The objective of this study was to clone the CDS region of SERPINA1 gene in Xinong Saanen dairy goats (Capra hircus),conduct the bioinformatics analysis using biological software and online prediction tools,and detect the mRNA expression level of SERPINA1 gene in different tissues of Xinong Saanen dairy goats by Real-time quantitative PCR.Specific primers were designed by Primer Premier 5.0 according to the CDS sequence of SERPINA1 gene in Capra hircus (GenBank accession No.:XM_018066209.1),the SERPINA1 gene CDS was amplified by RT-PCR and analyzed by bioinformatics after prokaryotic expression vector was constructed.Collecting tissues such as heart,liver,spleen,lung,mammary gland,kidney,muscle,rumen and small intestine,mRNA was extracted from these tissues and reversed transcription as cDNA,specific primers were designed for Real-time quantitative PCR to detect the expression differences of SERPINA1 gene mRNA in different tissues.The results showed that the CDS region of SERPINA1 gene in Xinong Saanen dairy goats (Capra hircus) was 1 326 bp in length,encoding 441 amino acids.Homology alignment analysis showed that the nucleotide sequences of Xinong Saanen dairy goats with Capra hircus,Ovis aries,Bos taurus and Mus musculus were 100%,98.6%,95.7% and 71.6%,respectively,and the goat was the most closest species with dairy goat,the second was sheep.The SERPINA1 protein had the molecular weight of 48.71 ku,the isoelectric point was 5.71,and was a hydrophilic protein.Amino acid sequence of SERPINA1 had 62 phosphorylation sites,with 3 transmembrane structure.Tissue expression analysis showed that SERPINA1 gene was highly expressed in liver of Xinong Saanen dairy goats (P<0.05),followed by the mammary gland,and was the least expressed in lung.The results provided a theoretical basis for further exploration of the role of SERPINA1 gene in milk protein synthesis and metabolism of dairy goats.

This experiment was aimed to obtain the genetic information of reindeer and the transcriptome characteristics of antler tissue by high-throughput sequencing technology,and further explore the key gene information related to the economic traits of antler.RNA was extracted from antler tissue in reindeer,and then the purity,integrity and contamination of RNA was detected.Qualified RNA was used for library preparation.The library preparation needs to be tested,after the test,transcriptome sequencing analysis of antler in reindeer using the Illuminanovaseq platform.Functional annotation,gene abundance analysis and other analysis of the transcriptome sequences of antler in reindeer were performed using software such as Diamond,HMMER,KAAS and Blast2GO.The results showed that 47 818 202 raw reads were obtained by sequencing,46 964 478 clean reads were obtained after filtering and quality control,and 49 171 Unigenes were obtained after assembly,indicating that the high-quality antler transcriptome was obtained by sequencing.Based on sequence identity analysis,Unigenes were compared with NR,Swiss-Prot,KOG,KEGG,Pfam and GO databases,the annotations accounted for 89.54% successfully,with a total of 44 029 sequences.After clustering of Corset,49 171 Unigenes were obtained,and 13 795 SNPs and 18 446 SSRs were found in Unigenes.22 955 Unigenes annotated with GO terms,accouting for 46.68% of the total annotated results.Compared with the KEGG database,49 171 Unigenes might be involved in the metabolic pathway,and 20 285 Unigenes were annotated to 32 KEGG metabolic pathways.In addition,there were some high-abundance genes in transcriptome results,such as OPN, TMSB4 and TMSB10.Their function might be related to anlter in reindeer.In summary,this results not only supplement the genomic data of reindeer,but also reveal the genetic characteristics of antler in reindeer in the growing season,which provided a theoretical basis for molecular genetics research,resource conservation and utilization and restoration of population resources.

In order to study the structure and function of Small-tailed Han sheep leukocyte antigen (OLA) class Ⅰ tetramer light chain β₂-microglobulin (β₂m).A pair of specific primers based on the published sequence of this gene was designed and the cDNA of full coding region for β₂m precursor was obtained by RT-PCR from the Small-tailed Han sheep whole blood.The Small-tailed Han sheep β₂m gene was cloned into the pMD18-T vector and the positive clones of pMD18T-OLAⅠ-β₂m were selected.After double digestion,the β₂m gene in positive clones was further ligated into the pET-28a(+) expression vector and transformed into E.coli BL21(DE3) to construct the recombinant strain of pET-28a(+)-OLAⅠ-β₂m.Then the targeted protein was detected by SDS-PAGE within induction of IPTG.The secondary structure of the OLAⅠ-β₂m protein was predicted and analyzed by SOMPA software.The PCR results showed that the length of OLAⅠ-β₂m was about 357 bp which was consistent with the theoretical value.The amplified fragment was successfully cloned into pMD18-T vector,and the positive clones were verified by EcoR Ⅰ and Hind Ⅲ digestion and DNA sequencing analysis.The cut β₂m gene from pMD18T-OLAⅠ-β₂m was inserted into pET-28a(+) and transformed into E.coli BL21(DE3) successfully.After IPTG induction,the expressed protein was detected by western blotting and SDS-PAGE,the results showed that the target protein (17.3 ku) was expressed efficiently with inclusion body.Finally,the secondary structure α-helix,extended strand and random coil of the target protein was 22.03%,22.03% and 55.93%,respectively.In this study,the recombinant tetramer precursor of OLAⅠ-β₂m light chain was constructed into pET-28a(+) expression line successfully,and the secondary structure of the OLAⅠ-β₂m protein was predicted and analyzed by SOMPA software,which would lay a solid foundation for constructing the OLAⅠtetramer of Small-tailed Han sheep.

This study was aimed to investigate the effects of melatonin on proliferation,cell cycle and apoptosis of antler cartilages cultured in vitro.The chondrocytes were identified by toluidine blue,alizarin red S and alexin blue staining,the exogenous addition of melatonin was used at different concentrations (0,400,800,1 200,1 600 and 2 000 pg/mL),velvet antler chondrocytes were treated at different times (24,48 and 72 h),and cell proliferation was detected by CCK-8 method.The chondrocyte was treated with 800 pg/mL melatonin for 24 h.The cell cycle distribution and apoptosis were detected by flow cytometry.The testosterone content in the cell culture medium was determined by ELISA kit.The results showed that after treatment with different concentrations of melatonin for different time,the cell viability of velvet cartilage cells treated with 800 pg/mL melatonin for 24 h was extremely significantly increased (P<0.01).After treatment with melatonin in chondrocytes,the proportion of G1 phase in melatonin-treated group was significantly lower than that in control group (P<0.05).The proportion of G2 phase was extremely significantly increased (P<0.01),and the cells were arrested in G2 phase.There was no significant change in the early apoptotic rate of melatonin treatment group (P>0.05),and the late apoptotic rate was significantly decreased (P<0.05).The testosterone secretion level was significantly increased by ELISA (P<0.05).In conclusion,the addition of 800 pg/mL melatonin could promote the proliferation of antler chondrocytes and inhibit the late apoptosis of chondrocytes to promote the secretion of testosterone,which provided a reference for elucidating the mechanism of melatonin involved in the growth and development of antler.

Intestinal microecology can affect the intake,storage and utilization of nutrients by the body,and has significant influence on animal growth and development.Establishment of an individual's microbial ecosystem is influenced by various factors,such as birth mode,diet,antibiotics,diseases and living environment.With deepening interest in microbial symbioses,increasing attention has been paid to the roles of intestinal flora.A dynamically balanced intestinal microbial ecosystem is required for the healthy growth of the host.Piglets take time to establish a balanced intestinal microbial ecosystem.Prior to establishing this balance,they are susceptible to external factors that can cause growth retardation,low feed conversion rate,and even occurrence of diseases.Regulation of intestinal flora is an effective technical means to reduce the occurrence of animal diseases and increase feed conversion rates.The author reviews research progress in the intestinal microecology of piglets in China and other countries.It starts with the microbial composition and functions of the intestinal microbial ecosystem,and expounds upon processes used at different development stages to establish a healthy intestinal microecosystem in piglets,and on current regulatory measures,with the goal of providing a theoretical reference for studies of the formation and maturation of intestinal microbial ecosystems,and its effects on the growth and development of piglets.

