In geese breeding, due to the frequent influence of drugs and environmental and other factors, geese are extremely prone to oxidative stress, which adversely affects growth and development, geese meat quality, down production, and severely affects the development of the geese industry. Ferulic acid from plant extracts can be used as a feed additive, which is safe and non-toxic, and it can exert certain therapeutic effects on oxidative stress in geese. This experiment investigated the effect of ferulic acid on the growth performance, organs indices, and intestinal oxidative indices of Jilin white geese under lipopolysaccharide-induced oxidative stress. Geese were randomly divided into six groups: C (blank control), L (lipopolysaccharide control), F1 (60 mg/kg ferulic acid), F2 (120 mg/kg ferulic acid), F3 (180 mg/kg ferulic acid), and F4 (240 mg/kg ferulic acid). Groups L and F1-F4 were injected intraperitoneally with 0.5 mg/kg lipopolysaccharide and group C with an equivalent volume of normal saline on days 14,17 and 20, and 10 animals from each group were randomly selected for slaughter on day 21. The results showed that: 1) On day 14, the final body weight and average daily feed intake were significantly higher in group F3 than in group L, and on day 21, the final body weight was significantly higher in group F3 than in group L. 2) The thymus index was significantly higher in group F4 than in group L. 4) In the duodenum, MDA activity was reduced in group C compared with that in group L. 5) In the jejunum and ileum, MDA was significantly lower in group F3 than in group L. These results show that the addition of 180 mg/kg of ferulic acid to the diet can promote the growth of geese and alleviate the damage caused by oxidative stress in all intestinal segments.
The changes in the composition of intestinal microbiota and metabolites have been linked to digestive disorders in calves, especially neonatal calf diarrhea. Bovine rotavirus (BRV) and bovine coronavirus (BCoV) are known to be the primary culprits behind neonatal calf diarrhea. In this study, we analyzed changes in the fecal microbiota and metabolites of calves with neonatal diarrhea associated with BRV and BCoV infection using high-throughput 16S rRNA sequencing and metabolomics technology. The microbial diversity in the feces of calves infected with BRV and BCoV with diarrhea decreased significantly, and the composition changed significantly. The significant increase of Fusobacterium and the reductions of some bacteria genera, including Faecalibacterium, Bifidobacterium, Ruminococcus, Subdoligranulum, Parabacteroides, Collinsella, and Olsenella, etc., were closely related to diarrhea associated with BRV and BCoV infection. Metabolites in the feces of BRV and BCoV-infected calves with diarrhea were significantly changed. Phosphatidylcholine [PC; 16:1(9 Z)/16:1(9 Z)], lysophosphatidylethanolamine (LysoPE; 0:0/22:0), lysophosphatidylcholine (LysoPC; P-16:0) and LysoPE (0:0/18:0) were significantly higher in the feces of BRV-infected calves with diarrhea. In contrast, some others, such as desthiobiotin, were significantly lower. BRV infection affects glycerophospholipid metabolism and biotin metabolism in calves. Two differential metabolites were significantly increased, and 67 differential metabolites were significantly reduced in the feces of BCoV-infected calves with diarrhea. Seven significantly reduced metabolites, including deoxythymidylic acid (DTMP), dihydrobiopterin, dihydroneopterin triphosphate, cortexolone, cortisol, pantetheine, and pregnenolone sulfate, were enriched in the folate biosynthesis, pantothenate and CoA biosynthesis, pyrimidine metabolism, and steroid hormone biosynthesis pathway. The decrease in these metabolites was closely associated with increased harmful bacteria and reduced commensal bacteria. The content of short-chain fatty acids (SCFAs) such as acetic acid and propionic acid in the feces of BRV and BCoV-infected calves with diarrhea was lower than that of healthy calves, which was associated with the depletion of SCFAs-producing bacteria such as Parabacteroides, Fournierella, and Collinsella. The present study showed that BRV and BCoV infections changed the composition of the calf fecal microbiota and were associated with changes in fecal metabolites. This study lays the foundation for further revealing the roles of intestinal microbiota in neonatal calf diarrhea associated with BRV and BCoV infection.
This study was conducted to identify the biological characteristics of pathogen responsible for the death of Tibetan sheep in Nagarze County, Tibet, China. For this purpose, samples were collected from diseased animals and for bacterial culture and isolation. The isolated strains were subjected to several tests which included gram in-vitro drug sensitivity and in-vivo pathogenicity tests. The results revealed that 7 strains of gram-positive cocci were isolated from Tibetan sheep, named TS-1, TS-2, TS-3, TS-4, TS-5, TS-6, and TS-7. These strains exhibited specific biochemical characteristics consistent with Enterococcus faecalis. Whereas, PCR amplification results were consistent with the expected outcomes on target band of approximately 1500 bp. Genetic evolutionary analysis revealed a significant homology (96.0%99.9%) between the isolates and Enterococcus faecium. In-vitro drug sensitivity tests demonstrated that all the isolates exhibiting multiple drug resistance. Furthermore, the isolated strains displayed varying degrees of pathogenicity in mice. This study confirms that Enterococcus faecium is the causative agent for the deaths of Tibetan sheep. These findings enhance our understanding of the disease and suggest valuable insights for its prevention, control, and future research.
