This study was aimed to construct the eukaryotic expression vector of circadian locomotor output cycles kaput (CLOCK) gene in goats, and systematically analyze the biological characteristics of CLOCK protein in goats.Total RNA extracting from ovary tissues in goats was reverse transcribed into cDNA, the coding sequence (CDS) of CLOCK gene in goats was amplified by PCR using the cDNA template.PCR product was ligated to pcDNA3.1-Puro-N-3HA vector by homologous recombination.The recombinant plasmid was identified by PCR, restriction enzyme digestion and sequencing, and the positive plasmid was named pcDNA3.1-3HA-gCLOCK.The pcDNA3.1-Puro-N-3HA and pcDNA3.1-3HA-gCLOCK plasmids were transfected into HEK293T cells, respectively.The expression of CLOCK gene was examined by Real-time quantitative PCR and Western blotting analysis.Meanwhile, several bioinformatics analysis methods were performed systematically on CLOCK gene in goats.The results showed that the length of CLOCK gene in goats were 2 538 bp, the PCR fragment was linked to the linearized pcDNA3.1-Puro-N-3HA and identified by restriction enzyme digestion and sequencing, the eukaryotic expression vector of pcDNA3.1-3HA-gCLOCK was successfully constructed.Real-time quantitative PCR and Western blotting results showed that the expression of mRNA and protein of CLOCK gene in goats transfected with pcDNA3.1-3HA-gCLOCK in HEK293T cells were extremely significantly higher than that in pcDNA3.1-Puro-N-3HA group (P<0.01).Bioinformatics analysis showed that the similarity of CLOCK gene CDS sequence in goats with Ovis aries, Bos taurus and Equus caballus was 99.4%, 98.7% and 95.6%, respectively.CLOCK protein in goats was an unstable protein with certain hydrophilicity, and no transmembrane region and signal peptide was observed.The secondary structure of CLOCK protein consisted mainly of alpha helix, extended chain, beta turn and random coil, and the tertiary structure was highly similar to CLOCK protein in mouse and human.In this study, the eukaryotic expression vector of CLOCK gene in goats was successfully constructed, and bioinformatics analysis was carried out, which provided materials for the further study on the biological functions of CLOCK gene and the transcriptional regulation mechanism of circadian clock system in goats.

The purpose of this experiment was to clone the CIDEa gene sequence of Bama Xiang pig, predict its structure and function, and analyze its tissue expression.According to the CIDEa gene sequence of Sus scrofa published by NCBI (accession No.: NM_001112696.2), primers were designed with Oligo 7.0 software, and the CIDEa gene sequence of Bama Xiang pig was amplified and cloned by RT-PCR.The nucleotide sequence and the hydrophobicity, physicochemical properties, transmembrane domain, secondary structure, tertiary structure and interaction proteins of the encoded proteins were analyzed by bioinformatics software.The expression of CIDEa gene in subcutaneous fat, liver, kidney, lung, spleen, longissimus dorsi muscle and heart of Bama Xiang pigs were detected by Real-time quantitative PCR.The results showed that the CDS regions of CIDEa gene in Bama Xiang pig was successfully obtained, which was 660 bp in length, encoded 219 amino acid.The nucleotide similarity with Sus scrofa, Bos taurus, Equus caballus, Canis lupus familiaris, Homo sapiens, Macaca mulatta and Mus musculus in GenBank were 99.4%, 84.7%, 81.5%, 80.9%, 79.7%, 78.9%, and 76.1%, respectively.Compared with the nucleotide sequence of Sus scrofa CIDEa in GenBank, there were four base mutations, of which A609G and T627C were synonymous mutations, and C173T (Pro→Leu) and C631T (Arg→Cys) were missense mutations.The molecular weight of CIDEa protein in Bama Xiang pig was 24 483.57 u, the isoelectric point was 9.48, and the instability index was 52.86, indicating that the protein was unstable.The protein contained the highest Leu (13.2%) and the lowest Trp (0.5%).CIDEa protein in Bama Xiang pig was an extramembrane hydrophilic protein, which contains a superfamily domain (CIDE-N).The secondary structure prediction of the protein showed that the percentage of alpha helix, beta turn, extended chain, random coil of CIDEa protein in Bama Xiang pig was 45.66%, 5.94%, 14.61% and 33.79%, respectively.The predicted results of the tertiary structure were consistent with those of the secondary structure.Protein interaction analysis showed that CIDEA protein of Bama Xiang pig interacted with PPARG, PPARGC-1, COX8H, PRDM16, DIO2, TMEM26, ELOVL3, COX7A1, CIDEC, DFFB proteins.The results of Real-time quantitative PCR showed that the expression of the CIDEa gene was the highest in subcutaneous fat of Bama Xiang pig and significantly higher than that in other tissues (P<0.05), and the lowest expression in heart.The results provided a theoretical basis for further exploring the regulatory mechanism of CIDEa gene on fat deposition in Bama Xiang pig.

The aim of this study was to clone the cooperative hemolysin protein (CAMP) gene of Riemerella anatipestifer, and analyze its prokaryotic expression and bioinformatics. CAMP gene was amplified and cloned from RA Guizhou serotype 2 strain(RA-SS-8), and the recombinant prokaryotic expression plasmid pET-28a-CAMP was constructed.The plasmid was transformed into Escherichia coli Rosetta(DE3) competent cells.After identification, the recombinant bacteria were induced to express and purified with IPTG.SDS-PAGE and Western blotting were used to analyze the characteristics of the expressed protein.CAMP gene was analyzed by bioinformatics analysis software.The results showed that the size of CAMP gene was 1 026 bp, and the sequence of CAMP gene was 99.7% simarility with 10 RA reference strains published in GenBank, such as RA-LZ01 (CP045564.1).By BamH Ⅰ and Xho Ⅰ double digestion, the size of 5 369 and 1 026 bp fragments were obtained, indicating that the pET-28a-CAMP recombinant plasmid was successfully constructed.SDS-PAGE and Western blotting analysis results showed that the expressed recombinant protein was about 37 ku in soluble form, and could react with His-tag monoclonal antibody at 37 ku, which was consistent with the expected results.Bioinformatics analysis results showed that the molecular formula of CAMP was C₁₆₇₀H₂₆₅₉N₄₃₇O₅₀₀S₁₂, containing 341 amino acids, the theoretical isoelectric point (pI) was 5.62, and the coefficient of instability was 40.71, indicating that CAMP was an unstable and weakly acidic protein.The results of hydrophobicity prediction showed that CAMP protein was hydrophilic.CAMP protein had no transmembrane domain, no signal peptide, 3 O-glycoylation sites, no N-glycoylation sites, 34 phosphorylation sites and a total of 17 epitopes.The secondary and tertiary structures showed that the protein was dominated by alpha helix and random coil.The above results might provide reference for further study on the function of CAMP protein and the development of Riemerella anatipestifer vaccine.

The object of this study was to explore the bioinformatics characteristics of miR-18a-5p and its changes in tissue expression in different growth stages of chickens.In this study, Suqin No.3 was used as the experimental material to detect the tissue expression of miR-18a-5p in chicken at different growth stages by Real-time quantitative PCR.The miR-18a-5p sequence of vertebrates was retrieved by references and miRBase.The position of miR-18a-5p in the genome was determined using the Ensembl database.Phylogenetic tree was constructed based on the mature sequence.The target genes of miR-18a-5p were predicted using miRmap, microT, miRanda and TargetScan websites, and GO and KEGG analysis was carried out.The results of tissue expression analysis showed that the expression of miR-18a-5p sequence of chicken in heart, spleen, kidney and hypothalamus was significantly higher than that in other tissues except brain (P<0.05).Compared with 3-day-old chicks, the expression of miR-18a-5p in heart, spleen, kidney, leg muscle and hypothalamus increased significantly at 90-day-old chicks (P<0.05).A total of 52 miR-18a-5p sequences were found in 50 vertebrates, and almost all species had only one mature sequence.A total of 52 miR-18a-5p sequences were found in 50 vertebrates, and almost all species had only one mature sequence.Gene mapping analysis showed that chicken miR-18a-5p was located in the gene spacer on chromosome 1.The multiple sequence alignment analysis showed that the mature sequences of miR-18a-5p in different species had high homology and were more conservative among species.Phylogenetic tree analysis showed that chicken miR-18a-5p clustered with other birds such as pigeon and zebra finch, which showed that miR-18a-5p was conservative in the evolution process.Target genes prediction and functional analysis revealed that there were 121 target genes in miR-18a-5p.GO analysis showed that the target genes were mainly enriched in protein ubiquitination, cell cycle arrest, negative regulation of interleukin-6 production and so on.KEGG pathway analysis showed that the target genes were mainly enriched in cell cycle, Wnt and FoxO signaling pathways.Multiple genes were related to muscle growth and cell proliferation were enriched on the related pathways.In conclusion, chicken miR-18a-5p was a miRNA which was widely expressed in different tissues, which might regulate muscle growth and cell proliferation and differentiation through Wnt and FoxO signaling pathway.This study provided a reference for the in-depth research of miR-18a-5p functions and regulatory mechanisms.

The purpose of this study was to clone C-C chemokine receptor type 5 (CCR5) gene in Chinese Holstein dairy cows, analyze its bioinformatics, and explore the expression in inflammatory and healthy tissues of dairy cows.The full-length CDS sequence of CCR5 gene in Holstein dairy cows was amplified by PCR and cloned, and the pMD18-T vector was connected and transformed into Escherichia coli DH5α competent cells.The positive clones were screened and sequenced by blue-white blot method, and sequence similarity alignment and phylogenetic tree construction were carried out.The encoded proteins were analyzed by a variety of online bioinformatics softwares.Real-time quantitative PCR was used to detect the expression of CCR5 gene in the mammary tissues of healthy and inflammatory dairy cows.The results showed that the total length of CDS region of CCR5 gene in Chinese Holstein dairy cows was 1 059 bp, which encoded 352 amino acids.The results of similarity and genetic evolution analysis showed that the genetic distance of CCR5 gene was 96.0% between Chinese Holstein dairy cows and Ovis aries, and 61.0% between Chinese Holstein dairy cows and Gallus gallus.CCR5 genes were highly conserved among different species.The molecular weight of CCR5 protein was 40.235 ku, and the theoretical isoelectric point (pI) was 9.30.CCR5 protein was hydrophobic protein but not secreted protein, which mainly existed in cytoplasm.In the secondary structure, alpha helix and random coil accounted for 51.14% and 32.95%, respectively.The tertiary structure prediction results were consistent with that of the secondary structure.Real-time quantitative PCR results showed that the expression of CCR5 gene in normal mammary tissue was extremely significantly lower than that in mammary tissue of dairy cows with mastitis (P<0.01), suggesting that CCR5 might be involved in the occurrence of mastitis in dairy cows.The results provided a theoretical basis for further study of the function of CCR5 protein in dairy cows, and had important significance for exploring the regulatory function of CCR5 gene in dairy cows mastitis.