In order to study the influence of environment and diets on the rumen microflora,the study applied high throughput sequencing technology to analyze the change of Qinghai yak rumen bacterium group after their introduction to Shandong.The results showed that the difference between the Observed species and Chao1 index of Qinghai yak tumor gastric juice and that of Shandong were extremely significant (P<0.01),the ACE index was significantly different(P<0.05).It indicated that bacteria abundance of Qinghai yak rumen microflora was significantly higher than that of Shandong.The shannon index and simpson index showed no significant difference (P>0.05),which indicated that there was no significant change in bacteria diversity of yaks rumen microflora between migration after and before.The bacteria with higher abundance in yak rumen fluid came successively as Bacteroidetes,Proteobacteria,Firmicutes,Verrucomicrobia,Tenericutes,Fibrobacteres.After the migration,Planctomycetes and Armatimonadetes,Oscillibacter and Papillibacter,were among those significantly changed phyla and genera (P<0.05),in terms of composition and proportion.According to the analysis of sequencing,bacteroidetes and Unidentified _Rikenellaceae played an important role in Qinghai yaks rumen liquid,while Prevotellaceae,Succinivibrionaceae and Aeromonadales were crucial part in that of Shandong yaks.Changes in the environment and diet could lead to changes in rumen microflora and related functions.This study provided a theoretical basis for environment and diet affected changes in the yak's rumen microflora structure.

Glycosylation is one of the most important post-translational modifications of proteins in life and plays a very important role in life.Oligosaccharides on glycoproteins can serve as binding sites for lectins,so lectins can be used to identify and analyze sugar chain structures,as an important complement to glycosylation analysis methods such as mass spectrometry.The purpose of this study was to establish and optimize the fluorescence lectin-based immunohistochemical method to study the glycosylation modification in the pig intestines,and to apply this method to study the differences in glycosylation modification in different intestinal segments and the effect of weaning on intestinal glycosylation modification in piglets.The results showed that:①The optimal conditions for fluorescence lectin-based immunohistochemical method were:deparaffinized sections were blocked with carbohydrate blocking solution for 30 min,sections were then incubated with 5 μg/mL FITC-lectin for 1 h at room temperature,and mounted with DAPI mounting medium.This method was characterized with high sensitivity,good specificity and more diversification regarding quantification.②Different glycosylation modification patterns were observed in the ileum and colon of growing pigs.The ileum was enriched with fucose and N-acetylglucosamine,while the colon was enriched with N-acetylglucosamine,fucose and mannose.Except that mannose was distributed in the non-goblet cells in the intestinal epithelium and lamina propria,fucose and N-acetylglucosamine were mainly distributed in the surface of intestinal villi and goblet cells.③The content of N-acetylglucosamine in the ileum increased along with the age of 22-28 days old piglets (P<0.05),and the content of N-acetylglucosamine in the ileum of piglets was significantly reduced by weaning at day 28 (P<0.05).In conclusion,a method to investigate glycosylation modification in pig intestine was successfully established in this study,and it was found that N-acetylglucosamine in the ileum could be used as a biomarker to indict a health intestinal barrier function in piglets by this method.

The objective of this study was to investigate the optimization of ultrasonic to improve the fiber structure of ammoniated rice straw and its effect on rumen degradation characteristics.The response surface analysis of four factors and five levels of central composite was used to design and optimize the ammoniated rice straw fiber modified ultrasonic conditions.Based on the neutral detergent fiber (NDF),acid detergent fiber (ADF) and acid detergent lignin (ADL) content as response values,a multivariate quadratic regression mathematical model was established to obtain the best ultrasound level.The contents of nutrients in ammoniated rice straw under the optimal ultrasonic level and untreated ammoniated rice straw were determined.The degradation rates of dry matter (DM),NDF and ADF in the rumen of cows were determined by nylon bag method,and the dynamic degradation parameters of each nutrient component were obtained.The results showed that ultrasonic power was 112 W,ultrasonic time was 17.3 min,liquid-solid ratio was 10.7 to 1,and ultrasonic power density was 1.317 W/mL,which was the best ultrasonic condition for ammoniating straw.At this ultrasonic level,the minimum contents of ADF and ADL of ammoniated rice straw were 43.75% and 7.95%,respectively,which were significantly lower than those before ultrasonic (P<0.05).The actual results were close to the model prediction,indicating that the model was feasible.Compared with before ultrasound,ammoniated rice straw ADF decreased by 6.64% and ADL decreased by 11.38%.After ultrasonic treatment,the slow degradation part (b),potential degradation part (d) and effective degradation rate (ED) of DM,NDF,ADF of the ammoniated rice straw were significantly improved (P<0.01),the slow degradation parts of which increased by 36.52%,30.06% and 22.19%,respectively,the potential degradation parts increased by 27.15%,28.56% and 21.35%,respectively,and the effective degradation rates increased by 25.09%,24.98% and 24.00%,respectively.In conclusion,appropriate ultrasonic treatment could reduce the content of ADF and ADL in the ammoniated rice straw and improve the effective degradation rate.

In order to study the regulation and repair of intestinal flora and intestinal mucosal damage by single-flavor Chinese medicine,the intestinal flora of broiler chickens caused by Escherichia coli was selected from Chinese herbal medicines of Honeysuckle,Forsythia and Herba violet.It provided a reliable theoretical basis for improving the beneficial flora of intestinal bacteria and protecting intestinal mucosal barrier.100 male broilers of 1 day old were selected and divided into five groups,20 in each group.The blank group was fed normally.The model group and the Chinese medicine group were intraperitoneally injected with E.coli O₇₈ (1.0×10⁶ CFU/mL) at 15-17 days,1 mL each.The Chinese medicine group began to drink traditional Chinese medicine on the 18th day for 7 consecutive days.The dose of each Chinese medicine was 1 g/kg.On the 7 and 14 d after administration,6 chickens in each group were treated with blood stasis.D-lactic acid (D-LA),diamine oxidase (DAO) and endotoxin (ET) were performed according to the Kit instructions by ELISA.Jejunum and cecum were collected.The relative expression levels of jejunal tight junction proteins (ZO-1,Occludin,and Claudin-1) were detected by Real-time fluorescent quantitative PCR.Cecal delivery and detection company was sequenced to detect the influence of traditional Chinese medicine on the diversity of cecal flora.The results showed that Honeysuckle,Forsythia and Herba violet could reduce the levels of serum DAO,D-LA and ET on the 7th and 14th day after administration.On the 7th day after administration,the relative mRNA expression of ZO-1 and Occludin in Honeysuckle group was significantly increased (P<0.05).The relative mRNA expression of Claudin-1 and Occludin in the Forsythia group was significantly increased (P<0.05).On the 14th day after administration,the relative mRNA expression levels of ZO-1 in Honeysuckle group were significantly increased (P<0.05),while the relative mRNA expression levels of ZO-1 and Claudin-1 in Forsythia group and Herba violae were significantly increased (P<0.05).Honeysuckle,Forsythia and Herba violet promoted the proportion of Firmicutes on day 7 after administration (P<0.05),while Honeysuckle,Herba violet promoted the proportion of Bacteroidetes on day 14 after administration (P<0.05).Honeysuckle,Herba violet inhibited the proportion of Proteobacteria at 7 and 14 d after administration,and Herba violet and Forsythia inhibited the growth of Actinobacteria at 7 d after administration (P<0.05).Forsythia,Honeysuckle can significantly increase the proportion of Lactobacillus 7 and 14 d after administration,and Herba violet can significantly increase the proportion of Lactobacillus 7 d after administration (P<0.05).The proportion of the uncultured_bacterium_f_Lachnospiraceae on 14 d after administration was significantly increased by Herba violet.Honeysuckle,Herba violet and Forsythia can reduce the content of serum DAO,D-LA and ET in broilers.Honeysuckle could improve the relative mRNA expression of ZO-1,Occludin and Forsythia could contribute to the relative mRNA expression of ZO-1,Claudin-1 and Occludin.The relative mRNA expression levels of ZO-1 and Claudin-1 were increased by Herba violae.Honeysuckle,Herba violet and Forsythia could increase the number of intestinal OTU,promote the reproduction of beneficial bacteria,inhibit the growth of pathogenic bacteria,and regulate the structure of intestinal flora.