【Objective】This study was aimed to further understand the evolutionary dynamics and epidemic characteristics of H9 subtype Avian influenza virus（AIV）in Guangxi,which could provide data support for disease prevention strategy constituted and modified．【Method】3 600swabs samples of poultry throat,cloaca and environment were collected in several area markets of Guangxi from 2016to 2021,then were inoculated in 9-11days age SPF chicken embryos to isolate virus,the hemagglutinin（HA）and neuraminidase（NA）genes of H9 subtype AIV were amplified,sequenced and genetic evolutionary drynamic analyzed based on hemagglutinin and hemagglutinin-inhibit test,and Real-time quantitative RT-PCR detection assays．【Result】46 HA and NAgenes sequence were acquired,the length of HAgene was 1 683bp,coding for 560amino acids and 4 NAgene was 1 410bp,coding for 469amino acids except 42isolates were 466amino acids with absence of 3amino acids.BLAST analysis indicated that isolates shared the highest similarity with 97.62%-99.88% and 93.13%-99.79% to HAand NAgenes in this study were from Vietnam and several provinces of China,and hosts were not identical,which suggested that the genetic of HA and NA genes was diversity.The nucleotide and amino acid similarity of Guangxi isolates intra were 93.0%-99.1% and 94.6%-99.1%,83.9%-98.2% and 86.1%-98.2%,respectively,which indicated that HAgene was more conserved,the similarity of Guangxi isolates with the reference strains of after 2016 was higher than strains of before,which manifested that the isolates were persisting evolution.The genetic analysis showed that HAand NAgenes of Guangxi isolates belonged to G57branch prevalent in China,the genetic distance of which and vaccine strains was farther.The evolutionary rate estimation of HAand NAgenes was3.99×10 -3 and 4.59×10 -3 substitution/sites/year,and the most recent common ancestor of which was 66.77and 58.24years,which suggested that the rate of HAgene was slower than NA gene.The recombination analysis of HA gene acquired from the chicken,duck and environment was positive signal,respectively.The major and minor parents strains were A/Beijing/1/2017and A/quail/Hainan/250/2012．【Conclusion】The HAand NAgenes of Guangxi isolates with higher similarity to those of Vietnam and provinces of China,the genetic of them was diversity,and evolution progress was close to the dominant gene type G57,the recombination of HAgenes was occurred between AIV and human-origin influenza strain,which could provide outbreak condition for novel subtype influence in future.
Individual identification technology was the primary requirement of automatic production techniques,including intelligence weighting,body condition scoring,body shape identification,and behavior monitoring.Individual cattle identification methods were categorized and elaborated in current review,that the research progress of different individual cattle identification methods including traditional,biometric,and deep learning methods were introduced,especially the difficulties of applying deep learning method in practice were discussed.Moreover,the advantages and disadvantages of different identification methods were analyzed.Traditional identification methods such as ear notching,ear tattooing,and hot branding identified cattle relies on manual labor,which had low identification accuracy and efficiency,and ignored animal welfare and mark persistence.Radio frequency identification techniques provided an automatic identifying protocol that elevated the efficiency but the data security could not be guaranteed.With the development of image recognition technology and deep learning methods,non-contact,safe and efficient cattle intelligent recognition had been realized with the technology based on biometric recognition and deep learning methods.However,the biometric identification method based on nose print,retinal blood vessels and iris had poor practicability,because the ideal image was difficult to obtain.Based on deep neural networks learning of image features,the deep learning method was more practical and promising in complex dairy farm circumstances.What’s more,different individual identification methods were compared,and the research on individual cattle identification technology was prospected in current review.