The objective of this study was to clone and bioinformatics analyze the CDS of porcine sentrin-specific protease 1 (SENP1) gene, and investigate the expression pattern of SENP1 gene in PK15 cells infected with Japanese encephalitis virus (JEV).The specific primers were designed according to the predicted sequence of porcine SENP1 gene (accession No.:XM_013997974.2) published in GenBank.The sequence of SENP1 gene CDS was cloned and sequenced by RT-PCR and T/A cloning technology, and bioinformatics analysis was carried out.Real-time quantitative PCR was used to analyze the expression pattern of SENP1 gene in JEV-infected PK15 cells at different time points (0, 12, 24, 36 and 48 h).The results showed that the full length of SENP1 gene CDS was 2 034 bp, encoding a total of 677 amino acids.The sequence alignment and phylogenetic tree analysis results showed that the similarity of amino acid sequence of porcine SENP1 with Capra hircus, Canis lupus, Homo sapiens and Bos taurus was 94.8%, 94.2%, 93.9% and 93.8%, respectively.The results indicated that porcine SENP1 was highly conserved among these species.The genetic relationship of SENP1 between Sus scrofa and Canis lupus was close.The results of bioinformatics analysis showed that the relative molecular weight of SENP1 protein was 76 825.76, the isoelectric point was 8.53, the half-life in vitro was 30 h, the instability coefficient was 53.59, and the total average hydrophilic index was －0.622.The porcine SENP1 protein contained no signal peptide sequence and transmembrane domain.The ratio of alpha helix, random coil, extended chain and beta turn in secondary structure was 31.31%, 46.68%, 16.25% and 5.76%, respectively.The tertiary structure analysis showed that there were 8 alpha helix and 7 beta turn regions in porcine SENP1 protein.Real-time quantitative PCR results showed that the expression of SENP1 gene was up-regulated in JEV infected PK15 cells, and the expression of SENP1 gene at 48 h after infection was significantly higher than control group (P<0.05).This study provided reference materials for further study of porcine SENP1 gene function.

In order to illustrate the transcriptomic profile of spleen in mice upon Pasteurella multocida infection, the mouse model of P.multocida infection was established by intraperitoneal injection.Transcriptome sequencing technology was performed to obtain spleen transcriptome data of infected and normal mice.Furthermore, COG, KOG, eggNOG, GO, KEGG database were utilized to accomplish functional annotation and the differentially expressed genes (DEGs) analysis.In order to screen out the genes that played the key role in immune pathways, DEGs which were significantly enriched in several immune pathways, were used to perform protein interaction analysis with STRING software and KEGG mapper.Finally, 10 key genes were randomly selected for Real-time RT-PCR verification.Transcriptome analysis results showed that compared with the normal group, 3 380 DEGs were screened in the infection group (P<0.01, log₂|FoldChange|≥0.5), of which 1 691 DEGs were up-regulated and 1 689 DEGs were down-regulated.Gene function enrichment analysis showed that DEGs in spleen of the infected group mainly played the function of signal transduction.The cytokine-cytokine receptor interaction pathway, chemokine signal pathway, HIF-1 signal pathway and TNF signal pathway were significantly enriched in the context of P.multocida infection.Protein interaction analysis screened out 28 core DEGs.After verification with Real-time RT-PCR, the expression of 9 genes were consistent with sequencing results, namely C3, Cd4, Cxcl13, Lck, Gnai1, Grap2, IL-6, Cxcr6 and Serping1 genes.This study demonstrated that the spleen played the essential immune role in resisting the invasion of P.multocida and could provide valuable information on the molecular mechanism of the interaction between goat-derived P.multocida and the host.

Single base editor (BE) is a new generation of gene editing technology based on CRISPR/Cas9.Compared with the traditional gene editing technology, single base editing has the unique advantages of high efficiency, safety and less by-products.It is gradually replacing the traditional techniques such as zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, clustered regularly interspaced short palindromic repeats/Cas (CRISPR/Cas) and become the mainstream gene editing technology.Single base editing technology has greatly promoted the development of livestock gene editing and human disease treatment and other research fields.With the continuous optimization of deaminase and Cas nuclease, the accuracy of single base editing is improving and the targeting range is expanding, so that the technology can be more widely used in the field of life science.The advantages and disadvantages of previous gene editing technology were compared.The main function structure, editing principle and optimization process of single base editing technology were reviewed.The advantages and disadvantages of single base editing technology were analyzed.This review focuses on the application of single base editing technology in the improvement of important economic traits such as disease resistance, meat yield, wool yield and fecundity of livestock and the treatment of human gene diseases.In this review, the further application of single base editing technology in improving multiple economic traits of livestock and overcoming the problem of pig gamete cryopreservation was proposed.

The purpose of this study was to investigate the effect of histone demethylase (JHDM2A) on porcine fibroblast induced pluripotent stem cell efficiency, and to explore its intrinsic molecular mechanism.Porcine fibroblasts were used as materials, doxycycline (DOX)-inducible lentiviral vectors were used to produce iPSCs, and JHDM2A was overexpressed on this basis.The effect of JHDM2A on induction efficiency was detected by alkaline phosphatase (AP) staining and induction time axis.The pluripotency of the iPSCs was determined by PCR.The protein expression of the pluripotent factors was detected by immunofluorescence.The effects of JHDM2A on the expression of pluripotent factors and histone methylation related genes after clonies formation and during clonies differentiation were detected by Real-time quantitative PCR.The results showed that the cells of the JHDM2A overexpressed group deformed on the 3rd day and formed iPSCs clonies on the 8th day, which were 1 and 2 d earlier than those of control group.The results of AP staining showed that compared with the control group, the morphology of iPSCs in the JHDM2A overexpressed group was significantly improved, the AP staining was deeper, and the induction efficiency was increased by 8 times.Conventional PCR results showed that endogenous Oct4, Sox2, Klf4, c-Myc and Nanog were expressed in JHDM2A overexpressed group, control group and porcine fibroblasts (PFF), and the expression of Oct4 in iPSCs was higher than that in PFF.Immunofluorescence results showed that Oct4, Sox2, Stat3 and JHDM2A were expressed in iPSCs of JHDM2A overexpressed group, while SSEA1 and SSEA4 were weakly expressed.Real-time quantitative PCR results showed that compared with control group and PFF, the expressions of iPSCs genes Sox2, Klf4, c-Myc, Nanog, Oct4 and Tcl1 in JHDM2A overexpressed group were significantly increased (P<0.05), and the expressions of Tfcp2l1 and Zfp57 were significantly decreased (P<0.05).After DOX was removed from P5 iPSCs culture medium in the JHDM2A overexpressed group, with the increase of generation, the expressions of Sox2 and Nanog gradually decreased, the expressions of Klf4 and c-Myc first increased and then decreased, and the expressions of Oct4, Tcl1, Tfcp2l1 and Zfp57 first decreased and then increased.These results suggested that overexpression of JHDM2A could improve the induction efficiency of porcine fibroblasts by promoting histone demethylation.

In order to investigate the regulation effect of puerarin on the adipogenic differentiation of precursor adipocytes in Songliao Black pig, 0, 10, 20, 40, 60 and 80 μmol/L puerarin were added into the cell induction solution to induce adipogenic differentiation, respectively.In order to determine the effect of lipid deposition and differentiation, oil red O staining and triglyceride assay were used to detect the accumulation of lipid droplets and triglyceride content during adipocyte differentiation, and to determine the best concentration of puerarin.The mRNA expression levels of lipid marker genes cell peroxisome proliferator-activated receptor γ(PPARγ), CCAAT-enhancer binding protein α(C/EBPα) and genes related to adipogenic differentiation like acetyl-CoA carboxylase (ACC), fatty acid binding protein(FABP4), stress protein(TRIB) and forked frame protein O1 (FOXO1) were detected by Real-time quantitative PCR in the control group and the optimal puerarin concentration group.The protein expression levels of PPARγ and C/EBPα were detected by Western blotting.The results showed that compared with control group, 20, 40 and 60 μmol/L puerarin significantly increased the content of lipid droplets and triglycerides (P<0.05), and 40 μmol/L puerarin had the best effect.Real-time quantitative PCR results showed that compared with control group, 40 μmol/L puerarin significantly up-regulated the expression of adipogenic marker genes PPARγ and C/EBPα and adipogenic differentiation genes ACC, FABP4, FOXO1 and TRIB (P<0.05).Western blotting results showed that compared with control group, 40 μmol/L puerarin significantly increased the expression of PPARγ protein (P<0.05).In conclusion, 40 μmol/L puerarin could promote the adipogenic differentiation and lipid deposition of preadipocytes in Songliao Black pigs.

Microribonucleic acid (microRNA, miRNA) is a type of endogenous non-coding RNA, which have a wide range of gene expression regulation.They can regulate the expression of corresponding proteins by affecting target genes at the post-transcriptional level, and then regulate cell life activities.miRNA is expressed in mammalian follicular granulosa cells and regulate the apoptosis of granulosa cells.Granulosa cells, as the most numerous cell group in ovarian follicles, play a vital role in the process of follicular development.They not only provide nutrients for oocytes, but also regulate their development and maturity.Granulosa cell apoptosis is an important cause of follicle atresia, which affects the number and quality of follicles and thus affecting the reproductive performance of female animals.The apoptosis of granulosa cells is regulated by a variety of factors.This paper briefly describes the regulatory effect and mechanism of miRNA on ovarian granulosa cell apoptosis, including miRNA regulating granulosa cell apoptosis by regulating hormone secretion and the expression of apoptosis-related factors, the effect of miRNA on granular cell apoptosis related signal pathway, miRNA regulating ovarian-derived diseases caused by granular cell apoptosis, and summarizes the miRNA that regulate granular cell apoptosis and the potential role of miRNA in disease diagnosis and treatment, in order to provide guidance and reference for the pathogenesis and treatment of subsequent related ovarian diseases and improve the reproductive performance of female mammals.

