillin, Its Major Metabolites, and Ampicillin Residues in Chicken Tissues.

A method for the simultaneous analysis of amoxicillin (AMO), amoxicillin metabolites, and ampicillin residues in edible chicken muscle, liver, and kidney samples via high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS) was developed and verified. The extraction and purification procedures involved the extraction of the sample using a liquid-liquid extraction method with acetonitrile to eliminate the proteins. The chicken tissue extract was then injected directly onto an HPLC column coupled to a mass spectrometer with an ESI(+) source. The HPLC-ESI/MS/MS method was validated according to specificity, sensitivity, linearity, matrix effects, precision, accuracy, decision limit, detection capability, and stability, as defined by the European Union and Food and Drug Administration. The linearity was desirable, and the determination coefficients (r2 values) ranged from 0.9968 and 0.9999. The limits of detection and limits of quantification were 0.10-2.20 µg/kg and 0.30-8.50 µg/kg, respectively. The decision limits were 57.71-61.25 µg/kg, and the detection capabilities were 65.41-72.50 µg/kg, and the recoveries of the four target analytes exceeded 75% at the limits of quantification and exceeded 83% at 25, 50, and 100 µg/kg (n = 6 at each level), confirming the reliability of this method for determining these analytes and providing a new detection technology. For real sample analysis, this experiment tested 30 chicken tissue samples, only one chicken muscle, liver, and kidney sample were contaminated with 5.20, 17.45, and 7.33 µg/kg of AMO values, respectively, while other target compounds were not detected in the 30 tested chicken tissue samples.

Development of an Accelerated Solvent Extraction-Ultra-Performance Liquid Chromatography-Fluorescence Detection Method for Quantitative Analysis of Thiamphenicol, Florfenicol and Florfenicol Amine in Poultry Eggs.

A simple, rapid and novel method for the detection of residues of thiamphenicol (TAP), florfenicol (FF) and its metabolite, florfenicol amine (FFA), in poultry eggs by ultra-performance liquid chromatography-fluorescence detection (UPLC-FLD) was developed. The samples were extracted with acetonitrile-ammonia (98:2, v/v) using accelerated solvent extraction (ASE) and purified by manual degreasing with acetonitrile-saturated n-hexane. The target compounds were separated on an ACQUITY UPLC® BEH C18 (2.1 mm × 100 mm, 1.7 µm) chromatographic column using a mobile phase composed of 0.005 mol/L NaH2PO4, 0.003 mol/L sodium lauryl sulfate and 0.05% trimethylamine, adjusted to pH 5.3 ± 0.1 by phosphoric acid and acetonitrile (64:36, v/v). The limits of detection (LODs) and limits of quantification (LOQs) of the three target compounds in poultry eggs were 1.8-4.9 µg/kg and 4.3-11.7 µg/kg, respectively. The recoveries of the three target compounds in poultry eggs were above 80.1% when the spiked concentrations of three phenicols were the LOQ, 0.5 maximum residue limit (MRL), 1.0 MRL and 2.0 MRL. The intraday relative standard deviations (RSDs) were less than 5.5%, and the interday RSDs were less than 6.6%. Finally, this new detection method was successfully applied to the quantitative analysis of TAP, FF and FFA in 150 commercial poultry eggs.

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.

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.

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.

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.

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.

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.

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.

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.

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.