【Objective】The effects of fermented hybrid Broussonetia papyrifera on growth performance,meat quality,digestive enzyme activity and intestinal flora of Hu sheep were studied to provide scientific data for the application of hybrid Broussonetia papyriferain mutton sheep breeding．【Method】Seventy-two Hu sheep with good health and similar weight were randomly divided into 4groups,with 6replicates in each group and 3sheep in each replicate.The sheep in control group（CG）was fed with basic diet,while the sheep in experimental groups（EG1,EG2and EG3）was fed with 16%,24% and 32% fermented hybrid Broussonetia papyrifera respectively.The pre-feeding period was 14days and the experimental period was 90days.After the experiment,the weight of Hu sheep was measured,and one sheep was slaughtered randomly from each repetition.Femoral muscles of hindlegs and longissimus dorsi,contents of the middle jejunum and cecum were taken to determine the meat quality,digestive enzyme activity and intestinal flora changes．【Result】Compared with the control group,（1）There was no significant difference in average daily feed intake,average daily gain and feed-to-weight ratio of the sheep fed with different proportions of fermented hybrid Broussonetia papyrifera（P＞0.05）．（2）In femoral muscles of hindlegs,the shear force of EG3group was significantly reduced（P＜0.05）,the cholesterol content in EG2and EG3groups decreased significantly（P＜0.05）,the crude fat content of the three experimental groups decreased significantly（P＜0.05）,while the hydraulic capacity increased significantly（P＜0.05）,and there was no significant difference in pH,crude ash,crude protein and amino acid content among three groups（P＞0.05）.The contents of palmitic acid,oleic acid,linoleic acid andα-linolenic acid in EG1,EG2and EG3groups were significantly increased（P＜0.05）,and the contents of Ca,Fe,Zn and Se in EG2and EG3groups were also increased（P ＜0.05）.In longissimus dorsi,the shear force,pH,crude fat and cholesterol contents in EG2and EG3groups were significantly decreased（P＜0.05）,while the crude ash content in EG2group was significantly decreased（P＜0.05）.There was no significant difference in hydraulic power,crude protein and amino acid content among three experimental groups（P＞0.05）.The contents of palmitic acid,oleic acid,linoleic acid,α-linolenic acid and arachidonic acid,and Ca and Zn contents in EG1,EG2and EG3groups increased significantly（P＜0.05）.Se content increased significantly in EG2and EG3groups,and Fe content increased significantly in EG3 group（P ＜0.05）．（3）Intestinal trypsin and lipase activities increased significantly（P ＜0.05）,while the concentration of total protein in intestine decreased significantly（P ＜0.05）in EG2and EG3groups．（4） Adding fermented hybrid Broussonetia papyrifera could significantly improve the diversity and richness of intestinal flora of experimental sheep,increase the relative abundance of probiotics such as Firmicutes,and decrease the relative abundance of harmful bacteria such as Proteobacteria．【Conclusion】Adding 24%-32%fermented hybrid Broussonetia papyrifera in the diet could improve the meat quality and digestive enzyme activity of Hu sheep,and improve the richness and diversity of intestinal flora which was conducive to promoting the absorption of nutrients by animals.
【Objective】The purpose of this survey was to investigate the distribution of magnesium contents in various feed materials from different regions in China,so as to provide scientific basis for reasonable supplement of magnesium in the diet．【Method】A total of 4 054samples of 37kinds of feed materials for livestock and poultry were collected from 31provinces,municipalities and autonomous regions in China.After pretreatment,microwave digestion was performed and then the magnesium contents were determined by an ion chromatography-inductively coupled plasma-mass spectrometry（IC-ICP-MS）.The One-Way ANOVA was used to analyze the magnesium content in different feedstuffs from the same category and some feedstuffs from different provincial（district）with SAS software,respectively.The differences in magnesium contents among different feedstuffs and different regions were compared．【Result】The distribution regulation of magnesium contents in various categories of feed materials was as follows:Mineral feeds（average magnesium contents were 12 740mg/kg） ＞ vegetable protein feeds（average magnesium contents were4 335mg/kg）＞cereal by-products（average magnesium contents were 3 275mg/kg）＞forage feeds（average magnesium contents were 2 482mg/kg）＞ straw feeds（average magnesium contents were2 346mg/kg）＞animal protein feeds（average magnesium contents were 1 436mg/kg）＞cereal seeds（average magnesium contents were 1 220 mg/kg）.There were significant differences in magnesium contents among different feed materials from the same categories（P＜0.05）.In the cereals,the magnesium content in wheat or barley（1 358mg/kg）was the highest,while that in rice（1 115mg/kg）was the lowest.In the cereal by-products,the highest（5 791 mg/kg）and lowest（387 mg/kg）magnesium contents were observed in rice bran and broken rice,respectively.In the vegetable protein feeds,the highest（6 664 mg/kg）and lowest（2 605 mg/kg）magnesium contents were detected in cottonseed meal and expanded soybean,respectively.As for animal protein feeds,the highest（3 479mg/kg）and lowest（269mg/kg）magnesium contents were detected in fish meal and dried blood cells,respectively.As for straw feeds,the highest（4 024mg/kg）and lowest（1 136mg/kg）magnesium contents were found in sweet potato vine and wheat straw,respectively.As for pasture feeds,the highest（2 996 mg/kg）and lowest（1 617 mg/kg）magnesium contents were found in alfalfa and Leymus chinensis,respectively.As for mineral feeds,the highest（18 290mg/kg）and lowest（3 118mg/kg）magnesium contents were found in calcium hydrogen phosphate and shell powder,respectively.A comparison was made among the magnesium contents of corn,wheat or soybean meal in different provinces（regions）,and the results showed that there were significant differences in the magnesium contents of the same feed materials from different provinces（regions）（P＜0.05）.According to 152feed formulas commonly used in swine and chickens all over the country,the calculated magnesium contents in the diets were ranged from 1 848to 2 100 mg/kg.In accordance with the magnesium requirements suggested by feeding standards of swine and chicken（2004）in China or the NRC（1994）of the United States for swine and chickens,about 1/3of the magnesium contents in the diets could meet all the nutritional requirements of magnesium for swine and chickens,but the above estimations had not considered the availabilities of magnesium in different feed materials．【Conclusion】The content of the mineral element magnesium varies greatly among regions and various types of feed ingredients in China.The magnesium contents in the commonly used diet formulas in the country could provide all the magnesium nutritional requirements of swine and chickens.Therefore,the actual production should fully consider the total magnesium content of the ration and its utilization rate in different regions,and reduce the addition of magnesium and the waste of resources under the premise of ensuring the health of livestock and poultry and efficient production.