The purpose of this study was to evaluate the variation in gastrointestina1 structure and function of Hu lambs pre and post-early weaning lambs so as to provide theoretical basis for early breeding of lambs.22 male lambs with similar birth weight(3.81 kg士0.55 kg) were selected.Lambs were fed breast milk from 1 to 7 days old, separated from ewes at 8-day-old, and began to be fed milk replacer (MR) (at 2% of BW measured on day 8, three equal amounts) and starter feeds (ad libitum).MR feeding was stopped at 35 days of age.Six lambs were slaughtered at 8, 21 and 42 days of age, respectively, the weight of rumen, reticulum, omasum and abomasum, and the length and weight of duodenum, jejunum, ileum, cecum, colon and rectum were measured.The rumen and small intestine tissues were collected, sliced and stained with eosin and hematoxylin.The rumen nipple length, width, circular muscle thickness, villus height, width and recess depth of small intestine were detected, and the villus to recess ratio (villus height/recess depth) was calculated.The contents of rumen, duodenum and jejunum were collected to detect the activities of amylase, cellulase, lactase, trypsin and lipase and the concentrations of ammonia nitrogen (NH₃-N) and volatile fatty acids (VFA).The results showed that the weight of rumen, reticulum, omasum and the proportion of rumen to total stomach weight increased significantly with the increase of day age (P<0.05), and the proportion of abomasum weight to total stomach weight decreased with the increase of day age (P<0.05).The length and weight of duodenum, jejunum, ileum, cecum, colon and rectum increased with age (P<0.05), the ratio of duodenum and ileum at 42 days old was significantly higher than that at 8 and 21 days old (P<0.05), the height, width and thickness of rumen papilla increased significantly with age (P<0.05), the villus height, villus width and recess depth of duodenum and jejunum decreased with the increase of age.The activities of duodenal amylase, trypsin, lipase, jejunal amylase and trypsin increased significantly with age (P<0.05), The activity of amylase and the concentrations of acetic acid, propionic acid and butyric acid in rumen of 42-day-old lambs were significantly higher than those of 21 day old lambs (P<0.05).The above results showed that compared with before weaning (21-day-old), the rumen development of weaned lambs was enhanced, the tissue morphology of abomasum and small intestine became smaller, and the activities of main digestive enzymes in rumen and small intestine were enhanced.

In order to find out the pollution status of mycotoxins and its impact on animals in silage corn of China, 251 samples (90 copies of whole plant corn and 161 copies of silage corn) were collected from differernt large-scale pastures located in 28 counties of 9 provinces (autonomous regions), including Northeast (Heilongjiang), Northwest (Inner Mongolia, Shanxi), Huang-Huai-Hai (Henan, Hebei), Central (Jiangsu), Southwest (Guizhou, Sichuan) and Tibet.The contents of 12 types of mycotoxins, such as aflatoxin (AFs), vomiting toxin (DON), fumonisin B (FB) and zearalenone (ZEN) were measured and the animal health risk was assessed by exposure assessment.The results showed that:①With referencing to feed hygiene standard of China, the over-standard rate of ZEN was 1.19%.When silage corn was used as the main roughage for lactating dairy cows, the over-standard rate of FB and AFs were 23.90% and 13.94%, respectively, referencing to the feed safety standards of FDA and EC accordingly.②There was no significant difference in the content of mycotoxins between whole plant corn and whole plant corn silage (P>0.05).③The detected type and contents of mycotoxins in Huang-Huai-Hai area, Central area and Southwest area were higher, and the content was lower in Tibet.④Among different animals, the order of animal health risk was lamb>calf>dairy cow, and the order of mycotoxin risk in lamb was AFs>ZEN>DON>FB when fed with the polluted corn silage.In summary, the over-standard rates of mycotoxins in whole plant corn and whole plant corn silage in China were low.The relatively polluted areas were Huang-Huai-Hai, Central and Southwest China.In these areas, when silage corn was used as the main roughage for lactating dairy cows, the over-standard risk was higher;When corn silage was used as the main roughage for young animals, AFs contamination might bring animal health risks.

The purpose of the experiment was to study the effects of castration at different months on the content of muscle fatty acids in three high-grade parts eye muscle, sirloin steak and upper brain of Yanbian Yellow cattle.50 healthy 6-month-old Yanbian Yellow cattle with similar body weight (150 kg±7.5 kg) were randomly divided into 5 groups, with 10 cattle in each group.The experimental group was castrated at 6, 8, 10 and 12-month-old (recorded as YG6, YG8, YG10 and YG12 groups), and the control group was not castrated (recorded as WYG).The experimental period was 24 months.The cattle were fed uniformly and freely.At the age of 30 months, the muscle samples of eye muscle, sirloin steak and upper brain were taken to determine the content of fatty acids.The results showed that:①Compared with WYG group, in eye muscle, the contents of C14∶1cis-9 in YG8 and YG12 groups were extremely significantly increased (P<0.01), while in YG6 and YG10 groups were significantly increased (P<0.05).The content of C18∶1cis-9 in YG6 group was significantly increased (P<0.05).The contents of C16∶0 in YG6 and YG10 groups were significantly decreased (P<0.05).The content of total saturated fatty acids was decreased in test groups (P>0.05), and the contents of total unsaturated fatty acids and total monounsaturated fatty acids were significantly increased in YG6 and YG10 groups (P<0.05).The content of total polyunsaturated fatty acids was extremely significantly increased in YG10 group (P<0.01), and were significantly increased in YG6, YG8 and YG12 groups (P<0.05).②Compared with WYG group, in sirloin steak, the contents of C14∶1cis-9, C16∶1cis-9 and stearic acid were significantly increased and the content of C16∶0 was significantly decreased in test groups (P<0.05).The content of C18∶1cis-9 was significantly increased (P<0.05) and the content of total saturated fatty acids was significantly decreased in YG6 group (P<0.05).The contents of total unsaturated fatty acids and total monounsaturated fatty acids were significantly increased in YG6 and YG10 groups (P<0.05).③Compared with WYG group, in upper brain, the content of C14∶0 in YG8 group was significantly decreased (P<0.05).The content of C16∶0 was extremely significantly increased (P<0.01) and the contents of total unsaturated fatty acids and total monounsaturated fatty acids were significantly increased in YG6 and YG10 groups (P<0.05).The content of total saturated fatty acids was significantly decreased in YG6 group (P<0.05).In conclusion, castration could increase the content of unsaturated fatty acids and reduce saturated fatty acids in high-grade parts of Yanbian Yellow cattle.The ratio of unsaturated fatty acids to saturated fatty acids in 6-month-old castration group was the highest.

The effect of 5-hydroxytryptamine (5-HT) on feed intake has been studied for decades.Tryptophan(Trp), an essential amino acid, is the precursor of 5-HT in the body, which is synthesized in the brain and intestine, and plays an important role in the body.However, 5-HT can not pass through the blood-brain barrier, so the 5-HT in central nervous system and peripheral are two independent systems, and play their roles in dependently.At present, a large number of studies have shown that a variety of central 5-HT receptors regulate animal feeding in different ways, mainly including 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C, 5-HT3A, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7.For example, lorcaserin, a clinically approved anti-obesity drug, is a 5-HT2C receptor agonist.In addition, central 5-HT can cooperate with peripheral hormones such as glucagon-like peptide-1, ghrelin and cholecystokinin to regulate animal feeding behavior.Peripheral 5-HT can also induce satiety by mediating glucagon-like peptide-1 and cholecystokinin.The increase in the synthesis of 5-HT in animals occurs primarily by the addition of tryptophan to the feed in livestock production.However, the research on the regulatory mechanism of tryptophan on livestock feed intake is not perfect at present.And the study showed that different concentrations of tryptophan had different effects on animal feed intake, and the concentration of optimal tryptophan in different livestock feed.Whereas tryptophan mainly regulates animal feeding through its metabolite 5-HT.Based on a large number of worldwide latest research, this paper is to summarize the research progress of 5-HT in regulating animal feeding behavior through central and peripheral pathways, hoping to provide a theoretical basis for the application of tryptophan metabolite 5-HT to animal feed regulation.

The resource utilization of food waste (FW) has received widespread attention.Incineration, burying and composting, anaerobic digestion are increasingly criticized for their problems of environmental sustainability.Feed processing technology can convert FW into high-value feed, reducing solid waste emissions, breeding costs, and environmental pollution.Feed technology is in line with the principles of reduction, reuse and recycling of organic waste treatment.The authors reviewed the feed technology of FW and its application in animal production.Feed technology mainly includes heat treatment, enzyme treatment, algae culture, insect breeding, and fermentation technology.Heat treatment can ensure the safety of microorganisms, which is the pretreatment technology of all technologies.Algae culture and insect breeding technology can transform waste sugars and amino acids into plant and insect proteins and avoid homologous pollution.However, it is not clear whether algae and insects can accumulate pollutants in the process of FW utilization.Enzyme treatment and fermentation technology can transform macromolecules into small molecules.The catalytic performance and stability of enzymes are easily affected by external conditions, so it is necessary to consider special enzyme preparations.Fermentation has a high degree of mechanization and resource utilization.The core of fermentation technology is microorganism, which should be effective and able to make full use of FW.The application of FW in animal production is less at present.In the production and application, it is need to pay attention to the dosage and use time of FW feed.In order to make FW feed really enter the food chain as animal feed, researchers must study the growth performance and product characteristics of different animals (include different growth stages) to ensure that animal health and their products have no negative impact on human health.