In ruminants,the rumen is an important digestive and absorptive organ,and a certain amount of degraded nutrients can be directly absorbed across the rumen epithelium and then utilized by the host.Therefore,the development degree of rumen is closely related to the production performance of ruminants,and the well-developed rumen of young ruminants is essential for the optimal performance in adulthood.However,to perform its normal functions,the undeveloped rumen requires a complex transformation process from the non-ruminant phase to the ruminant phase through the extra stimulation,such as solid feed and weaning.At present,how to utilize the developmental pattern of the rumen to conduct the early weaning of young ruminants and concurrently ensure the well-development of the rumen have become one of the urgent problems in the modern ruminant industry.In this paper.The authors summarized the research progress on the evolution of rumen microbiota,rumen histomorphology and metabolic development and its regulation mechanism.Moreover,the authors comprehensively summarized the development rules of rumen from the physiological structure to the function,and clarified the related factors affecting the rumen development and the possible regulation mechanism.This paper aimed to further enrich the theoretical basis related to rumen development,provide theoretical reference for developing nutritional strategies that promote rumen development and tapping the production potential of young ruminants.

The experiment was conducted to investigate the effect of supplemental selenium (Se) on growth performance and meat quality of broilers from 22 to 42 days of age.A total of 380 one-day-old male Arbor Acres broilers were fed the same corn-soybean meal diet (containing 0.47 mg/kg Se) from 1 to 21 days of age.At 22 days of age,336 broilers with similar body weight were selected and randomly divided into 6 dietary treatments with 7 replicate cages of 8 broilers per cage for each treatment in a completely randomized design,and were fed a Se-unsupplemented corn-soybean meal basal diet (control group,containing 0.015 mg/kg Se) or the basal diet supplemented with 0.10,0.20,0.30,0.40 and 0.50 mg/kg Se from sodium selenite.The experiment lasted for 21 days.The results showed that supplemental different levels of Se had no effect on average daily gain,average daily feed intake and feed to gain ratio of broilers from 22 to 42 days of age (P>0.05).Supplemental different levels of Se had also no effect on the percentages of dressing,eviscerated yield,breast muscle,thigh muscle and abdominal fat of broilers at 42 days of age (P>0.05).Supplemental different levels of Se had significant effect on the shear force in thigh muscle and L^* value in breast muscle of broilers at 42 days of age (P<0.05).The addition of 0.50 mg/kg Se treatment group had the lowest shear force in thigh muscle,and the addition of 0.20 and 0.50 mg/kg Se treatment groups had the lowest L^* value in breast muscle.But supplemental Se level did not affect other indices of meat quality (P>0.05).In conclusion,supplemental different levels of Se had no significant effect on growth performance,carcass traits and most of meat quality indices in broilers fed corn-soybean meal diet from 22 to 42 days of age,however,dietary supplemental 0.50 mg/kg Se partly improved the shear force in thigh muscle and L^* value in breast muscle of broilers.

The objective of this study was to investigate the effects of sodium nitrate on methane production and fatty acid hydrogenation process of buffalo was studied by in vitro batch fermentation.Three buffaloes weighted (650±50) kg with permanent rumen fistula were selected as the donors of rumen fluid.The ratio of concentrate to forage of substrate was 40:60.Four groups (5 replicates in each group) were set up,each group was added with 0.25 mg/mL α-Linolenic acid,and the level of sodium nitrate was 0,1,2 and 3 mg/mL respectively.The gas production and methane production were measured at 3,6,9,12 and 24 h respectively,and the fermentation parameters and fatty acid content were measured at the end of 24 h respectively.The results showed that:① Adding sodium nitrate significantly reduced the total gas production,CH₄ content and the ratio of methane/total gas production in rumen culture medium for 24 hours (P<0.05).Adding 1,2 and 3 mg/mL sodium nitrate reduced the methane content by 89.62%,91.20% and 91.75% respectively.② The pH and NH₃-N content of rumen culture medium added with sodium nitrate were significantly higher than that of the control group (P<0.05),the MCP content of 1 mg/mL sodium nitrate was also significantly higher than that of the control group (P<0.05),and the difference of other groups was not significant (P>0.05);The difference of acetic acid concentration added with sodium nitrate was not significant (P>0.05),but the concentrations of propionic acid,butyric acid,isobutyric acid,valeric acid and isovaleric acid were significantly lower than that of the control group (P<0.05),the acetic acid/propionic acid was significantly higher than that of the control group (P<0.05),and the total volatile fatty acid added with 3 mg/mL sodium nitrate was significantly lower than that of the control group (P<0.05).③ The content of C18:2 cis-9,trans-11,C18:2 trans-10,cis-12,C20:1,UFA and UFA/SFA in the group with 1 mg/mL sodium nitrate was significantly higher than that in the other groups (P<0.05);the content of C18:2n6c,C18:1n9t,C20:5n3 (EPA) and C22:6n3 (DHA) in the group with 1 mg/mL sodium nitrate was the highest,but the difference was not significant (P>0.05).The contents of C18:3n3,C18:2n6c and C18:1n9c in sodium nitrate groups were higher than those in the control group (P>0.05).It could be seen that the addition of sodium nitrate in vitro significantly reduced the total gas production and methane content,increased the pH and NH₃-N content,decreased TVFA content,increased acetic acid/propionic acid by significantly reducing propionic acid content.The addition of 1 mg/mL sodium nitrate significantly increased the content of CLA and UFA in rumen fluid,and inhibited methane,it could reduce the hydrogenation degree of unsaturated fatty acids at the same time.

In order to analyze the sequence features of piRNAs and the expression difference of piRNAs between anestrus and estrus season in ovine ovary,Solexa high-throughput sequencing technology was used in ovary tissue of Tan sheep during the anestrous and estrus season (include follicular and luteal phases).Then piRNAs in ovine ovary were screened,and the length distribution,the first base bias,genomic distribution,chromosome distribution,strand specificity and ping-pong signature of piRNAs and the types of repetitive sequence which piRNAs mapped to were analyzed by bioinformatics methods.The differentially expressed piRNAs (DE piRNAs) in ovary of Tan sheep between anestrus and estrus season were screened and the target gene of DE piRNAs were subjected to GO and KEGG analysis.The results showed that the first base of different size piRNAs in ovine ovary were various.All of the piRNAs from three stages were mainly mapped to intron,coding sequence and lncRNA while little mapped to repeat sequences.The distribution of piRNAs in ovary were uneven among the chromosomes.piRNAs in ovine ovary had strong strand specificity.piRNAs from three stages had no obvious ping-pong signature.The KEGG enrichment analysis showed that the target genes of DE piRNAs were mainly enriched in those pathways that were correlated with ovine reproduction,such as RNA transport pathway,purine metabolism pathway,focal adhesion,PI3K-Akt signaling pathway and Hippo signaling pathway and so on.In conclusion,the characters of piRNAs from ovine ovary during different reproductive stages were revealed,the results suggested that piRNAs in sheep ovarian tissue might participate in regulation of seasonal reproduction through target genes such as BAD and pathways such as focal adhesion,which provided some reference for further analysis of the molecular mechanism of sheep seasonal estrus.