【Objective】 The purpose of this experiment was to study the lipid profile of duck breast muscle and investigate the differences between the breast muscle lipidome of Pekin duck and Liancheng White duck.【Method】 28 Pekin ducks and 28 Liancheng White ducks were selected at 6-week-old and their breast muscle tissues were collected to detect the lipid profile used lipidomics techniques based on liquid chromatography-mass spectrometry（LC-MS）.The results were analyzed by multivariate statistical analysis, and the differential lipids were screened by fold change, t-test and partial least squares discriminant analysis（PLS-DA）.【Result】 A total of 950 lipids were identified in duck breast muscle, covering 6 categories, 395 glycerophospholipids（GP）,365 glycerolipids（GL）,86 sphingolipids（SP）,78 fatty acyls（FA）,23 sterol lipids（ST） and 3 prenol lipids（PR）;The most abundant lipid subgroup was triglycerides（TG） with 283 species.Compared with Pekin duck, 18 lipids were screened in Liancheng White duck, 7 were upregulated and 11 were downregulated; Among them, TGs were the most abundant and high in Pekin duck, which were rich in saturated and monounsaturated fatty acids, while these GP containing very long chain polyunsaturated fatty acid（VLC-PUFA） were found to be high in Liancheng White duck.【Conclusion】 In this study, a comprehensive and detailed lipid profile of duck breast muscle was obtained, and 18 differential lipid markers were screened for Pekin duck and Liancheng White duck, this study provided a scientific and theoretical basis for intramuscular lipid metabolism, meat quality research and quality breeding of duck.
【Objective】 The purpose of the experiment was to prepare a new antibacterial peptide-Shengtaicin post-medicated bath solution, and characterize its effectiveness and safety.【Method】 The optimal formulation of Shengtaicin post-medicated bath solution was screened by one-way effect test and L 9 （3）~3 orthogonal test.In vitro bactericidal test was used to characterize the bactericidal efficacy of the post-medicated bath solution.The safety was verified by hemolysis test, cytotoxicity test, skin irritation test in single-administered mice, skin irritation test in multiple-administered mice and skin sensitization test in mice.【Result】 The orthogonal test results showed that, the film solution was configured with 7% film-forming agent PVA1788 as the substrate, and glycerol had the greatest experimental impact, followed by Tween 80.The optimal formulation of the Shengtaicin post-medicated bath solution was as follow: The optimal addition volume fraction of the plasticizer glycerol was 4%,the optimal addition volume fraction of the surfactant Tween 80 was 1%,and the optimal addition mass percentage concentration of the thickener pullulan polysaccharide was 0.4%.More than 99.999% of the tested Staphylococcus aureus,Staphylococcus epidermidis,Streptococcus agalactiae and Streptococcus dysgalactiae were killed within 30 min.The bactericidal effect was comparable to that of povidone iodine.In addition, the hemolytic and cytotoxic properties of Shengtaicin post-medicated bath solution were lower than those of the post-bath solution of povidone-iodine.The results of single and multiple skin irritation test and skin sensitization test in mice showed that Shengtaicin post-medicated bath solution was non-irritating and non-sensitizing to the skin of mice.【Conclusion】 The antimicrobial peptide Shengtaicin post-medicated bath solution was a validity, green and safe bath, which could effectively prevent the occurrence of mastitis in cows, and it would ensure the quality and safety of raw milk and promote the healthy development of the dairy industry.