Corn straw is one of the most abundant lignocellulosic biomass in crop residues.It is mainly composed of cellulose, hemicellulosic and lignin through hydrogen bonds, other chemical bonds and molecular bonds to form polymer compounds with complex polymerization structure, which makes its low availability as animal feed.Steam explosion is a technology which applies steam ejection principle to pretreat biomass in explosion process.The steam explosion technology was applied to the treatment of corn straw, which could effectively separate cellulose and had obvious depolymerization effect on corn straw, which was beneficial to improve the utilization degree of corn straw as feed.In this paper, the principle and parameters of steam explosion technology were introduced, and the effects of steam explosion technology on fiber structure, chemical composition, enzymatic hydrolysis sugar production, nutrient digestibility of corn stalk and microbial attachment in rumen were reviewed.Existing studies have shown that steam explosion can make steam molecules penetrate into plant tissues, and transform internal energy into mechanical energy through instantaneous release, acting on the cell layers of biomass tissue to achieve the purpose of raw material decomposition.The content of hemicellulose and lignin can be decreased by changing the fiber structure of corn straw, and the content of cellulose can be increased.With the increase of steam blasting pressure and maintaining pressure time, the content of hemicellulose can be decreased significantly, and the increase of material moisture content will lead to the increase of cellulose content.With the increase of steam burst pressure and maintenance time, the reduction sugar yield of maize straw in vitro fermentation increased.The in vitro gas production, gas production rate, dry matter and acid detergent fibre of corn stalks were significantly increased, the digestibility and utilization rate of ruminant corn stalks were improved, the cellulose of corn stalks was fully utilized, and the nutritional value of corn stalks was improved.By destroying the surface structure of corn stalks, rumen microorganism can be attached to corn stalks, thus promoting the degradation of corn stalks.To sum up, steam explosion technology can improve the utilization rate of corn straw as feed.

This experiment was aimed to use resveratrol (Re) and melatonin (MT) to improve the efficiency of electroporation transfection of sheep fetal fibroblasts, and optimize the in vitro maturation system of oocytes, in order to improve the production efficiency of transgenic animals.Sheep fetal fibroblasts were divided into control group, high, medium and low concentrations of resveratrol groups (R^-5, R^-6 and R^-7), resveratrol and melatonin combined groups (R^-5M^-5and R^-6M^-7), 6 groups in total.The optimization scheme of electrotransfer system was determined by electrotransfection efficiency and cell apoptosis.The mechanism was studied by detecting cell cycle, mitochondrial membrane potential and reactive oxygen species (ROS).Sheep oocytes were divided into control group and combined addition group of resveratrol and melatonin (R^-5M^-5, R^-6M^-7), the effect of ovine oocytes on in vitro maturation was detected by cumulus cell expansion, first polar body excretion and cleavage rate of in vitro fertilized embryos.The results showed that the P3 generation of isolated sheep fetal fibroblasts proliferated vigorously, and the cell growth curve showed a typical S shape.When compared with control group, the electrotransfection efficiency of sheep fetal fibroblasts in all experimental groups was significantly increased (P<0.05), R^-5M^-5 group had the highest electrotransfection efficiency, R^-6M^-7 group significantly decreased the cell apoptosis rate (P<0.05), R^-5, R^-6, R^-5M^-5 and R^-6M^-7 groups significantly promoted the cell proliferation (P<0.05), R^-6, R^-7, R^-5M^-5 and R^-6M^-7 groups increased the proportion of G1 phase cells, while S phase cells decreased relatively, but the difference was not significant (P>0.05).ROS level was significantly decreased in R^-6, R^-7, R^-5M^-5 and R^-6M^-7 groups (P<0.05), and mitochondrial membrane potential was significantly increased in R^-6, R^-5M^-5 and R^-6M^-7 groups (P<0.05).R^-6M^-7 group significantly promoted the expansion of cumulus cells, and significantly increased the rate of first polar body extrusion and the development ability of in vitro fertilization embryo (P<0.05).In conclusion, resveratrol and melatonin could optimize the electroporation transfection system, improve the in vitro maturation efficiency of oocytes, and provide references for the application of resveratrol and melatonin in the production of transgenic animals.

The aim of this study was using transcriptome to analyze the deposition mechanism of porcine intramuscular fat (IMF) content, in order to find the pathways and candidate genes related to IMF deposition.The whole sib individuals with large differences in IMF content were selected for double terminal chain specific transcriptome sequencing, and the related genes were screened by edgeR software.ClusterProfiler software was used to perform GO function enrich and KEGG pathway analysis on differential genes, and molecular module (MCODE) analysis was used to mine the intramuscular fat-related candidate genes.The results showed that a total of 474 differentially expressed genes were screened, of which 329 were significantly up-regulated and 145 were significantly down-regulated, enriched and involved in 20 biological processes.Combined with GO network analysis, a total of 15 IMF-related processes were screened, mainly including:Regulation of ion transmembrane transport, regulation of glucose transmembrane transport membrane potential and fatty acid metabolism.MCODE analysis results showed that the differentially expressed genes PPP1CB, CLCA1, COX4I1, SGK1, PAK, PC, KRAS, MAST1, VWC2, CACNG family genes, especially PPP1CB, CLCA1, COX4I1, KRAS and CACNG family genes, might be key genes for regulating IMF, and could be used as IMF candidate gene for further study.In conclusion, this study had identified some new pathways for the action of IMF and screened out candidate genes related to IMF content, which could provide materials for the study of IMF deposition mechanism and reference for the breeding of high-quality meat quality in pigs.

In order to explore the impacts of non-genetic factors on test-day lactose percentage(LP) in Holstein cows, and estimate the heritability of test-day LP and its genetic correlations with somatic cell score (SCS) and milk yield (MY), the test-day DHI records of 11 554 lactating cows from 6 herds were collected from 2017 to 2019 in Beijing.A mixed linear model was used to analyze the impacts of influencing factors affecting test-day LP, and the repeatability model were employed to estimate the genetic parameters for test-day LP, SCS and MY using the DMUAI module of DMU software.Furthermore, the genetic trend of test-day LP was also analyzed.The results showed that the average of test-day LP was 5.10% in Holstein cows in Beijing.The parity, lactation stage, herd, test year and test season had extremely significant impacts on LP (P<0.01).The heritability estimate for test-day LP was 0.097, which was higher than that of SCS, and the repeatability estimate was 0.162 in Holstein cows in Beijing.In general, an increasing trend was observed according to estimated breeding value of test-day LP from 1994 to 2019.The genetic correlations of LP with SCS (-0.49) and MY (0.28) were moderate to low, which indicated the cows with higher LP would have the higher MY and the lower somatic cell count.In this study, the results were beneficial for in-depth genetic modeling analysis for test-day LP, and the genetic foundation of test-day LP was initially revealed.

In order to investigate the polymorphism of METTL23 gene and its association with reproductive traits in Songliao Black pig.178 Songliao Black sows were selected as the research objects.The single nucleotide polymorphisms (SNPs) of exons 1-5 of METTL23 gene were screened by Sanger direct sequencing, and the correlation between SNP of METTL23 gene and reproductive traits (total number born, number born alive, birth weight, 3-week weight, weaning weight, weaning piglet number and nipple number) were analyzed by using SPSS 19.0 software.The results showed that only one SNP site (named A62G) was found in exon 4 of METTL23 gene in Songliao Black pig.There were three genotypes (AA, AG and GG) in the research population.GG and G were dominant genotype and allele, which was in Hardy-Weinberg balanced (P>0.05).The genetic homozygosity (Ho) of this locus was 0.6516 which was higher than that of genetic heterozygosity (He) of 0.3484, indicating that it had little variation in the population of Songliao Black pigs.The polymorphism information content (PIC) was 0.2877, which belonged to moderate polymorphism (0.25<PIC<0.5), indicating that the genetic marker could provide a certain amount of genetic information.Correlation analysis results showed that the total litter size, live litter size and weaned piglets of GG genotype were significantly or extremely significantly higher than those of AA genotype (P<0.05; P<0.01), and the total litter size was also significantly higher than that of AG genotype individuals (P<0.05).The number of weaned piglets in AG genotype was significantly higher than that in AA genotype (P<0.05).There were no significant differences among genotypes in birth weight, 3-week-old weight, weaning weight and nipple number (P>0.05).In conclusion, there was a SNP site A62G in exon 4 of METTL23 gene, which was significantly correlated with the litter size and might be a genetic marker for litter size in Songliao Black pig molecular breeding.

This study was aimed to explore the polymorphism of myostatin (MSTN) gene and its effect on the growth traits in East Friensian sheep.DNA was extracted from a total of 324 East Friensian sheep, PCR amplification was performed on the exon sequence and UTR of MSTN gene.SNP were identified by the first-generation sequencing technology and its genetic diversity parameters were analyzed.At the same time, the association analysis between genotypes and growth traits at different loci was carried out.The results showed that no mutation was detected in exon of MSTN gene in East Friensian sheep, but there was a G→A single-base mutation at 272 bp in 3'-UTR region, which contained AG and AA genotypes.The frequency of AA genotype was higher than that of AG genotype.A mutation of C→A was detected at 176 bp of 5'-UTR, which contained CC and AC genotypes, and the frequency of CC genotype was higher than that of AC genotype.The results of genetic diversity parameter analysis showed that the homozygosity of 3'-UTR-272 and 5'-UTR-176 of MSTN gene in East Friensian sheep were 0.827 and 0.887, and the heterozygosity were 0.173 and 0.113, respectively.The polymorphisms of the two loci were low (PIC<0.25), and the genotype distribution accorded with Hardy-Weinberg equilibrium (P>0.05).Through association analysis, the body oblique length of AG genotype was significantly higher than that of AA genotype at 3'-UTR-272 (P<0.05), the chest circumference of CC genotype was significantly higher than AC genotype at 5'-UTR-176 (P<0.05), and there was no significant difference in other traits (P>0.05).This results suggested that the 3'-UTR and 5'-UTR mutation of MSTN gene in East Friensian sheep could be considered as molecular markers for the selection of target traits.