This study was aimed to investigate the genetic polymorphism of uncoupling protein 2 (UCP2) gene and its correlation with growth traits in Qianbei Ma goats,in order to provide theoretical basis for breed improvement of Qianbei Ma goats.This experiment took 200 Qianbei Ma goats aged 6 months of age as the research object,SNP of UCP2 gene in Qianbei Ma goats was detected by direct sequencing,and the correlation between UCP2 gene and the growth traits of Qianbei Ma goats was analyzed.The expression of UCP2 gene in different tissues of Qianbei Ma goats was detected by Real-time quantitative PCR.The results showed that there were 4 SNPs in UCP2 gene,which were g.29614172 A>G,g.29619320 G>T,g.29619721 C>T and g.29620037 A>G,all of which produced 3 genotypes.Among them,g.29619320 G>T and g.29620037 A>G were missense mutations,g.29619320 G>T mutation caused glycine (Gly) to cysteine (Cys),and g.29620037 A>G mutation led to threonine (Thr) was mutated to alanine (Ala).The polymorphic information content showed that 4 SNPs were all moderate polymorphism (0.25 < PIC < 0.5).χ² test results showed that g.29614172 A>G,g.29619721 C>T and g.29620037 A>G mutation sites were in Hardy-Weinberg equilibrium in Qianbei Ma goats population (P>0.05),g.29619320 G>T mutation site extremely significantly deviated from Hardy-Weinberg equilibrium (P<0.01).The linkage disequilibrium analysis showed that g.29619320 G>T and g.29619721 C>T sites had strong linkage effects.Real-time quantitative PCR results showed that UCP2 gene was expressed to varying degrees in all tissues of Qianbei Ma goats.The highest expression level was in lung,followed by spleen,liver and heart,and the other tissues had lower expression level.The results of association analysis showed that the body weight,body oblique length,chest width,chest depth,bust and tube circumference of AG and GG genotypes at g.29614172 A>G were significantly higher than that of AA genotype,the chest depth,bust and tube circumference of TT genotype at g.29619320 G>T were significantly higher than that of GT and GG genotypes,the body height of CT genotype at 29619721 C>T was significantly higher than that of CC and TT genotypes,the body weight,chest depth and tube circumference of GG genotype at g.29620037 A>G were significantly higher than that of AG and AA genotypes (P<0.05).The results initially revealed that UCP2 gene had significant effects on some growth traits of Qianbei Ma goats,and 4 SNPs could be used as genetic markers for economic trait selection of Qianbei Ma goats.

In this study,a selected principal component evaluation model for the udder traits was established by analyzing the correlation between udder traits of different parities and the correlation between udder traits and lactation performance in Mediterranean buffaloe.The study was helpful to breed high-yielding buffalo with excellent udder characteristics.A total of 123 Mediterranean buffalo with 1 107 records were employed in this study,the correlation analysis was performed using SAS 9.2 CORR process,and the selected principal component evaluation model was established using PRINCOMP process.For 1-4 parities,the 270 d milk yield increased with the increasing parity.At 1 to 2 parities,the front teat length were extremely significantly positively correlated to the hind teat length (P<0.01).There was a significant positive correlation between the front teat length and the hind teat length at 3 parities and above (P<0.05).At 1 to 4 parities,the teat area was extremely significantly positively correlated with the front teat distance,front and hind teat distance,as well as hind teat distance (P<0.01).Additionally,the front teat distance was extremely significantly positively correlated with hind teat distance (P<0.01).At more than 4 parities,the front and hind teat distance was significantly positively related to the milk yield and milk protein (P<0.05),and extremely significantly positively related to the milk fat (P<0.01),at the same time,the area of the teat was significantly positively related to the lactation performance (P<0.05);At 1st to 2nd and 4th parities and above,there was a very significant positive correlation among milk production,milk protein and milk fat (P<0.01),and there was a significant positive correlation among lactation performance at 3rd parity (P<0.05).The selected principal component evaluation model was Y=0.2631x1+0.1186x2+0.0862x3+0.0796x4+0.1039x5+0.0042x6-6.9689,in formula,x1,Front teat length;x2,Hind teat length;x3,Front teat distance;x4,Front and hind teat distance;x5,Hind teat distance;x6,Teat area.This study further revealed that there was a strong correlation among udder traits in different parities of Mediterranean buffalo,and the correlation between teat length and lactation performance was affected by parities.

In order to find out the single nucleotide polymorphism (SNP) of fibroblast growth factor 23 (FGF23) gene and lay a foundation for further research on the genetic variation of FGF23 gene,SNP of FGF23 gene was screened by direct sequencing of PCR products in Wumeng Crested chickens,and the genetic characteristics,linkage disequilibrium,haplotype,diplotype and bioinformatics were analyzed.The results showed that 3 moderately polymorphic SNPs were detected in the promoter region of FGF23 gene in Wumeng Crested chickens,which were g.73424341 C>A,g.73424417 A>G and g.73424701 T>A,respectively,each SNP produced 3 genotypes.χ² test results found that only g.73424417 A>G extremely significantly deviated from the Hardy-Weinberg equilibrium (P<0.01).The results of linkage disequilibrium,haplotype and diplotype analysis showed that there was only strong linkage disequilibrium between g.73424417 A>G and g.73424701 T>A among 3 SNPs,and a total of 4 haplotypes and 8 diplotypes were found,diplotype H1H2 had the highest frequency,followed by H3H4,and the lowest frequency was H2H2.Bioinformatics analysis results found that the core promoter region of FGF23 gene was likely to be in the range of -400 to -300 bp,3 SNPs were not in the core promoter region in this study.The total number of transcription factors were 293 and 300 of g.73423055-g.73425055 region before and after mutation,respectively.The transcription factor was unchanged (all were ICSBP) of g.73424341 C>A before and after mutation.g.73424417 A>G caused the transcription factor to change from GR to C/EBPalp.There was no transcription factor of g.73424701 T>A before and after mutation.It was speculated that SNPs might have an important influence on the regulatory elements of the promoter.

Intrauterine growth retardation (IUGR) refers to the injury of development of embryos and organs characterized by retardation or stagnation of development of offspring during pregnancy of sow.Low or very low birth weight is the main characteristic of IUGR piglets.IUGR seriously affects the survival and later growth of newborn offsprings.The main causes of inducing IUGR include insufficient maternal nutrient intake,disease,environmental stress and abnormal uterine and placental functions during pregnancy.As multi-fetus domestic animals,pigs are most affected by IUGR,mainly because sows can't provide enough nutrients to meet the needs of normal growth and development of all fetuses in the uterine horn during pregnancy.The normal transport and metabolism of nutrients in placental tissue,the only organ connected between mother and fetus,is a key factor affecting the development of fetal pigs.Carbohydrate is the most important energy substrate in fetal pig development.Abnormal glucose transport and metabolism of placenta are crucial to the formation of IUGR.In the process of glucose metabolism,glucose,pentose phosphate and fructose can regulate intrauterine development of piglets through glucose transporters,placental trophoblastic cells and angiogenesis.In this review,the regulation effect of placental glucose metabolism on intrauterine growth retardation in sows was discussed,which would provide scientific significance to reduce the occurrence of IUGR and improve the growth and development of IUGR piglets.

In order to promote the application of crossbreeding technology and improve the production efficiency of Longlin goats in Guangxi,this study focused on Nubia goats,Longlin goats and Nulong hybrid F1 generations (Nubia goats♂×Longlin goats♀),all the goats were raised and managed at the same level.Three groups of 205 goats were selected to measure the growth performance and body size of goats at 1,3 and 6 months old.6 parents and F1 hybrids of 6 months old goats were slaughtered and their longissimus dorsi muscles were taken for meat quality analysis.The results showed that there was no significant difference between the lamb rate and lamb survival rate of the F1 generation of Nulong hybrid and the parent generation (P>0.05).The birth weight,body size index of different month ages were extremely significantly different from that of the parent generation (P<0.01).The weight,weight of carcass,net meat weight,slaughter rate and net meat rate of Nulong hybrid F1 were significantly improved compared with Longlin goats (P<0.05).The muscle crude protein content,muscle fat content,essential amino acids,flavor amino acids,free fatty acids of Nulong hybrid F1 generation all had different degrees of improvement when compared to parents.Therefore,Nubia goats had good comprehensive performance in improving body shape,growth rate and meat quality of Longlin goats.

Embryonic stem cells (ESCs) differentiate into germ cells in vitro can be used to treat infertility and provide the best model for revealing the molecular mechanism of species from generation to generation.The study aimed to discuss the effect of retinoic acid (RA) on the differentiation of cESCs into male germ cells.ESCs were isolated from the X-stage chicken embryos of fresh breeding eggs by trypsin digestion,and then cultured in vitro with the chicken embryo fibroblast (CEF) cells as trophoblast.The morphogenesis,alkaline phosphatase(AKP) staining and SSEA-1 detection were used to identified the obtained ESCs.The results showed that typical cESCs clones were obtained as nests or islands,and the AKP cells were blue-purple,indicating high endogenous AKP activity.The result of SSEA-1 was positive,indicating the pluripotency of cESCs cloning.10^-5 mol/L RA was used to induce cESCs to differentiate into male germ cells (MGCs),the cell morphological changes were observed by microscopic,and total RNA was extracted at 0,2,4,6,8 and 10 day of induction cells,respectively,then the RNA was reverse transcribed into cDNA for Real-time quantitative PCR detection of germ cell marker genes expression.The results showed that ESCs marker genes Nanog,Sox2 expression decreased significantly in the induction,while the expression levels of germ cell-specific genes Dazl,Stra8,c-kit and integrin α6 all showed continuous upward trend.The specific gene-related proteins were observed of positive clones by immunocytochemistry test.In this study,cESCs were successfully isolated,which could be cultured in vitro and remained undifferentiated and pluripotent.10^-5 mol/L RA could induce the expression of corresponding genes in germ cells and promote the differentiation of cESCs into MGCs,laying a theoretical foundation for further study on formation and regulation mechanism of MGCs.