【Objective】 The purpose of this study was to establish an ELISA method for detection of IgA antibody against Porcine epidemic diarrhea virus（PEDV）.【Method】 In this study, PEDV epidemic strain CH/HNPJ/2017（MF152604.1）was used as the biological material, and the S2 gene（2 368—4 170 bp） was amplified by RT-PCR and inserted into prokaryotic expression vector pET-24a to transform the competent cells of Escherichia coli BL21（DE3）.It was induced by IPTG and purified by nickel column.Western blotting was used to verify the reactivity and specificity of recombinant protein S2 with PEDV positive serum.Using it as the coating antigen, an IgA antibody ELISA method based on recombinant full-length S2 protein of PEDV variant strain was established.The chessboard method was used to determine the optimal detection conditions, and its sensitivity, specificity and repeatability were determined.It was preliminarily applied to the detection of 130 porcine serum and 40 porcine oral mucus.【Result】 The S2 gene was amplified by RT-PCR,and the recombinant expression vector pET-24a-S2 was successfully constructed.The recombinant S2 protein was obtained after being transformed into Escherichia coli and expressed and purified by induction.SDS-PAGE results showed that the purified S2 protein had specific bands and no heterobands.Western blotting test showed that the protein had good reactivity with PEDV positive pig serum.The optimal coating conditions for recombinant S2 protein were 0.5 μg per well at 4 ℃ overnight, the optimal serum reaction conditions were 1∶160 dilution at 37 ℃ for 60 min, the best reaction conditions for HRP-conjugated antibody were 1∶5 000 dilution at 37 ℃ for 30 min, and the optimal color rendering time of TMB was 10 min under ambient temperatures.The sample was positive when the S/P value ≥ 0.04,it was negative when the S/P value ≤ 0.02,and it was suspicious when the S/P value was between 0.02 and 0.04.The ELISA method established in this study was used to detect the standard positive serum of swine fever virus, porcine circovirus, porcine reproductive and respiratory syndrome virus, pseudorabies virus and porcine deltacoronavirus.The S/P values were all less than 0.02,indicating that the method had good specificity.When the PEDV positive serum was diluted by 640 times, it was still positive by this method, indicating that this method had good sensitivity.This method was used for inter assay and intra assay, and the coefficient of variation were all less than 8%（within 10%）,which shows that this method had good repeatability.130 pig sera were detected by this method, and the coincidence rate between the results and IDEXX IgA antibody detection kit was 90.77%.The detection results of 40 samples of pig oral mucus showed that the positive detection rate was 90.0% and the negative detection rate was 93.3%.【Conclusion】 In this study, the recombinant plasmid pET-24a-S2 was successfully constructed, and the recombinant S2 protein with high purity was obtained, which was used as coating protein to establish an ELISA method for IgA antibody based on full-length S2 protein.The method could effectively detect both serum and pig oral mucus type samples, and had strong sensitivity, high specificity, stability, and repeatability, which was of great clinical practical significance to the clinical diagnosis and prevention and control of PEDV.
【Objective】 This study was aimed to better understand the molecular characteristics of complete genomes of waterfowl infected with H3 subtype Avian influenza virus（AIV） in the coast area of Guangxi.【Method】 Two strains of H3 subtype AIV named A/duck/Guangxi/S11359/2020（H3N1） and A/duck/Guangxi/S11374/2020（H3N8） were acquired by one-step RT-PCR amplification of genome segment, then conducted sequencing and evolutionary analysis further in this study.【Result】 The amino acid cleavage sites of HA protein of both H3 subtypes AIV were 340 PEKQTR↓GLFG 349 each, which was in accordance with the molecular characteristics of low pathogenic AIV,and the 226th and 228th amino acid sites correlated to receptor tropism were Q and T,indicating that were poultry receptor as possible.The BLAST analysis result showed that M and NP genes of H3N1 and PA gene of H3N8 with the highest similarity to H7 subtype AIV was 99.5%,98.7% and 98.9%,respectively, which suggested that genes recombination was occurred between H3 and H7 subtypes AIV.The genes of both H3 subtype AIV shared the highest similarity with that of waterfowl-origin H3,H4,H6,H7 and H9 subtypes AIV from Vietnam, Bangladesh and South Korea countries around China, which indicated that the isolates possessed the same evolutionary source.Genetic analysis indicated that all genes of both H3 subtype AIV isolates belonged to the Eurasian branch, the genetic distance of those was the closest to waterfowl-origin influenza virus strains, closer to canine and feline-origin strains, close to human and porcine-origin strains, but in addition to NS gene of H3N1 isolate which got the closest genetic distance to A/Aichi/2/1968 derived from Japanese earlier, suggesting that genes exchanged could occurred between them.Recombination sign of HA gene of H3N8 isolate was detected, the recombinant parent strains of that were A/duck/Beijing/40/04 and A/canine/Beijing/20121215-34/2012 strains, with the similarity was 93.1% and 97.1%,respectively.【Conclusion】 The source and genetic characteristics of genes of both sea duck-origin H3 subtype AIV from Guangxi was complicated and divergent.
【Objective】 The purpose of this experiment was to identify and master the epidemiological characteristics and biological characteristics of Bovine viral diarrhea virus（BVDV） in Liaoning province, and to provide theoretical basis and technical support for the prevention and treatment of BVDV.【Method】 The tissues suspected of BVDV infection from a cattle farm in Liaoning province were collected, Infectious bovine rhinotracheitis virus（IBRV）,Bovine respiratory syncytial virus（BRSV）,and Bovine parainfluenza virus type 3（BPIV3） and BVDV gene-specific primers were identified by RT-PCR,and MDBK cells were inoculated with interstitial fluid to isolate the virus.After cytopathic observation of the cells, the virus was identified again by electron microscopy, indirect immunofluorescence assay（IFA）,and the whole genome sequence of the virus was amplified by PCR and analyzed for genetic evolution.【Result】 The identification result of BVDV specific primer RT-PCR was positive.There was a band at 280 bp, which was consistent with the expected band size.The other pathogen specific primers did not amplify the band, which proved that other pathogens were not infected.The isolated strain did not show obvious cytopathic effect in MDBK cells.After virus purification, virus particles with a diameter of about 50 nm were observed by electron microscopy.The results of IFA showed that bright green fluorescence could be observed in the MDBK cells inoculated with the isolated virus strain, while no fluorescence was observed in the control cells.The isolated virus was named LN-1 strain.The size of the whole genome amplification sequence was 12 269 bp.Genetic evolution analysis showed that LN-1 strain was in the same branch as XC,SD-15 and ZM-95 strains, and the nucleotide similarity with XC strain was 96.2%,belonging to BVDV-1m type.【Conclusion】 In this study, one strain of 1m BVDV strain was successfully isolated, which was of great significance for the follow-up vaccine research and development, epidemiological investigation and pathogenesis research.