The purpose of this study was to explore the expression differences of BTNA1A1、XDH、PLIN1 and PLIN2 genes which were lipid droplet formation related genes in the mammary tissue of Dehong dairy buffalo with different milk fat contents.216 healthy Dehong dairy buffaloes with the same breeding community were selected in this experiment.Milk samples were collected, and milk fat contents were detected by milk composition analyzer.According to the determination result of milk fat contents, the experiments were divided into low milk fat content group (L group, <7.0%), medium milk fat content group (M group, 7.5%±0.5%) and high milk fat content group (H group, >8.0%).Three Dehong dairy buffaloes with second fetuses, similar age and in the middle lactation stage were selected in each group.After slaughter, the breast tissue was collected and the total RNA was extracted.The mRNA expression levels of lipid droplet formation related genes in mammary tissue were detected by Real-time quantitative PCR, and the correlation between the lipid droplet formation related genes expression and milk fat contents, fatty acid contents were analyzed.The results showed that the same expression trend of BTN1A1, XDH, PLIN1 and PLIN2 genes were fallow as:group H>group M>group L.The mRNA expression levels of BTN1A1, XDH, PLIN1 and PLIN2 genes in the mammary tissue of Dehong dairy buffalo in group H were extremely significantly higher than those in group M and group L (P<0.01).The expression level of BTN1A1 gene was no significant difference between group M and group L (P>0.05), but the mRNA expression levels of XDH, PLIN1 and PLIN2 genes in group M were extremely significantly higher than those in group L (P<0.01).Correlation analysis showed that the expression levels of BTN1A1, XDH, PLIN1 and PLIN2 genes were significantly positively correlated with milk fat contents, total fatty acids, long chain fatty acids and unsaturated fatty acids contents (P<0.01).The results of this study indicated that the expression of BTN1A1, XDH, PLIN1 and PLIN2 in mammary tissue might have a positive effect on milk fat synthesis and secretion in Dehong dairy buffalo, which would provide a reference for further explanation of regulation mechanisms on lipid droplet formation related genes during lactation.

The purpose of this study was to estimate the correction coefficient and parity adjustment coefficient of standardized 305-day milk yield corresponding to days in milk (DIM) in different regions, by analyzing the test-day data from scaled dairy farms.Wood model (Incomplete Gamma Function) was used to fit the lactation curves of 683 160 test-day milk yield records from 11 749 Holstein dairy cows from 9 reference population in different temperature zones in China from 2010 to 2020.The parameters of lactation curve were estimated, and the correction coefficients corresponding to the lactation days from 1 to 305 days of primiparous and multiparous cows were calculated.The differences between the standardized 305-day milk yield and actual accumulated milk yield at 305 days of reference group and validation group were analyzed respectively.Mixed linear model in SAS 9.2 was used to estimate the parity effects for cows with at least the first five lactations standardized 305-day milk yield of 1-6 parities, and the multiplicative adjustment coefficients of 1-6 parities were calculated.The difference between the conventional adjustment coefficient and new adjustment coefficient were compared.The results showed that:①For primiparous and multiparous cows of reference population, the goodness of fit (R²) ranges of lactation curve equation were 0.4593 to 0.4913 and 0.5796 to 0.6341, respectively.The peak days of lactation were 79 to 85 and 53 d, the peak milk yield was 33.1 to 34.4 and 46.0 to 48.6 kg, respectively.②In the reference population, the adjustment coefficients were basically consistent when DIM more than 90 d of primiparous and DIM more than 30 d of multiparous cows.Standardized 305-day milk yield for DIM more than 60 d was close to the actual accumulated 305-day milk yield, the difference of milk yield was lower than 100 and 200 kg, for primiparous and multiparous cows, respectively.③For the standardized 305-day milk yield of validation population, adjustment coefficients of DIM more than 150 d of primiparous and DIM more than 180 d of multiparous cows yielded better fitness, the ratio of standardized 305-day milk yield and actual accumulated 305-day milk yield was higher than 79%.④Adjustment coefficients of 1-6 parities were 1.2121, 1.0380, 1.0063, 1.0000, 1.0220 and 1.0290, respectively.⑤Compared with the conventional adjustment coefficient, new adjustment coefficient of DIM derived from current study suited better to adjust the lactation effect, difference between 305-day milk yield standardized by new adjustment coefficient and the actual accumulated 305-day milk yield of primiparous and multiparous cows were within 900 and 700 kg, respectively.Using the new adjustment coefficient, milk production of 2-4 parity cows could be adjusted to 5th lactation to mature equivalent more accurately.The results indicated that updating the adjustment coefficients of DIM and parity for standardized 305-day milk yield regularly was necessary, milk yield of lactating cows with different DIM and parities could be accurately adjusted to the same benchmark.Thus, the comparison of milk performance among individual dairy cows would be more efficient and provided fair reference for management in dairy farm.

The aim of this study was to investigate the association between long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) gene single nucleotide polymorphism (SNP) and the growth traits in Yanbian Yellow cattle.DNA direct sequencing and high resolution melting curve (HRM) were used to analyze the polymorphism of MEG3 gene in 90 Yanbian Yellow cattle at 24 months of age, and association analysis was performed combined with 13 growth traits such as chest width, thurl width and perimeter of abdomen.The results showed that MEG3 gene exon 5 contained two mutations of g.65745819 G>A and g.65745850 C>A, both of them had two alleles (G, A and C, A), the allele frequency were 0.661, 0.339 and 0.778, 0.222, respectively, and produced three genotypes(GG, GA, AA and CC, CA and AA), the genotype frequency were 0.422, 0.478, 0.100 and 0.611, 0.333, 0.056, respectively.χ² test showed no deviation from Hardy-Weinberg equilibrium (P>0.05).The genetic homozygosity, heterozygosity, effective number of alleles and polymorphism information content (PIC) of the two loci were 0.552, 0.448, 1.812, 0.348 and 0.286, 0.346, 1.528, 0.286, respectively, which were in moderate polymorphism (0.25<PIC<0.5).Association analysis showed that the chest width of GA genotype at g.65745819 G>A was significantly higher than that of AA genotype (P<0.05), and the thurl width of AA genotype was significantly higher than that of GG and GA genotypes (P<0.05).The perimeter of abdomen trait of AA genotype at g.65745850 C>A was significantly higher than that of CA genotype (P<0.05), but there was no significant difference among other genotypes and traits (P>0.05).The results indicated that the polymorphism of lncRNA MEG3 gene was associated with some growth traits of Yanbian Yellow cattle, and could be used as a candidate gene for molecular marker assisted breeding of Yanbian Yellow cattle.

Red deer is a very important species resource.Due to habitat loss and human interference, the number of wild red deer has decreased sharply, while most domestic red deer have been improved, so the number of pure red deer has decreased sharply.The study of molecular genetics of red deer can not only deepen people’s understanding of the origin and speciation of red deer, but also help to carry out genetic diversity protection research.With the rapid development of high-throughput sequencing technology, molecular biology and bioinformatics, the origin and evolution of red deer have been progressed to the genome-wide level.The study on the origin and evolution of red deer has gradually developed from the study of body appearance and chromosome karyotype to the study of DNA sequence and physiological indexes.This paper reviews the research progress on the origin, evolution and genetic diversity of red deer at home and abroad in recent years.It not only reveals the evolutionary history of red deer from the aspects of origin time, origin place and migration route, but also introduces the different molecular markers for paternal, maternal and autosomal genetic diversity selection of red deer in order to further reveal the genetic variation, differentiation, migration route and phylogenetic relationship of red deer population, provides important references for the utilization and protection of genetic resources and the healthy development of red deer industry.

Melanin, produced in melanocytes, is a water-insoluble polymer formed by the oxidative polymerization of phenols or indoles.It widely exists in microorganisms, animals and plants.It has the physiological functions of eliminating free radicals, antioxidation, inhibiting virus infection, activating immune system, improving body immunity and delaying aging.Under the effect of tyrosinase, tyrosine generates dopamine, dopamine forms melanin through a complex series of biological reactions.Existing studies have found that there are many signaling pathways (α-MSH/MC1R/cAMP signal pathway, Wnt/β-catenin signaling pathway, PI3K/Akt/GSK3β signal pathway, MAPK signal pathway, etc.) and related regulatory factors (MITF, MC1R, TYR, etc.) are involved in the formation of melanin.It is generally believed that mammalian melanocytes are mainly distributed in skin, hair follicles and other tissues, while a large number of melanocytes also exist in the visceral tissues of Black-bone sheep, and the formation mechanism of melanocytes was not clear yet.The character of Black-bone sheep is an important economic character, which is closely related to its nutritional and medicinal value and is affected by the degree of melanin deposition in body tissues.In this paper, the physical and chemical properties, biological functions, synthetic route and signaling pathways of melanin are reviewed, the research status of candidate functional genes affecting the deposition and distribution of melanin in Black-bone sheep are summarized.It is expected to provide reference for in-depth study of the regulation mechanism of melanin deposition and the application of candidate functional genes in production practice.

The purpose of this experiment was to study the regulatory effect of Salmonella Typhimurium chaperone protein Hfq-dependent sRNA GcvB on spaP and invC gene mRNA.λ-Red homologous recombination technology was used to construct spaP and invC genes LacZ fusion strains, P₂₂ phage transduction technology was used to construct GcvB and Hfq genes single deletion and double deletion strains and GcvB functional region deletion strains.β-galactosinosidase activity experiment was performed to determine the protein expression activity of spaP and invC genes in chaperone proteins Hfq, sRNA GcvB and Hfq & GcvB deletion strains.Real-time PCR was used to determine the mRNA expression of spaP and invC genes.The results showed that P₂₂ phage transduction technology successfully constructed ΔGcvB (696 bp), ΔHfq (2 369 bp), ΔGcvBΔHfq (2 369/696 bp), GcvBΔR1 (757 bp), GcvBΔR2 (951 bp), GcvBΔR3 (986 bp) deletion strains.β-galactosinosidase activity test results showed that compared with wild-type strains, the β-galactosinosidase activity of spaP gene was extremely significantly down-regulated in the deleted strains GcvBΔR2 and GcvBΔR3 (P<0.01), and the β-galactosinosidase activity of the invC gene was extremely significantly up-regulated in the deleted strains ΔGcvB, ΔHfq, ΔGcvBΔHfq and GcvBΔR1 (P<0.01).The invC gene was extremely significantly down-regulated in the deletion strain GcvBΔR2 and GcvBΔR3 protein activity (P<0.01), and the GcvBΔR1 protein activity was significantly down-regulated in the deletion strain (P<0.05).The protein activity in the deletion strains ΔGcvB, ΔHfq and ΔGcvBΔHfq was extremely significantly up-regulated (P<0.01).Real-time PCR results showed that compared with the wild-type strain, the transcription level of the spaP gene in ΔHfq, ΔGcvB, ΔGcvBΔHfq and GcvBΔR1 deletion strains was extremely significantly up-regulated (P<0.01), while the transcription level of GcvBΔR2 deletion strain was extremely significantly down-regulated (P<0.01).The transcription level of GcvBΔR3 deletion strains was significantly down-regulated (P<0.05), the transcription level of invC gene ΔHfq, ΔGcvBΔHfq and ΔGcvB deletion strains was extremely significant up-regulated (P<0.01), GcvBΔR1 and GcvBΔR2 deletion strains were all extremely significantly downward-regulated (P<0.01), GcvBΔR3 deletion strains were all significant down-regulated (P<0.05).According to the above experimental results, sRNA GcvB and chaperonin Hfq had a certain regulatory effect on invC and spaP genes.This experiment provided a theoretical basis for the regulation of sRNA GcvB and chaperonin Hfq, and enriched the number of target genes of GcvB.