The purpose of the experiment was to study the expression patterns of three kinds of TMEM219 gene spliced antler at different weights and the effect of TMEM219 gene expression on the weight of velvet antler,in order to explore the regulatory mechanism of TMEM219 gene on the growth and development of velvet antler.The Real-time quantitative PCR technology was used to detect the relative expression levels of TMEM219 gene and its three spliced variants in different tissues of the same weight group and the same tissue of different weight groups of velvet antler.Simultaneously,the serum levels of insulin growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) were measured and compared of Sika deer serum with different antler yields.The results showed that three kinds of spliceosome of TMEM219 gene were expressed in reserve mesenchyme and precartilage (RP),transition zone (TZ) and cartilage (C) of Sika deer antler.The relative expression of TMEM219-918 was extremely significantly higher than that of TMEM219-1005 and TMEM219-1960 (P<0.01),and there was no significant difference in the relative expression of TMEM219-1005 and TMEM219-1960 (P>0.05).The expression of TMEM219 gene in the high weight group was significantly higher than that in the low weight group (P<0.05).At the same time,the concentration of IGF-1 in the serum of high weight group was significantly higher than that of low weight group (P<0.05),while the IGFBP-3 concentration was significantly lower than that of low weight group (P<0.05).The results suggested that the high expression of TMEM219 gene might promote the growth of velvet antler and increase the weight of velvet antler.It was speculated that the possible mechanism was that TMEM219 competitively binded to IGFBP-3,reduced its IGF-1 binding ability,and strengthened the IGF-1 and IGF-1R affinity,which in turn enhanced the growth promotion effect of IGF-1 on velvet antler.TMEM219 gene would become a candidate gene that affected the growth and development of velvet antler,providing a theoretical basis for improving the growth of velvet antler.

The effect of different concentrations of mirabilite on the proliferation and apoptosis of donkey skin fibroblasts in vitro was evaluated in this study.The skin fibroblasts were cultured in vitro by the tissue block method,and identified by HE and Masson staining.Then,the fibroblasts were cultured with high (2 000,1 500 μg/mL),medium (1 000,500 and 250 μg/mL) and low concentration (100,50 and 10 μg/mL) of mirabilite.The proliferation rate and apoptosis rate of fibroblasts were measured by the methods of MTT and Caspase-3 respectively.Finally,the expression of genes and proteins that affected the collagen synthesis of donkey skin fibroblasts were detected by Real-time fluorescence quantitative PCR and ELISA in different concentrations of mirabilite media (1 500,250 and 10μg/mL).The results showed that the fibroblasts of donkey skin began to climb out from the edge of the tissue mass at the 5th day of culture,and grew up in the whole culture dish at the 25th day.HE staining showed a typical spindle shape,and the collagen fibers of donkey skin fibroblasts were blue by Masson staining.The proliferation rate of fibroblasts in the medium concentration group was significantly higher than that in the high and low concentration groups (P<0.05),correspondingly,apoptotic factor Caspase-3 activity was significantly lower in the medium concentration group (P<0.05).Compared with 24 h,the mirabilite could significantly increase the expression of genes related to collagen synthesis after 48 h (P<0.05),and the effect was best with 250 μg/mL of mirabilite.In summary,the method of tissue block culture was easy to isolate and culture donkey skin fibroblasts.250 μg/mL of mirabiliten could significantly improve the proliferation rate of donkey skin fibroblasts after 48 h and the expression of genes related to collagen synthesis.This result could provide theoretical basis for the study of the effects of mirabilite on the proliferation of donkey skin fibroblasts and the mechanism of wound healing.

The purpose of this study was to establish a set of identification system for parent-child relationship of Texel×Kazakh sheep.In the experiment,11 microsatellite marker sites were selected for combined amplification.By optimizing the primer concentration,annealing temperature,and reaction system of the combination of microsatellite marker sites,4 sets of multiplex PCR systems were established.Capillary electrophoresis was performed after multiplex PCR to perform genotyping to identify.The genotyping results were read by PROSize 3.0 software,and the gene-tic diversity of the population was analyzed by Cervus 3.0 software.The parent-child relationship between Texel×Kazakh progressive F2 generation (Texel×Kazakh progressive F2) and cross F2 generation (Texel×Kazakh cross F2).The results showed that the alleles of the Texel×Kazakh progressive F2 and cross F2 were 180 and 140,the average numbers of alleles were 16.364 and 12.727,the average observed heterozygosity were 0.533 and 0.544,the average expected heterozygosity were 0.807 and 0.831,and the average polymorphism information content were 0.783 and 0.803.Parent-child relationship identification was performed at 11 microsatellite marker sites of 70 candidate parents and 64 candidate progenies of the Texel×Kazakh progressive F2 and cross F2 two breeds.The results showed that:When the parental genotype was unknown,the combined exclusion probability (CE-1P) was 0.9995 and 0.9997;when the parental genotype was known,the combined exclusion probability (CE-2P) was all reached 1.0000;And the combined exclusion probability of both parents reached 1.0000.It indicates that the 11 selected microsatellite marker sites had high polymorphism and high probability of exclusion,which was suitable for genetic analysis and paternity identification of individuals.The parent-child identification system of Texel×Kazakh sheep established by microsatellite markers provided a theoretical basis for analyzing genetic diversity of sheep populations and assisting breeding work.

To observe the correlation between the expression of the IGFBP-3 gene and growth traits in chickens,the expression profiles of the IGFBP-3 gene of pectoral muscle and liver in two yellow-feathered broiler breeds (Huashan partridge chickens and Qingyuan partridge chickens) differing in growth rate were detected by Real-time quantitative PCR.Results showed that IGFBP-3 mRNA could be detected in pectoral muscle and liver at 9 embryonic day.The expression level of IGFBP-3 mRNA in pectoral muscle in both two chicken breeds was higher than that in liver at the same embryo age.After hatching,the expression level of IGFBP-3 mRNA in liver in two chicken breeds increased rapidly while the expression level of IGFBP-3 mRNA in pectoral muscle decreased.The expression level of IGFBP-3 mRNA in liver in both two chicken breeds was significantly higher than that in pectoral muscle at the same day old (P<0.01).The correlation analysis showed that in both two chicken breeds the mass of body,pectoral muscle and liver were significantly positively correlated with the expression level of IGFBP-3 mRNA in liver (P<0.05;P<0.01),and significantly negatively correlated with the expression level of IGFBP-3 mRNA in pectoral muscle(P<0.01).These results indicated that the expression level of the IGFBP-3 gene in chickens varied in breed,age and organ.The liver produces most IGFBP-3 after hatching,and the expression level of IGFBP-3 mRNA in liver might be important for the body mass and pectoral mass in chickens.