【Objective】 This study was aimed to investigate the drug resistance of animal-derived Escherichia coli（E.coli） isolates in Hebei and the genetic relationship among the drug-resistant strains after the prohibition of resistance, so as to provide a scientific basis for the effective prevention and control of E.coli drug resistance.【Method】 297 fecal samples（48 from chickens and 249 from pigs） of healthy animals were collected from a farm in Hebei province.The isolates were identified by bacterial isolation and purification and PCR amplification, and the resistance of the isolates were determined by drug susceptibility test.Drug resistance gene detection, serotype identification, phylogenetic group analysis, plasmid replicon typing and genotyping were performed.【Result】 The isolates appeared as pink round colonies on MacConkey medium.A total of 129 strains of E.coli were isolated and identified by 16S rDNA sequencing analysis, of which 92 strains of E.coli showed multi-drug resistance, mainly 3-4 resistance, and the detection rate of the drug resistance genes were the highest with floR,tetA,qnrS and strB.The multidrug-resistant E.coli was dominated by O132 serotype, and the phylogenetic groups were mainly distributed in groups A and B1,and the main prevalent plasmids were IncFIB and IncN,27 ST types were obtained by MLST typing, mainly ST93 type, of which 12 sequence types were unknown type.【Conclusion】 The phenomenon of multi-drug resistance of E.coli in the intestine of healthy animals in Hebei was relatively serious, with numerous serotypes and diverse molecular characteristics, and it was still necessary to strengthen the monitoring of drug resistance and molecular characteristics of E.coli of animal origin.
【Objective】 The purpose of this experiment was to clone and bioinformatically analyze the gene of the rate-limiting enzyme aralkylamine-N-acetyltransferase（AANAT） for N-acetyl-5-methoxytryptamine（MT） synthesis in American mink（Neovison vison）,so as to provide a reference for biological functions in mink.【Method】 DNA was extracted from the caudal vein of mink, the sequence of AANAT gene was cloned by homologous recombination, the sequence of CDS region was analyzed, the amino acid sequence of AANAT gene was deduced for similarity alignment and phylogenetic tree construction, and the AANAT protein was analyzed by bioinformatics.【Result】 The sequence of AANAT gene in mink was about 1 631 bp, and the sequence of CDS region was 504 bp, which could encode 167 amino acids.The amino acid sequence similarity of AANAT gene in mink was 98.2% with Mustela putorius furo,and the phylogenetic tree analysis also showed that it was the closest relative to Mustela putorius furo.Bioinformatics analysis results showed that AANAT protein in mink was a hydrophobic protein without transmembrane domain and signal peptide, mainly distributed in the cytoplasm, and had 5 antigen-binding sites as a non-secreted protein.AANAT protein contained multiple post-translational modification sites, such as phosphorylation and glycosylation.AANAT protein contained a conserved domain of the N-acyltransferase superfamily, which was closely related to mammalian circadian rhythms.The secondary structure of AANAT protein was dominated by random coil, and the tertiary structure contained several conserved catalytic residues.Protein network interaction analysis results showed that AANAT protein could interact with multipleserotonin synthesis-related proteins.【Conclusion】 The sequence of AANAT gene in mink was successfully obtained.The structure and specific modification sites of AANAT protein might participate in the regulation of MT biosynthesis by localizing acetyl-CoA and 5-hydroxytryptamine.The reults provided a reference for research on the regulation mechanism of AANAT gene on embryonic diapause and improving fecundity in mink.