In order to facilitate the storage and transportation of recombinant Lactococcus lactis after fermentation, microcapsules were prepared by recombinant Lactococcus lactis pAMJ399-LFBA/MG1363, which could express lactoferrin peptide.The preparation conditions of microcapsules were optimized, and the related biological characteristics of the encapsulated recombinant bacteria were detected.In order to select the best wall material and its concentration, the embedding amount was measured by controlling the single variable experiment of different wall materials and wall material concentrations.The release rate of microcapsules was compared through simulated gastric and intestinal fluid environment experiment, and the shelf life experiment of different wall materials was carried out to compare the lowest viable bacteria number.The response surface experiment was used to determine the optimal process conditions, and the microcapsules were prepared under the optimal process conditions for verification.The optimized results were as follows:Sodium alginate and chitosan were finally selected as wall materials.Under the conditions of sodium alginate was 2.49%, chitosan was 0.96%, CaCl₂ was 6.67% and solidification time was 57 min, the microcapsule effect was the best, and the prediction embedding amount was 7.85×10⁸ CFU/g.Microcapsules existed stably in simulated gastric juice, and could completely break in simulated intestinal juice, and could release more than 95% of lactic acid bacteria in microcapsules.After the sodium alginate-chitosan microcapsules were stored at 4 ℃ for 3 weeks, the number of viable bacteria in microcapsules was 1.41×10⁷ CFU/g.The results showed that the embedding amount of microcapsules was 8.08×10⁸ CFU/g.After being stored at room temperature for 2 weeks, the viable bacteria count could still reach 3.89×10⁶ CFU/g.The lactoferrin peptide expressed by recombinant Lactococcus lactis in microcapsules was detected by ELISA, and the antibacterial assay showed that the lactoferrin peptide expressed by recombinant Lactobacillus lactis in microcapsules still had significant inhibitory effect on Salmonella and Staphylococcus aureus.These results indicated that the recombinant Lactococcus lactis expressing lactoferrin peptide could be prepared into microcapsules with low cost, long shelf life and gastric juice resistance, which laid a foundation for the further application of recombinant lactic acid bacteria in production.

In order to preliminarily identify and explore the lncRNA-mRNA regulatory network of ORF3 protein of swine Hepatitis E virus (HEV) affecting the riboflavin metabolism signal pathway of HepG2, in this study, we constructed the Adenovirus overexpression vector to prepare the high titer Adenovirus, and mediated the overexpression of swine HEV-ORF3 in HepG2 cells.After the overexpression of ORF3 protein was verified by Western blotting, lncRNA high-throughput sequencing was used to screen the differentially expressed lncRNA and predict the targeted genes.GO function and KEGG pathway enrichment analysis was performed for the differentially expressed lncRNA targeted genes, and the lncRNA-mRNA regulatory network related to riboflavin metabolism signal pathway was preliminarily identified.Western blotting showed that the target band appeared at about 12 ku, indicating the successful overexpression of swine HEV-ORF3 in HepG2 cells mediated by Adenovirus.High throughput sequencing of lncRNA showed that 102 differentially expressed lncRNAs were up-regulated and 80 differentially expressed lncRNAs were down regulated.GO function and KEGG pathway enrichment found that lncRNA(MSTRG.13995.2) and lncRNA (MSTRG.1960.1) might be differentially expressed lncRNAs related to riboflavin metabolism signaling pathway, which affected riboflavin metabolism signal pathway by cis regulating their target genes APC-5 and FLAD1, respectively.The results showed that lncRNA(MSTRG.13995.2)-APC5 and lncRNA(MSTRG.1960.1)-FLAD1 might be the lncRNA-mRNA regulatory network that affected the riboflavin metabolism signal pathway of HepG2.

This experiment was conducted to investigate the host innate immune response to different origins of Infectious bronchitis virus (IBV) infection in SPF chicken.One hundred and forty one-day-old SPF White Leghorn chickens were randomly divided into four groups, including three challenge groups and one control group.The challenge groups were inoculated 3 IBVs (including chicken originated IBV virulent strain, chicken originated IBV attenuated strain, and pheasant originated IBV strain) by intranasal eye drops, and the control group was mock inoculated with phosphate-buffered saline (PBS) by the same route.From each of these infected groups five chickens were humanely sacrificed to collected their bursa of Fabricius, kidney and trachea at 12 hour post infection (hpi), 36 hpi, 72 hpi, 7 day post infection (dpi) and 14 dpi.The remaining chickens in each group were continuously observed for clinical symptoms, morbidity and mortality before being killed.The expression of IBV viral load, Toll-like receptors (TLRs) and some cytokines of interleukin (IL) and interferon(IFN) were evaluated by Real-time quantitative PCR.The results showed that SPF chickens infected with chicken origined IBV virulent strain developed clinical sympotoms, the infected SPF chickens showed depression, wing ptosis and other classical symptoms.A total of 7 chickens died during the period of infection (5-10 d), the mortality was 20%.In contrast, for chickens infected with pheasant origined IBV strain, chicken origined IBV attenuated strain and control group, no respiratory clinical signs and no gross lesions were observed during the experimental period.Chickens infected with chicken origined IBV virulent strain demonstrate enlarged kidneys, urate deposits and variegated lesions.Viral loads were detected in bursa of Fabricius, spleen, kidney and trachea of chicken infected with chicken originated IBV virulent.In addition, viral loads were detected in some tissues of the chicken originated IBV attenuated group, but were not detected in the tissues of control and pheasant origined IBV group.The expressions of TLR1, TLR2, TLR3, TLR5, TLR7 and TLR15 genes in bursa of Fabricius were dramatically up-regulated in chicken originated IBV virulent strain group at 72 hpi compared with other groups (P<0.05), IL-6 and IFN-β were released to participate in stronger antiviral immune response at 72 hpi.In chicken originated IBV attenuated strain challenged group, the expressions of TLR2, TLR3, TLR15, TLR21, IL-6 and IL-18 genes in kidney were significantly increased at 7 dpi (P<0.05).The expression of IFN-γ gene in bursa of Fabricius was significantly up-regulated at 36 hpi after infection with pheasant originated IBV strain (P<0.05).In summary, chicken originated IBV virulent strain was highly pathogenic to SPF chicken, the chicken origined IBV virulent strain could dramatically up-regulated the expression of immune-related cytokines in SPF chicken.The above results revealed that the different pathogenicity of IBV virus from different origins to SPF chickens was related to the different immune response.

To investigate the epitope and function of Porcine epidemic diarrhea virus (PEDV) S protein, by optimizing the codon of S1D gene, the recombinant prokaryotic expression plasmids pET-S1D which was not optimized and pET-ΔS1D which was optimized were constructed.The expression of S1D protein was respectively induced and ultimately purified.SDS-PAGE and Western blotting were used to verify the correct expression of S1D and ΔS1D proteins in Escherichia coli.The expression levels of S1D and ΔS1D proteins were scanned by the software Image J, and the difference was analyzed by t-test.The purified ΔS1D protein was used as antigen to immunize BALB/c mice.Monoclonal cell strain was obtained by cell fusion, screening and subcloning.Induction method in vivo was used to prepare ascites with anti-PEDV S1D protein.ELISA, Western blotting and IFA were used to detect the titer and specificity of ascites.SDS-PAGE and Western blotting results showed that both S1D and ΔS1D proteins had the correct target band at 34 ku.t-test result showed that the protein expression levels of S1D and ΔS1D were extremely significantly different (P<0.01).ELISA result showed that the antibody titer of ascites reached 1∶1 000 000.The monoclonal antibody reacted positively with PEDV and purified ΔS1D protein, but negatively with PEDV N protein, pET-32a(+) empty vector protein and Porcine reproductive and respiratory syndrome virus (PRRSV), Transmissible gastroenteritis virus (TGEV), Classical swine fever virus (CSFV), Porcine deltacoronavirus (PDCoV) and Swine acute diarrhea syndrome coronavirus (SADS-CoV).Western blotting result showed that ascites and ΔS1D protein, ascites and PEDV S protein had specific bands at 34 and 180 ku, respectively, but no band with pET-32a(+) empty vector protein and normal Vero cell protein.IFA indicated that the ascites and the positive control group could make the cells show a specific green fluorescent signal, while the blank and negative control groups had no green fluorescent signal.Codon optimization could significantly increase the expression level of the recombinant protein in the prokaryotic expression system.Based on high efficiency expression of ΔS1D protein, a monoclonal antibody capable of stable secretion and specific response to PEDV S1 protein was successfully prepared in this study.The preparation of S protein monoclonal antibody provided a good basis for further research on the PEDV S protein antigen epitope and protein function.