In order to further study the expression and immune effect differences of Japanese encephalitis virus (JEV) NS1 and NS1-2A proteins,eukaryotic expression plasmids of pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag were constructed by connecting T4 DNA ligase to pcDNA3.1(+),which contained NS1 and NS1-2A genes with Flag tag at C-terminal.Two eukaryotic expression plasmids were transfected into BHK-21 cells,respectively.RT-PCR,IFA and Western blotting were used to detect the expression of NS1 and NS1-2A proteins in vitro.BALB/c mice were immunized with pcDNA3.1-NS1-Flag,pcDNA3.1-NS1-2A-Flag and pcDNA3.1(+) to detect the expression difference of the two proteins in vivo.The results showed that the experiments successfully constructed the eukaryotic expression vectors pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag of NS1 and NS1-2A genes.The expression of NS1 and NS1-2A proteins were successfully identified by IFA and Western blotting.Recombinant plasmids pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag could induce specific humoral immunity after immunizing mice.The serum antibody titer and INF-γ cytokine secretion level of pcDNA3.1-NS1-2A-Flag combined immunization group were higher than that of pcDNA3.1-NS1-Flag immunization group.Moreover,the pcDNA3.1-NS1-2A-Flag group were extremely significantly different from pcDNA3.1(+) empty vector immune group (P<0.01).The final IFN-γ of mice immunized with pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag immunized group increased,and they showed a trend of first increase and then decrease.Mice immunized with pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag could stimulate JEV-specific antibodies secretion and enhance the body's cellular immune function,and the immune effect of NS1-2A combined gene was better than NS1 single gene.The results laid a foundation for further research on the function of non-structural proteins of JEV and the vaccines of NS1 and NS1-2A genes.

In order to reveal the molecular characteristics and phylogenetic analysis of Oma87 gene of Pasteurella multocida (Pm),the Oma87 gene in Pm8 (capsular serotype A) was amplified and cloned,the sequence and antigenicity was analyzed by bioinformatics software and conducted to construct phylogenetic tree in this study.The results showed that the length of the Oma87 gene was 2 376 bp,which encoded 791 amino acids.Amino acid sequence analysis found that the Oma87 protein was an acidic protein with hydrophilicity,which had high stability.It had a signal peptide but without transmembrane domain.The secondary and tertiary structure analysis found that the Oma87 protein was "N" type three-dimensional structure formed by random coils connected multiple extended strands.The nucleotide homology analysis result showed that the Oma87 gene of Xinjiang isolated strain Pm8 was highly homologous with bovine Pm (capsular serogroup A) isolated and part of swine Pm (capsular serogroup A) from other different geographic regions,but not highly homologous to other capsular serotype group.VaxiJen classified Oma87 protein as probable antigen because it had a value of 0.5478 which was higher than the normal threshold value of 0.4,proved that Oma87 protein could act as the immunogenic protein,which laid a foundation for further study on Oma87 protein as Pm vaccine candidate antigen.

This experiment was aimed to establish an efficient,fast and sensitive kit for fowl aviadenovirus serotype 4 (FAdV-4),the primers and probe were designed from penton gene of FAdV-4,and FITC was labeled in the downstream primer,Biotin was labeled in the probe.The nano PCR-LFD method for detection of FAdV-4 was established by using nano PCR technology and LFD technology,and the reaction and hybridization conditions were optimized.At the same time,the specificity,sensitivity,intra- and inter-batch repeatability,stability,shelf-life evaluation and clinical samples of the kit were tested.The specificity test showed that the kit could detect FAdV-4 specifically,while for other major viral and bacterial pathogens of poultry,such as infectious laryngotracheitis virus (ILTV),infectious bronchitis virus (IBV),infectious bursal disease virus (IBDV),chicken Marek's disease virus (MDV),egg drop syndrome virus (EDSV),duck plague virus (DPV),Newcastle disease virus (NDV),avian influenza virus H9 subtype (AIV (H9)),avian reovirus (ARV),FAdV standard strain (FAdV-1,FAdV-2,FAdV-8a,FAdV-8b and FAdV-11),E.coli,Staphylococcus aureus (SA),Salmonella Pullorum (SP) and Mycoplasma gallisepticum (MG) were all negative.The sensitivity detections suggested that the kit was so sensitive that it could detect the 56.0 copies/μL level and 10 folds higher than conventional PCR.The results of intra- and inter-repeatability test showed that it had good consistency and stability.The stability test results showed that the kit could withstand at least 20 times of repeated freezing and thawing,and still had a good detection effect.The shelf life test showed that the kit could be stored for at least 3 months at 4 ℃ and 12 months at -20 ℃.The kit realized the visualization of the detection results of FAdV-4,simplified the operation process and improved the detection efficiency.The positive rate of FAdV-4 was 17.95% (21/117).It could be used in the clinical diagnosis and molecular epidemiological investigation of FAdV-4,which provided an important technical support for the early detection of FAdV-4 infection in poultry farms and the formulation of comprehensive prevention and control measures.

In order to understand the resistance of avian-origin Staphylococcus chromogenes,a Staphylococcus chromogenes strain named GZQX2019 was isolated from Jinling chicken.The bacteria were isolated and cultured,Gram staining microscopy,biochemical test,16S rDNA sequence analysis,drug sensitivity test,partial resistance and enterotoxin gene detection and animal regression test.The bacteria separation results showed that the white colony with convex surface and neat edge appeared on the blood agar medium,and the Staphylococcus was detected by Gram staining.The results of biochemical tests showed that the reactions of sucrose,arginine hydrolase and xylose were positive,while urea,xylitol and maltose were negative.The phylogenetic tree showed that the isolation was in the same branch with Staphylococcus chromogenes.The drug sensitivity test showed the isolation was resistant to 6 kinds of antibacterial such as erythromycin,ceftazidime and doxycycline,and sensitive to 11 kinds of antibacterial such as cefuroxime,cefoperazone and neomycin.The results of drug resistance and enterotoxin gene detection showed that Alt,ermB,VIM,TEM and tetB could be detected.The results of animal regression tests showed that the isolates were not pathogenic or non-pathogenic to mice and chicks.The experiment successfully isolated one avian-origin Staphylococcus chromogenes,which enriched the cognition of avian-origin Staphylococcus chromogenes.

To understand the parasite infection situation and influencing factors of Elaphurus davidianus in Beijing Nanhaizi Milu park,53 faeces,9 soil samples,9 lake water and sludge samples were collected in April and October 2018.The parasite eggs in deer faeces and the soil were tested by the saturated salt water float method,precipitation accumulation,the improved anti-acid staining,the saturated magnesium sulfate floating,the test tube filter paper culture and the filter membrane culture.At the same time,the shellfish,which came from the water and sludge of the park,were classified and identified.The results showed that the parasite infection rate of Elaphurus davidianus was 100%.And the infected parasites were mainly nematodes,trematodes,tapeworm and protozoa,of which nematodes and trematodes were predominant,mostly mixed infections.In the deep soil of the park,90-95 cm,the detection rate of parasite eggs was 100%,which was higher than that in the middle soil (30-35 cm)and shallow surface layer (2-7 cm) 33%,but no hookworm and ascaris eggs were detected.In the water and silt,three kinds of medical mollusc were detected,of which the main was Bellamya jousseaume,which could be the intermediate host of Angiostrongylus cantonensis,Clonorchis sinensis and Echinotrematodes.This study indicated that there was widespread parasite infection among Elaphurus davidianus in Beijing Milu park,which was closely related to the living environment.The main factors that affected the prevalence of parasite were insufficient deinsectization and rotational grazing,no harmless treatment of faeces and ponds,and the eggs discharged with faeces became an important factor of circulating infection.So effective comprehensive control measures should be taken to ensure the healthy development of the Elaphurus davidianus population.

In order to study the immune effect of the recombinant protein of Mycoplasma suis eno gene,the Mycoplasma suis eno gene was linked with pET-15b vector and transformed into E.coli BL21 competent cells.The recombinant Mycoplasma suis eno protein (named rMseno) was used to immunize mice,and the immune response of the recombinant protein in mice was evaluated.Twenty four-week-old male Kunming mice were randomly divided into four groups:Group A,group B,group C and group D.Immune group A was inoculated with recombinant protein rMseno.Immune group B was inoculated with E.coli-Mseno induced by IPTG.Control group C was inoculated with the same amount of PBS.Control group D was inoculated with E.coli-Mseno which was not induced expression.The levels of specific antibody against Mycoplasma suis,IFN-γ and IL-4 cytokines in serum were determined by ELISA,and the proliferation of T lymphocyte in mice was detected by spleen lymphocyte test,the experimental data were analyzed statistically.The results showed that the target gene fragment of the recombinant pET-15b-eno plasmid was 1 632 bp,and the purified recombinant protein rMseno was 61 ku,which could be recognized by the mouse anti-Mycoplasma suis serum.The levels of anti-Mycoplasma suis specific antibody,IFN-γ,IL-4 cytokines and T lymphocyte proliferation index in the sera of mice in immune group A and immune group B were significantly or extremely significantly higher than those in control groups (P<0.05;P<0.01).The results showed that eno gene recombinant protein of Mycoplasma suis could induce high humoral immunity and cellular immune response in mice.