[目的]筛选牦牛卵泡发育过程中可能对靶向Smad家族成员4(Smad family member 4,Smad4)基因的bta-miR-146a具有"海绵吸附"作用的长链非编码RNA(long non-coding RNA,lncRNA).[方法]使用miRanda和RNAhybrid数据库对靶向牦牛bta-miR-146a的lncRNA进行预测;采集牦牛卵巢,分离健康、闭锁卵泡,用Trizol法提取RNA并反转录为cDNA,利用PCR检测所预测lncRNA的表达情况;利用实时荧光定量PCR法检测所筛选lncRNA和bta-miR-146a/Smad4在牦牛健康、闭锁卵泡中的表达情况;构建lncRNA-ENSBGRT00000000387.1的野生型和突变型双荧光素酶载体,将其与bta-miR-146a-mimics、mimics NC共转染至HEK293T细胞,检测双荧光素酶活性.[结果]试验共筛选出7个可能对靶向Smad4基因的bta-miR-146a具有"海绵吸附"作用的lncRNAs,其中的lncRNA-ENSBGRT00000000387.1在牦牛卵泡中表达量极显著高于其他lncRNAs(P＜0.01).实时荧光定量 PCR 检测发现,lncRNA-ENSBGRT00000000387.1、bta-miR-146a 和 Smad4 基因 mRNA 在牦牛健康和闭锁卵泡中共表达,且lncRNA-ENSBGRT00000000387.1和Smad4基因mRNA在牦牛健康卵泡中的表达量均显著高于闭锁卵泡(P＜0.05),bta-miR-146a在牦牛健康卵泡中的表达量极显著低于闭锁卵泡(P＜0.01).试验成功构建牦牛lncRNA-ENSBGRT00000000387.1野生型及突变型pmirGLO双荧光素酶报告质粒,双荧光素酶活性结果显示,bta-miR-146a mimics 对牦牛 lncRNA-ENSBGRT00000000387.1-WT 具有极显著下调作用(P＜0.01).[结论]lncRNA-ENSBGRT00000000387.1、bta-miR-146a 和 Smad4 基因 mRNA 可能在牦牛卵泡发育或闭锁过程中存在调控机制,并在体外初步证实lncRNA-ENSBGRT00000000387.1与bta-miR-146a具有"海绵吸附"作用,这为进一步研究lncRNA-ENSBGRT00000000387.1在牦牛卵泡中的功能机制提供依据.
【Objective】 The purpose of this study was to explore the maturation characteristics of raccoon dog oocytes, and to provide a basis for the in vitro maturation culture of raccoon dog oocytes and to select high-quality mature oocytes, so as to improve the in vitro fertilization efficiency of raccoon dog.【Method】 Fresh ovaries from 1 to 3 years old female raccoon dogs at reproductive stage were collected to prepare light and electron microscope samples to observe the changes of organelles during oocyte maturation of raccoon dog. X-ray microanalysis was used to explore the relationship between the changes of chemical elements in plasma membrane and maturation of oocyte.【Result】 The maturation and development of raccoon dog oocytes could be divided into 8 stages according to the layer number of granulosa cells, the diameter of follicles, the size of oocytes and the changes of the zona pellucida.Raccoon dog oocytes were surrounded by granulosa cells in the early developmental stage（stages Ⅰ to Ⅳ）,with the gradual appearance of the zona pellucida and the follicular cavity, the chromatin in the germinal vesicles became more and more compact.With the maturation and development of oocytes（stages Ⅴ to Ⅶ）,cortical granules and microvilli appeared and the number of organelles such as Golgi complex, mitochondria increased significantly and they gradually migrated to the cortex, while mitochondria and lipid droplets experienced morphologic variation.When the oocyte grew mature（stage Ⅷ）,the Golgi complex and the rough endoplasmic reticulum disappeared, and the cortical granules were arranged under the plasmalemma.After ovulation, the nucleolus became compact.The contents of sodium, chloride and sulfur in oocyte plasma membrane decreased with the increase of follicular cavity.The contents of sodium, chlorine and sulfur in oocyte plasma membrane in stage Ⅴ were significantly higher than those in stage Ⅶ（P＜0.05）,and the relative content of calcium was significantly lower than that of stage Ⅵ and Ⅶ oocytes（P＜0.05）.Potassium showed a low-high-low variation, and the content of potassium in oocyte plasma membrane in stage Ⅵ was significantly higher than that of stage Ⅴ and Ⅵ oocytes（P＜0.05）.【Conclusion】 After the raccoon dog oocytes developed to stage Ⅴ,the type and number of organelles increased, the plasma membrane chemical elements changed, and oocyte membrane potential was depolarized, and the oocytes began to enter the mature stage.At this time, the oocytes had the ability to mature in vitro.
【Objective】 The purpose of this experiment was to analyze the effective active ingredients of Radix Paeoniae Rubra on antioxidative stress and its molecular mechanism using network pharmacology and molecular docking technology.【Method】 Active ingredients and related targets of Radix Paeoniae Rubra were collected from TCMSP database and then converted into gene names using UniProt database.Targets related to oxidative stress were collected from GeneCards and OMIM databases, and the intersection targets of Radix Paeoniae Rubra and oxidative stress were obtained by Veeny online platform.Subsequently, protein-protein interaction（PPI） network map and a Radix Paeoniae Rubra-target-oxidative stress visualization network for topological analysis were established by STRING database and Cytoscape 3.8.0 software, and then key targets were identified.The active ingredients and antioxidant signaling pathways of Radix Paeoniae Rubra were determined by GO function and KEGG pathway enrichment analysis with Metascape database.AutoDock and PyMol softwares were used to perform molecular docking verification of the core target of Radix Paeoniae Rubra.【Result】 12 active ingredients in Radix Paeoniae Rubra were screened, including baicalein, beta-sitosterol, ellagic acid, stigmasterol,（+）-catechin, etc.There were 76 active ingredients targets in Radix Paeoniae Rubra and 4 873 oxidative stress targets, including 69 intersection targets, such as protein kinase B（AKT1）,activator protein 1（JUN）,cellular tumor antigen p53（TP53）,tumor necrosis factor（TNF） and caspase-3（CASP3）, etc.The results of GO function and KEGG pathway enrichment analysis revealed that antioxidative stress of Radix Paeoniae Rubra might be related to the phosphatidylinositol-3-kinase-threonine-protein kinase（PI3K-Akt）,neurofibromin-κB（NF-κB）,and other signaling pathways.Moreover, the results of molecular docking showed that the main active ingredients of Radix Paeoniae Rubra,baicalein and beta sitosterol, had good binding ability to the core targets.【Conclusion】 The active ingredients such as baicalein, beta sitosterol and ellagic acid in Radix Paeoniae Rubra might play an antioxidative stress role by regulating key targets in PI3K-Akt, NF-κB,interleukin-17（IL17） and other signaling pathways.