In order to explore whether truncated diphtheria toxin DT390 had immune enhancing effect on VP2 protein of Infectious bursal disease virus (IBDV), in this study, DT390 and VP2 were fused and expressed by Pichia pastoris expression system with diphtheria toxin resistance.The expressed fusion protein DT390-VP2 was identified by SDS-PAGE and Western blotting.Twenty four 21 days old SPF chicks were randomly divided into four groups:VP2 group, DT390-VP2 group, PBS (control group) and B87 group (positive control group).The chicks in each group were immunized on the the 0 and 14th days, respectively.On the 28th day after the first immunization, each chick was inoculated with IBDV BC6/85 strain of 100 BID₅₀.The serum VP2 specific antibody levels of chicks in each group at different time before and after immunization were detected by ELISA.HE staining and pathological damage score were used to evaluate the damage degree of Fabricius, and the immunogenicity and protective function of fusion protein DT390-VP2 in chicks.SDS-PAGE and Western blotting analysis showed that the expressed and purified target protein was glycosylated fusion protein DT390-VP2 (91.26 ku).The results of in vivo test showed that 21 days after the first immunization, the titer of VP2 specific antibody in DT390-VP2 group was significantly and extremely significantly higher than that in B87 group (P<0.05) and VP2 group (P<0.01), respectively.28 days after the first immunization, the titer of VP2 specific antibody in DT390-VP2 group was extremely significantly higher than that in VP2 group (P<0.01).Compared with VP2 protein, the fusion protein DT390-VP2 reduced the atrophy of bursal lymph follicles in chicks.In conclusion, truncated diphtheria toxin DT390 enhanced the humoral immunogenicity of VP2 by fusion expression, the fusion protein DT390-VP2 reduced the damage of tissue caused by IBDV BC6/85 strain to a certain extent.

In order to determine the causative agent of gosling gout in a goose farm in Yancheng city in 2020, the samples were deeply sequenced by the method of viral metagenomics, and the suspected pathogens were found through the alignment of the original data.The whole genome sequence was obtained by de novo assembly, and the homology and phylogenetic analysis were done.The pathogen was verified by the isolation and identification of the suspected virus and animal regression test.The results of viral metagenome sequencing showed that a total of 599 733 original reads were obtained.There were 278 593 virus reads, of which Goose astrovirus sequences accounted for 95%, no other suspected virus sequences were found.The whole genome sequence was obtained by de novo assembly, which was 7 137 nt in length with the typical genomic characterization of other known avastroviruses, the isolated virus was named YC20, GenBank accession No.:MW536497.The results of similarity alignment showed that the YC20 isolate shared the highest identity of 98.2% with the Goose astrovirus strain SD01 in terms of the whole-genome sequence.The results of phylogenetic analysis showed that YC20 clustered together with the new type of Goose astrovirus.The results of virus isolation showed that no goose embryos died during their first 3 passages but the mortality rate reached 100% after the fifth passages.Meanwhile, no death or pathological changes were found in SPF chicken or duck embryos, and PCR results were negative.After negative staining, the virus particles of about 25 nm were observed under electron microscope.In the goose regression experiment, the infected goslings experienced a significant peak of death during days 6 to 8, resulting in a mortality rate of 58.3%.At necropsy, uric deposits in heart, liver, and articular cavity, as well as severe hemorrhage and kidney swelling were observed in dead goslings.The results suggested that the pathogen of gout in this goose farm was a new type of the new Goose astrovirus.In this study, the combination of viral metagenomics with traditional virus isolation techniques confirms the pathogen of goose gout, which had guiding significance for the identification and study of the pathogen.

In order to understand the variation of gp85 gene of Avian leukemia virus (ALV) subgroup J and the carrying of virulence gene of Enterococcus faecalis (EF) in a farm in Guiyang city, etiological investigation and analysis were carried out on the cases of co-infection of ALV and EF in this farm.Samples were collected for virus nucleic acid detection, gp85 gene cloning and sequencing analysis, nucleotide sequence similarity analysis, bacterial isolation and identification, virulence gene detection, etc.The results showed that one ALV strain and one Enterococcus faecalis strain were successfully isolated and identified, which were named GZM52021 and GZEF2021 respectively.The specific amplification band of ALV only appeared at about 422 bp, indicating the existence of ALV-J infection.ALV gp85 gene was successfully cloned, and the similarity between GZM52021 strain and gp85 gene of subgroup J reference strain (GenBank accession No.:KM655820, KF796654, JN378888 and Z46390) was high, ranging from 97.9% to 98.2%.The construction results of genetic evolutionary tree of nucleotide sequence of gp85 gene were consistent with the similarity results.GZEF2021 strain grew round convex and opaque milky white colonies on LB solid plate, and there was obvious hemolysis on blood agar plate.Gram staining microscopy showed spherical or oval positive Streptococcus.16S rRNA sequencing showed that the similarity between GZEF2021 strain and Enterococcus faecalis (GenBank accession No.:KJ626240, MT611693, KY399248, MH919370, MN379663 and KU937389) was more than 99%.The results of drug sensitivity test showed that GZEF2021 strain was sensitive to cefoperazone, penicillin, piperacillin and carbenicillin, moderately sensitive to compound sulfamethoxazole, cefradine and cefazolin and resistant to neomycin, amikacin, doxycycline and minocycline.The detection of virulence genes showed that GZEF2021 strain carried ace, gelE, asa1, EF3314 and efaA five virulence genes, indicating that the Enterococcus faecalis isolated from chicken might be pathogenic.The co-infection of ALV subgroup J and Enterococcus faecalis existed in the dead chicken.The results of this experiment could provide some references for understanding the epidemic trend of ALV subgroup J and Enterococcus faecalis and the diagnosis and treatment of mixed infection in Guizhou.

The study was designed to develop an HPLC method for the determination of the content of the principal ingredient and related substances in eprinomectin (EPR) sustained-release injection.The external standard method and self-control method were utilized for calculating the content of the principal ingredient and related substances, respectively.The chromatographic column of the content and related substances was Phenomenex Luna 5 μ-C8-100A 250 mm×4.60 mm, 5 μm.The detection method was as followed:Acetonitrile-0.1% aqueous perchloric acid solution with gradient, the flow rate was 1.5 mL/min, the detection wavelength was 245 nm, the column temperature was 35 ℃, and the injection volume was 20 μL.The results showed that the principal component of EPR was well separated with the peak of related substances, the degree of separation between B1a and B1b was greater than 3.The linear relationship was good when the concentration of EPR was 1.5625-1 000 μg/mL.The detection and quantification limit of EPR were 0.01 and 0.34 μg/mL, respectively.RSD of the three kinds of precision was less than 2%, RSD of stability test was 0.28%, average recovery was 102.54%, and RSD was 3.14% (n=9).The robustness showed that the resolution of the principal components of EPR sustained-release injection was more than 1.5, and the number of theoretical plates was more than 4 500.The results showed that the detection conditions were durable.In 3 batches, average value of EPR was 102.60%, B1b accounts for 1.68% of the sum of B1a and B1b, total impurity were 2.57%.All the results suggested that the optimized HPLC method in this study was simple, specific, sensitive, accurate, and suitable for quality control of EPR sustained-release injection.

In order to study the drug resistant phenotype and genotype of Escherichia coli (E. coli) in waterfowl, a total of 1 505 duck feces, water and soil samples were collected from waterfowl farms and their surrounding environment in Guangdong, Fujian, Zhejiang, Jiangsu and Shandong provinces of China. E. coli were isolated by selective medium and identified by MALDI-TOF-MS mass spectrometry.Drug resistance phenotypes were detected by minimal broth dilution method, and the 25 drug resistant genes in E.coli were screened by PCR.The results showed that a total of 449 strains of E.coli were isolated and identified from 1 505 samples, the separation rate was 29.8% and 335 were from feces, 52 were from soil and 62 were from water, among these samples, Zhejiang (43.3%) and Guangdong (43.2%) were the highest, followed by Fujian (28.1%) and Jiangsu (23.8%), and Shandong (18.3%) had the lowest separation rate.449 strains of E.coli were multiple drug resistance.The resistance rates to ampicillin, ciprofloxacin, aureomycin, florfenicol and sulfonamides were 90.2%, 50.3%, 96.7%, 87.3% and 90.6%, respectively, while they were sensitive to tegacyclin and meropenem.E.coli from different provinces showed different resistance rates to different antibiotics.The PCR results showed that in 449 strains of E.coli, the detection rate of resistance gene tet(A) was the highest (88.2%).It was followed by floR (58.13%), sul2 (57.02%), tet(B)(53.9%), cmlA (39.2%) and sul1 (36.75%), the positive E.coli of qnrA, tet(M) and tet(X) were not detected.Among the 5 provinces, the distribution of all drug resistance genes was high in the samples of Shandong province, but low in the samples of Zhejiang and Jiangsu provinces.In conclusion, this study investigated the drug resistance status of E.coli in waterfowl and surrounding environment of 5 coastal provinces in Southeast China, providing a theoretical basis for the standard use of antibiotics in aquaculture.

In order to investigate the effect of selenium-enriched lactic acid bacteria on the apoptosis of chicken small intestinal epithelial cells (CSIEC) induced by aflatoxin B₁ (AFB₁), CSIEC cultured in vitro were divided into 5 groups:control group (C), AFB₁ group (300 μmol/L AFB₁), AFB₁+0.01 Se group (300 μmol/L AFB₁+0.01 μmol/L selenium-enriched lactic acid bacteria, in terms of Se), AFB₁+0.05 Se group and AFB₁+0.1 Se group.When the cells grew to 60%-70% confluence, the culture medium of AFB₁+0.01 Se, AFB₁+0.05 Se and AFB₁+0.1 Se groups was replaced with the culture medium containing the corresponding Se concentration, and C and AFB₁ groups were replaced with normal culture medium. After 8 h, 300 μmol/L AFB₁ was added to the AFB₁-containing groups, and the culture was continued for 24 h.CCK-8 was used to detect the antagonistic effect of selenium-enriched lactic acid bacteria on the cytoactive of CSIEC induced by AFB₁.ELISA was used to detect the change of lactate dehydrogenase (LDH) activity in the culture medium.TUNEL was used to observe the effect of selenium-enriched lactic acid bacteria against AFB₁ on cell apoptosis rate.The protein levels of Bcl-2/Bax, PERK, ATF4, CHOP, GRP78, p53 and p21 associated with mitochondrial and endoplasmic reticulum stress pathways were detected by Western blotting.The results showed that the cytoactive of CSIEC in the other groups was extremely significantly lower than that in control group (P<0.01), and those in selenium-added groups were extremely significantly higher than those in AFB₁ group (P<0.01).The LDH activities in the other groups were extremely significantly higher than those in control group (P<0.01), and LDH activities in selenium-added groups were extremely significantly lower than those in AFB₁ group (P<0.01).Compared with control group, the number of TUNEL positive cells in AFB₁ group was extremely significantly increased (P<0.01);and no significant difference were found between selenium-added groups and AFB₁ group (P>0.05).Compared with control group, the protein expression levels of Bcl-2/Bax in AFB₁ group were extremely significantly decreased (P<0.01), and that in AFB₁+0.05 Se group was increased (P<0.05).Endoplasmic reticulum stress pathway protein levels showed that the protein levels of PERK, CHOP, GRP78 and p53 in AFB₁ group were extremely significantly higher than those in control group (P<0.01), protein levels of PERK and GRP78 in selenium-added groups were significantly lower than those in AFB₁ group (P<0.01).Protein levels of CHOP and p53 in AFB₁+0.05 Se and AFB₁+0.1 Se groups were extremely significantly lower or significantly lower than those in AFB₁ group (P<0.01;P<0.05).Therefore, selenium-enriched lactic acid bacteria could alleviate the apoptosis of CSIEC induced by AFB₁.