This study aimed to isolate and identify the bovine viral diarrhea virus (BVDV) in imported fetal bovine serum.The virus RNA in fetal bovine serum was extracted and amplified by 5'-UTR nested PCR.The PCR products were linked to pMD19-T for sequencing.The fetal bovine serum samples were inoculated with MDBK cells for cell subculture.The sample was identificated by cell culture,direct immunofluorescent assay,nucleotide sequencing and bioinformatics analysis.DNAStar was used to compare the multiple sequences of BVDV 5'-UTR,N^Pro and BLAST virus reference strains published in GenBank,and Mega 6.0 was used for genetic evolution analysis.At the same time,the antibody of BVDV was detected by indirect ELISA with skimmed milk powder.The results showed that BVDV antigen and antibody were both positive in fetal bovine serum,and a new bovine BVDV strain was successfully isolated from fetal bovine serum,and was named BVDV-GC strain,which failed to cause cytopathic changes in MDBK cell proliferation.The results showed that the virus strain did not cause cytopathic changes when it was cultured on MDBK cells.The PCR amplification of 5'-UTR and N^pro was positive,and the size of the amplification products was consistent with the expectation.The direct immunofluorescence was positive.The virus titer was 10^-3.6TCID₅₀/0.1 mL.Genetic evolution analysis showed that the isolate was closely related to USMARC-60779 (BVDV-2) strain,the same strain belonged to BVDV-2.The test was carried out with skimmed milk powder and commercial ELISA kit.The results showed that there was BVDV antibody in skimmed milk powder.In this study,a BVDV-2 virus was isolated from BVDV fetal bovine serum,which showed that BVDV antigen and antibody contamination existed in both imported fetal bovine serum and skimmed milk powder,providing a reference factor for the follow-up cell research.

For the rapid and accurate evaluation of the IgA antibody level of porcine epidemic diarrhea virus (PEDV) in pig serum and milk,a specific PEDV IgG monoclonal antibody (MAb) 8A3 was screened from four strains of PEDV MAb,which could capture all virus particles (inactivated virus cell culture medium) of PEDV efficiently.In this method,the coating concentration of 6.0 μg/mL showed the optimal performance of MAb 8A3,the cut-off value (D_{450 nm}) was settled as 0.34,it had no cross-reactivity with the positive serums of common porcine viruses.Compared with immune-peroxidase monolayer assay (IPMA),the concordance rates of established ELISA for positive and negative serum detection were 98.7% (152/154) and 98.0% (145/148),respectively.For positive and negative samples of colostrum and milk,the concordance rates of the established ELISA compared with IPMA were 100% (60/60) and 95.8% (23/24),respectively.IgA levels in colostrum and milk samples during lactation detected by established ELISA were highly correlated with trends in neutralizing titers (kappa=0.835).Collectively,the indirect ELISA in this study had high sensitivity and specificity,it was a rapid and objective method suitable for large-scale detection of PEDV IgA in clinical samples.

The purpose of this research was to acquire the situation of drug resistance and resistance genes prevalence of Salmonella isolated from goats in Chongqing.185 fecal samples were collected from 5 goat farms.Salmonella were isolated by selective enrichment and PCR,and their serotypes were determined.Then,the antimicrobial susceptibility of the isolates to 28 antimicrobial agents was measured.The mutations of quinolone resistance-determining region (QRDR),plasmid-mediated quinolone resistance (PMQR) and extended-spectrum β-lactamases (ESBL) genes were also detected.A total of 11 Salmonella isolates were obtained from goats,and 10 of them were S.Derby.The isolated strains were more resistant to ampicillin,cefazolin,cefalexin,cefotaxime,tetracycline and pefloxacin,and 9 strains showed multi-drug resistant to 3-7 classes of antimicrobial agents.All strains had QRDR resistant mutations and carried bla_TEM gene,whereas,7 strains carried 1-5 PMQR genes.On the whole,Salmonella isolated from goats were resistant to common used antimicrobials.Meanwhile,drug resistant mutations and drug resistant genes were widespread in the isolates.

The purpose of the experiment was to establish a capillary gas chromatography (GC) method for the content determination of patchouli alcohol in Maxiangling oral liquid.The samples were determined by capillary GC.The chromatographic conditions were as follows:The chromatographic column HP-5 capillary column was used,and the column temperature was controlled by programming (the initial temperature was 150 ℃,held for 18 min,and then rose at a rate of 50 ℃/min up to 280 ℃ for 5 min).The injector temperature was 280 ℃.Carrier gas was high purity nitrogen.The flow rate was 1 mL/min.The injection volume was 1 μL,and the split ratio was 20:1.FID detector was used and the detector temperature was 280 ℃.Hydrogen flow rate was 40 mL/min and air flow rate was 370 mL/min.The system applicability test,specificity test,linear range test,determination of detection limit and quantitative limit,precision test,stability test,repeatability test,sample recovery test,durability test and content determination of ten batches of samples were conducted.The results showed that the solvent peak,impurity peak and main component peak in the specific solution could be separated effectively and the resolution of the chromatographic method met the requirements (R ≥ 1.5),which showed that the method had good applicability.No outstanding peak was detected in the negative control solution,indicating that other components in the sample did not interfere with the determination.Limit of quantitation(LOQ) and limit of detection(LOD) of patchouli alcohol were 2.842 and 0.812 μg/mL.Under the above chromatographic conditions,a good linearity was obtained in the range of 15.86-1 015 μg/mL (y=0.869x-10.45,R²=0.999,n=7);RSDs of precision,stability(intra-day precision and daytime precision) and reproducibility tests were 0.90%,1.63%and 1.83%,2.90%,respectively.RSDs were within controllable range (RSD ≤ 3%).The average recovery was 95.36%,with a RSD of 2.82%(n=6).The results showed that the established gas chromatography method had a good recovery rate for the determination of patchouli alcohol.The durability test was carried out to verify that the established chromatographic method could stably determine the content of patchouli alcohol.The methodological verification showed that the GC method was accurate,stable,convenient and feasible,and could be used as a method for the content control of patchouli alcohol in Maxiangling oral liquid.At the same time,it also provided an effective detection method for the quality control of this preparation.It was tentatively determined that the content of patchouli alcohol in this product was not less than 47.53 μg/mL.

Ketosis is a common disease of high-yield cows in early lactation,and classified as clinical ketosis (CK) and subclinical ketosis (SCK) by the clinical symptoms.Ketosis results in an increase of β-hydroxybutyric acid (BHBA) levels in blood and milk.At present,blood BHBA levels are used for the diagnosis of ketosis.CK has a significant impact on the performance of cows,so it attracts the attention of farmers.However,the symptoms of cows suffering from SCK are not obvious and often overlooked by farmers.Like CK and other transition diseases,SCK is also one of the main causes of economic and welfare losses in dairy cows.SCK increases the risk of production-related disorders,such as clinical ketosis,displacement abomasum,retained placenta,lameness,mastitis,and metritis,while reduced animal performance (lower milk production,poor reproductive performance,increased cull rates).SCK may start to affect production even before it is detected,causing huge financial losses in the dairy industry.Notably,the financial losses caused by SCK are more than CK,which are always underestimated by farmers.This review highlights the latest research advances in SCK in dairy cows,which will provide insight into the pathogenesis of ketosis and the adverse effects of SCK on productivity in dairy cows.In addition,early detection and prediction of SCK is presented to decrease the loss of animal welfare and the economy in dairy industry.