【Objective】 The aim of this study was to identify candidate genes and molecular markers affecting the contents of mineral elements in breast muscle of ducks by genome-wide association study（GWAS）,and analyze the genetic mechanism of mineral element traits.【Method】 The F2 generation resource population of Pekin ducks × Anas platyrhynchos was constructed, and the whole genome was resequenced.The breast muscles of ducks were collected for 8 weeks and the contents of 7 major mineral elements were determined according to the national standard method.Combined with the resequencing data and mineral element phenotypic data, the genes related to element content were identified by whole gene association analysis.【Result】 The phenotypic heritability of the contents of seven mineral elements was P（0.007）＜Fe（0.100）＜Mg（0.120）＜Na（0.140）＜K（0.150）＜Ca（0.190）＜Zn（0.350）.The results of correlation analysis showed that the correlation between Mg and K was the highest（r=0.62）.There was a high positive correlation between P and Mg as well as K in duck breast, r were 0.43 and 0.56,respectively.A SNP locus significantly related to Zn content were identified by GWAS and reached a significant association(-log 10 P-value=8.03) at 12 075 283 bp on chromosome 6. 39 SNPs highly correlated with the highest point SNP narrowed the candidate interval to 39 kb, in which there was only one gene SORCS3.Combined with gene functional annotation and transcriptome, SORCS3 was identified as a candidate gene for Zn content variation.The highest point of correlation between SNP and P content at the whole genome level was highly correlated with 260 SNPs（R~2＞0.4）,all of which were located at chromosome 4 between 17.1-18.6 Mb and contained 27 genes.Combined with correlation analysis, gene functional annotation and transcriptome, six candidate genes were highly related to P content were identified（TUSC3,MICU3,LOC101803915,MTMR7,SLC7A2 and ASAH1 genes）.【Conclusion】 The candidate gene SORCS3 of Zn content was identified by GWAS,and combined with transcriptome, six candidate genes related to P content were identified.These results enriched the knowledge system of mineral nutrition of poultry meat, and provided an important theoretical basis for breeding excellent livestock and poultry products.
[目的]研究茯苓多糖(Poria cocos polysaccharide,PCP)对脂多糖(lipopolysaccharide,LPS)诱导的猫肾细胞(crandell rees feline kidney,CRFK)炎症反应的保护作用及抗炎机制.[方法]通过MTT法检测不同浓度PCP(5、10、15、25、35、45 μg/mL)和 LPS(20、40、60、80、100、125、150 μg/mL)对 CRFK 细胞活力的影响;用不同浓度PCP处理LPS诱导的CRFK炎症模型,观察PCP干预后的细胞形态变化,通过硝酸还原酶法和流式细胞术检测CRFK中一氧化氮(NO)释放和细胞凋亡情况;利用实时荧光定量PCR和Western blotting检测细胞炎症和凋亡相关基因以及蛋白表达水平.[结果]5～25 μg/mL PCP对CRFK无毒性,且可逆转LPS刺激后CRFK细胞形态变化.25 μg/mL PCP可显著降低LPS刺激后CRFK内NO释放和细胞凋亡率(P＜0.05).实时荧光定量PCR结果表明,不同浓度 PCP 能够显著降低 LPS 诱导 CRFK 中 IL-6、TNF-α、Caspase3、Caspase8、Caspase9、Bid 和 Bax mRNA表达量,显著上调Bcl-2 mRNA表达量(P＜0.05).Western blotting结果显示,LPS诱导的CRFK中TLR4、NF-κB、Caspase3和Caspase9蛋白表达水平显著升高(P＜0.05),PCP处理能显著降低TLR4、NF-KB、Caspase3和Caspase9蛋白表达量(P＜0.05).[结论]PCP能够通过调控细胞凋亡抑制LPS诱导的CRFK炎症损伤,其作用机制可能与抑制TLR4-NF-κB信号通路有关.研究结果为PCP治疗CRFK炎症提供了新的靶点和试验依据.