The purpose of this test was to study the toxic effect of T-2 toxin on porcine kidney cells (PK15) and its oxidative stress response.The cells were treated with different concentrations of T-2 toxin.The cytotoxicity of the toxin was evaluated by MTT assay, intracellular lactate dehydrogenase (LDH) release rate and HE staining.The effects of different doses of T-2 toxin on the level of oxidative stress were evaluated by detecting the levels of reactive oxygen species (ROS), malondialdehyde (MDA), total glutathione (GSH) and total glutathione peroxidase (GPx).The expression levels of Nrf2, Keap1, GPx-1, Nqo1 and Hmox1 genes mRNA related to Nrf2-ARE signal pathway were detected, and the effect of T-2 toxin on this signal pathway was analyzed.The results showed that the activity of PK15 cells decreased in a toxin dose-dependent manner (P<0.05), and the release rate of LDH increased in a toxin dose-dependent manner (P<0.05).With the increase of T-2 toxin dose, the cell gap gradually increased, the cell pyknosis and the number of cells decreased.The rate of ROS positive cells increased in a dose-dependent manner, and when T-2 toxin was 100 nmol/L, the rate of ROS positive cells was significantly higher than that in other dose groups (P<0.05).The intracellular MDA content and GPx activity increased in a toxin dose-dependent manner (P<0.05), while the GSH content decreased in a toxin dose-dependent manner (P<0.05).20, 50 and 100 nmol/L T-2 toxin could significantly up regulate the relative expression of Keap1, GPx-1 and Nqo1 genes mRNA (P<0.05), and the relative expression of 50 nmol/L was the highest.T-2 toxin caused toxic effects on PK15 cells and induced oxidative damage, and the oxidative stress process was regulated by Nrf2-AER signal pathway.

The purpose of this experiment was to study the inhibitory effect of chlorogenic acid on fowl adenovirus type 4 (FAdV-4) and its effect on the expression of inflammatory factors and signal molecules in chicken embryo liver.9-day-old SPF chicken embryos were randomly divided into 7 groups:Control group, 5 chlorogenic acid groups with different concentrations and FADV-4 infection group.Chlorogenic acid groups were injected with different concentrations of chlorogenic acid through the allantoic cavity.48 h after hatching, chlorogenic acid groups and FAdV-4 infection group were inoculated with 0.2 mL FAdV-4, of which the ELD₅₀ was 1.36×10⁷ copies, and the control group was injected with equal volume of normal saline.The safe concentration (IC₅₀) of chlorogenic acid was detected by MTT method, and the content of chlorogenic acid inoculated into each chicken embryo was calculated.The liver of chicken embryos was collected for pathological observation 72 h after inoculation with virus, viral load was detected by Real-time quantitative PCR, the contents of liver cytokine interleukin-1β (IL-1 β), IL-6 and tumor necrosis factor-α (TNF-α) as well as signal molecules phosphatidylinositol-3-kinase (PI3K), nuclear transcription factor-κB(NF-κB) and NF-κBp65 were measured by ELISA method.The MTT results showed that the IC₅₀ of chlorogenic acid to chick embryo fibroblast was 28.6 μg/mL.Based on the IC₅₀, chlorogenic acid treated groups were inoculated with 0.2 mL of different doses of chlorogenic acid per egg containing 858, 429, 214.5(≈215), 107.25(≈107) and 53.125(≈53) μg, respectively.72 h after FADV-4 inoculation, the liver edge of chicken embryo in FADV-4 infection group was blunt, round, swollen, brittle, earthy yellow or yellow, and even necrotic.In chlorogenic acid group, with the increase of chlorogenic acid concentration, liver swelling became less obvious, necrotic foci and bleeding petechiae decreased.The virus proliferated significantly in liver, and the viral load was 7.8 log₂copies/mg, 107 μg chlorogenic acid could significantly inhibit the proliferation of FAdV-4 in chicken embryo liver (P<0.05).The detection results of inflammatory factors showed that compared with control group, FADV-4 could extremely significantly increase the content of IL-1β, IL-6 and TNF-α in chicken embryo liver (P<0.01), the contents of IL-1β and TNF-α were extremely significantly increased in 53 μg chlorogenic acid group (P<0.01), the contents of IL-6 and TNF-α were significantly increased in 107 μg chlorogenic acid group (P<0.05).Compared with FAdV-4 infection group, the contents of IL-1β and TNF-α were extremely significantly increased in 107 μg chlorogenic acid group (P<0.01), the content of IL-6 was significantly decreased in 215 and 429 μg chlorogenic acid group (P<0.05), and the content of IL-6 was extremely significantly decreased in 858 μg chlorogenic acid group (P<0.01).Signal molecules determination results showed that compared with control group, FAdV-4 could extremely significantly increase the contents of PI3K, NF-κB and NF-κBp65 (P<0.01), the contents of PI3K, NF-κB and NF-κBp65 were significantly increased in 53 μg chlorogenic acid group (P<0.05).Compared with FAdV-4 infection group, the contents of PI3K and NF-κBp65 were significantly decreased in 53 μg chlorogenic acid group (P<0.05), the content of NF-κB was significantly decreased (P<0.05) and the content of NF-κBp65 was extremely significantly decreased in 215 μg chlorogenic acid group (P<0.01).When chlorogenic acid reached 429 μg, the contents of PI3K, NF-κB and NF-κBp65 were extremely significantly decreased (P<0.01).These findings suggested that chlorogenic acid could inhibit the proliferation of FAdV-4, and inhibit the expression of inflammatory factors and signal molecules in a dose-dependent manner, which indicated that chlorogenic acid might be used for the prevention and control of antiviral and inflammatory diseases in chickens.

Nicarbazin (NIC) is a class of compounds composed of 4 4’-dinitrocarbanilide (DNC) and 2-hydroxyl-4, 6-dimethylpyrimidine (HDP), in which DNC is its residual marker.NIC is widely used in coccidiosis control of chickens and turkeys due to its safety and high efficiency.In recent years, its residue problem was high, which seriously threatened the safety of animal derived food and the safety of consumers’ life and health.At present, the analysis methods for NIC in poultry products and feed mainly include instrumental analysis methods and immunoassay methods.Among them, instrumental analysis methods, as confirmatory methods, have the advantages of high specificity, high sensitivity and reliable results, mainly including high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS).Immunoassay is the mainstream technology for rapid detection of NIC, which has the advantages of high inspection speed, low cost and strong practicability.It mainly includes enzyme-linked immunosorbent assay (ELISA), immunochromatography (IC), surface plasmon resonance (SPR), fluorescent immunoassay (FIA) and flow cytometry (FCM) etc.This paper summarized the detection and analysis methods of NIC in poultry products and feed at home and abroad in recent 40 years, so as to provide theoretical and practical guidance for NIC residue monitoring.

The aim of the present study was to explore the effect of dimethyl α-ketoglutarate (DMKG) on antioxidative stress injury of canine adipose-derived mesenchymal stem cells(ADSCs).The canine ADSCs were treated with DMEM/F12 basic medium which containing hydrogen peroxide (H₂O₂) at concentrations of 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L.After 1 h, the cell survival rate was detected by CCK-8 assay to screen the optimal concentration for building canine ADSCs oxidative stress model.In this experiment, the well-growing passage 3 canine ADSCs were divided into three groups: Blank control group, model group and DMKG group.The blank control group was cultured normally without treatment.In the model group, canine ADSCs were treated with the best concentration of H₂O₂ solution for 1 h. In DMKG group, canine ADSCs were pretreated with 1 mmol/L DMKG for 24 h, and then treated with the best concentration of H₂O₂ solution for 1 h.After the culture of each group, the cell survival rate of each group was detected by CCK-8 assay, the content of reduced glutathione was detected by visible spectrophotometry, the level of reactive oxygen was detected by DCFH-DA fluorescent probe, the apoptosis rate was detected by Annexin V/PI double staining, and the mRNA expression levels of superoxide dismutase(SOD), catalase (CAT), anti-apoptotic gene Bcl-2, pro-apoptotic genes Bax and Cleaved-Caspase3 were detected by Real-time quantitative PCR.The results showed that compared with the model group, DMKG pretreatment could effectively reduce the production of reactive oxygen species (P<0.05), increase the cell survival rate (P<0.01), and decrease the rate of cell apoptosis (P<0.05).Meanwhile, DMKG pretreatment could increase the content of reduced glutathione (P<0.01), up-regulate the expression levels of antioxidant enzyme-related genes SOD1, SOD2 (P<0.01)and CAT (P<0.05), down-regulate the expression levels of Cleaved-Caspase3 gene (P<0.05)and increase the rate of Bcl-2/Bax (P<0.05).In conclusion, DMKG pretreatment could improve the ability of anti-oxidation and anti-apoptosis of canine ADSCs, so as to increase the survival of canine ADSCs under oxidative stress.