In order to explore the antimicrobial activity and antimicrobial mechanism of antimicrobial peptide NZ2114 against Streptococcus agalactiae (S.agalactiae),S.agalactiae ATCC 13813 was used.The antimicrobial properties and mechanism of antimicrobial peptide NZ2114 were evaluated by means of minimum inhibitory concentration (MIC),time-killing curves,post antibiotic effect (PAE) and electron microscopy.The inhibition and elimination effects of S.agalactiae on biofilms and persister bacteria were further analyzed.The results showed that the MIC value of NZ2114 against S.agalactiae ATCC 13813 was 0.23 μmol/L,while the values of NZ2114 against CAU-FRI 1,2 and 3 strains,which were clinical isolated from mastitis cow,were all 0.11 μmol/L.The MIC values of vancomycin against the above-mentioned four strains were all 0.67 μmol/L,so the antimicrobial activity of NZ2114 were 2.91 and 6.09 times than that of vancomycin,respectively.Meanwhile,2×MIC and 4×MIC of NZ2114 could kill 99.9% S.agalactiae in 0.5 h and the pharmacodynamic time was up to 270 min.The result of scanning electron microscope (SEM) showed that the surface of the bacteria appeared to shrink or even break after the treatment of NZ2114,and the biofilms were eliminated.NZ2114 effectively inhibited early biofilms by 99.9% in 2×MIC,and eradicated mature biofilms by 99.9% in 32×MIC.At the same time,the bacteria (approximately 99.9%) in biofilm were efficiently killed by NZ2114 in 16×MIC,and that of the persisters which were resistant to vancomycin were eliminated by 99% in 0.5×MIC of NZ2114.Confocal laser scanning microscopy (CLSM) also demonstrated the potent antimicrobial and anti-biofilm activity of NZ2114 (biofilm from 28.48 μm reduced to 10.32 μm).The above results showed that NZ2114 had high bactericidal activity against S.agalactiae ATCC 13813 with fast speed,strong ability to inhibit/eradicate the biofilm,and high bactericidal activity against persisters in early and mature biofilms,and its effects were all significantly higher than those of vancomycin.NZ2114 had the potential to develop as a novel antibacterial agent for mastitis treatment caused by S.agalactiae.

The experiment investigated the effect of alkannin gel on wound healing of donkey in order to provide reference for the treatment of skin scars.In vitro cultured donkey macrophages were treated with different concentrations of alkannin,the cells mRNA and supernatant protein expression levels of TGF-β1,IL-10,TNF-α and IL-6 were detected respectively to determine the optimal concentration of alkannin.The skin wound model of donkey was established,and the skin wound was coated with seaweed gel.The wound healing was observed by taking photos and HE staining,and the expression levels of wound healing related genes and serum in the skin of donkey were detected.The results showed that compared with the control group,the addition of alkannin at different concentrations could reduce the expression of related inflammatory factors,among which the 10 μmol/L alkannin group had the best effect.On the 11th day,the wound shrinkage rate of the alkannin group was extremely significant higher than the control group (P<0.01).HE staining showed that the tissues in alkannin were gradually arranged on day 11. At 60th day,the thickness of the superficial cortex in the alkannin group decreased relatively. Real-time quantitative PCR showed that at the 11th day,the expression levels of TGF-β1 and IL-10 mRNA in the alkannin group were extremely significantly increased (P<0.01),while the expression levels of TNF-α and IL-6 mRNA were extremely significantly decreased (P<0.01).At the 3rd day,the serum concentrations of TGF-β1 and IL-10 in the alkannin group were increased,and the concentrations of TNF-α and IL-6 were extremely significantly decreased (P<0.01).At day 7,the concentration of TGF-β1 in the alkannin group was significantly decreased (P<0.05),the concentration of TNF-α was extremely significantly decreased (P<0.01),and the concentration of IL-10 was significantly increased (P<0.05).Therefore,the treatment of donkey skin wound healing with 10 μmol/L alkannin gel had certain anti-inflammatory effect,which promoted the situation of wound healing.

In order to understand the pathogenic characteristics and drug resistance of Lohmann Pink chickens in a farm in Guizhou province,a Gram-negative strain was isolated from dead chickens,named GZ2019,and the isolated bacteria were purified and cultured,biochemical test,16S rRNA sequence analysis,drug susceptibility test and animal regression test.The isolated bacteria showed round raised,smooth surface,translucent small gray colonies on the ordinary agar medium,and the colony morphology on the blood-nourishing agar medium was consistent with that on the ordinary agar medium,without hemolysis.Microscopic examination of Gram staining results showed that the isolates were diplicate negative coccidiobacteria,with occasional single presence or chain arrangement.The results of biochemical tests showed that citrate utilization and catalase reaction of isolated bacteria were positive,while nitric acid reduction,oxidase and glucose fermentation were negative,with no movement.The 16S rRNA sequence analysis results showed that the homology between the isolated strain and acinetobacter was between 72.6% and 99.9%,and the homology with Acinetobacter schindleri LUH 4760 (GenBank accession No.:AJ275041),SNSK 752 (GenBank accession No.:MG584984),MCDA01 (GenBank accession No.:KY385627) and RP1 (GenBank accession No.:MG461636) was 99.9%.The results of drug susceptibility test showed that the isolate were sensitive to ceftriaxone and furazolidone,moderately sensitive to five drugs,such as cefoperazone,cefuroxime and ceftazidine,and resistant to 17 drugs,such as neomycin,ciprofloxacin,carboxylbenzicillin.The results of the animal regression test showed that only one mouse in the experimental group died after 1 week of isolation,with a mortality rate of 20%,indicating that the pathogenicity of the isolates was weak.This study successfully isolated a strain of low pathogenicity Acinetobacter schindleri GZ2019,which could provide certain reference for the prevention and treatment of Acinetobacter schindleri in chickens.

The aim of the study was to investigate the grazing behavior and rumen microbiology profile of yak in different seasons,in order to provide the scientific basis for improving yak production performance and the animal production transformation efficiency.Nine healthy yaks with similar body weight were selected as the experimental animals,and the trail was conducted in January 2019 and September 2019,respectively.Rumen fluid was collected to analyze the rumen environmental parameters and rumen microbial diversity.The MOOnitor system was adopted to record the grazing behavior.The results showed that the grazing behavior and spatial distribution of yak varies with the season variations of forage yield and nutrition quality.The time spending for rest and rumination (13.378 h/d),grazing (5.174 h/d),and ruminating (8.160 h/d) of yak was significantly decreased in cold season compared with those in warm season (P<0.05),however,the walking time was significantly increased in cold season (P<0.05).The spatial distribution of grazing was more dispersed in cold season.The content of ammonia nitrogen,acetate,propionate,isobutyrate,valerate and total volatile fatty acids in rumen fluid,and the ratio of acetate to propionate were decreased significantly during cold season (P<0.05).However,microbial protein and isovaleric acid content in rumen were significantly increased in cold season (P<0.05).At the level of phylum,Bacteroidetes increased significantly (P<0.05) and Firmicutes decreased significantly (P<0.05) in cold season.At the genus level,cellulose-decomposing bacteria was dominate in rumen fluid of yak in cold season.In conclusion,in cold season,yak reduced the time for grazing,ruminating and rest,and increased the time for walking within a greater space to cope with the lower forage biomass and nutrition quality.In the meantime,the rumen digestion metabolism and microbiology were also changed in cold season in order to improve the energy intake and body utilization efficiency to cope with the deficiency of forage.

For further understanding of the prevalence and drug resistance of Staphylococcus aureus and Escherichia coli in livestock and poultry manure,the established loop-mediated isothermal amplification technique (LAMP) in livestock and poultry manure was used to detect S.aureus and E.coli in cattle,pig,chicken and duck manure in Shandong province.Then the strains were isolated from the positive samples,and the resistance genes of the multiple resistance strains in the drug sensitivity test were predicted.The results showed that the LAMP method to detect S.aureus and E.coli in livestock and poultry manure was completely consistent with the national standard method,with good repeatability and high specificity.Three strains of S.aureus isolated from 80 samples of livestock and poultry manure were resistant to penicillin.The drug resistance rate of 34 E.coli strains to florfenicol and rifampicin was more than 50%,and the proportion of drug resistance to ampicillin,tetracycline,doxycycline,sulfisoxazole,cephalotin and ceftiofur were more than 25%.The prediction results of drug resistance gene showed that four strains of E.coli in chicken,duck,cow and pig manure carried 10,8,20 and 15 drug resistant genes,respectively.One strain of S.aureus in chicken manure carried 11 drug resistant genes.It indicated that the drug resistance situation of S.aureus and E.coli in livestock and poultry manure of Shandong was severe,the strains were generally multi-drug resistant,and they carried a variety of drug resistance genes.