To evaluate the results of transcriptome sequencing analysis and explore circular RNA(circRNA) with potential function in fat deposition,10 circRNA derived from the peroxisome proliferator activated receptor γ (PPARG) were identified.Based on the obtained sequence,divergent primers were designed for RT-PCR and normal sequencing to obtain real circRNA.Real-time PCR was used to analyze the expression profile of circRNA in different tissues and adipose tissue of different parts in buffalo,RT-PCR was used to identify their expression in different species.In total,5 of the 10 PPARG-derived circRNA were identified,and their sequences were consist with those in transcriptome sequencing analysis.Specific primers for Real-time PCR were available for only 2 of the 5 circRNA,and the 2 circRNA were mainly expressed in adipose tissue and with different expression levels among adipose tissue depots.Further,3 circRNA were detectable in adipose tissues of cattle,yak,pig and mouse.Their length were consist with those in buffalo,and they were with high similarity in sequence among species.These results indicated that the results of transcriptome sequencing analysis were reliable,2 circRNA were mainly expressed in adipose tissue and conserved among species,which could be candidate circRNA for fat deposition of buffalo.

This study was aimed to explore the gene structure of cysteine-rich acidic and rich in cysteine-like 1 (SPARCL1) and its expression differences in tissues of Small-tail Han sheep.The total RNA was extracted from the liver tissue of Small-tail Han sheep.Primers were designed based on the sequence of SPARCL1 gene in sheep published in GenBank,and the SPARCL1 gene codings region (CDS) was amplified by PCR.The amplified product was constructed into pMD18-T vector for sequencing,and the sequence information of the complete CDS of SPARCL1 gene was obtained.The sequence and protein structure were analyzed by bioinformatics software.The expression of SPARCL1 gene mRNA in different tissues (liver,muscle,stomach,duodenum and small intestine) of Small-tail Han sheep was detected by Real-time quantitative PCR.The results showed that the length of SPARCL1 gene CDS was 1 962 bp,encoding 653 amino acids;The molecular weight was 74.39 ku,the theoretical isoelectric point was 4.64,which was a hydrophilic protein;SPARCL1 gene had a information peptide cleavage site and it was a secreted protein;The homology of SPARCL1 gene sequence in Small-tail Han sheep was 99.80% with that of sheep provided by NCBI,and there were 4 mutation sites of SPARCL1 gene CDS,but no amino acid changes were caused;There were 77 protein phosphorylation sites and 3 glycosylation sites of SPARCL1 protein.SPARCL1 protein expression was mainly localized in cytoplasm.The alpha helix,beta fold,beta turn and random coil of SPARCL1 protein in the secondary structure were 31.9%,6.4%,28.7% and 33.0%,respectively,the tertiary structure prediction result of SPARCL1 protein were consistent with the secondary structure.The relative expression of SPARCL1 gene in subcutaneous adipose tissue of Small-tail Han sheep was significantly higher than that in other tissues.In this study,the complete CDS sequence of SPARCL1 gene was successfully cloned,and its sequence components,protein physicochemical properties,structure and expression differences among tissues were analyzed,which provided a theoretical basis for studying the function of SPARCL1 gene and exploring its possible role of fat metabolism in Small-tail Han sheep.

This study was aimed to perform bioinformatics analysis and screening of T cell and B cell epitopes for bovine viral diarrhea virus (BVDV) NS3 protein.First,the amino acid sequence of the NS3 protein was found in GenBank database.The resulting sequence was analyzed for physicochemical properties using an Expasy ProtParam online server.Then TMHMM Server was used to analyze the transmembrane domain.The DNAStar software was used to predict the secondary structure of the NS3 protein.To more accurately predict the secondary structure of a protein,the SOPMA analysis software performed a second prediction.The Phyre2 online server was used to predict the tertiary structure of the NS3 protein,and Raswin software was used to identify the location of each element.Linear B cell epitopes of NS3 protein were analyzed by four predictive software (BCPREDS,ABCpred,BepiPred and SVMTriP),CD4⁺ T cell epitopes were predicted by NetMHCⅡpan,NetBoLApan and IEDB were used to predict CD8⁺ T cell epitopes.Finally,the results obtained were screened for T cell and B cell epitopes.The results showed that the NS3 protein consisted of 683 amino acids,the molecular weight was 75 ku,the theoretical pI value was 8.31,chemical formula was C₃₃₄₇H₅₃₄₆N₉₁₆O₁₀₀₉S₂₈,the instability coefficient was 35.93,the average value of hydrophilicity (GRAVY) was -0.267,it was indicated that NS3 was a stable and hydrophilic protein.The TMHMM results showed that the NS3 protein did not have a transmembrane domain.SOPMA results showed that in the secondary structure of NS3 protein,α-helix β-fold,β-turn and random coil accounted for about 30.75%,22.55%,8.35% and 38.35%,respectively.DNAStar software marked their respective position.Eight linear epitopes were screened using four different B cell prediction software:12-16,289-298,349-360,394-407,421-432,489-498,515-525 and 665-679 amino acids.Four T cell epitopes were screened using NetMHCⅡpan:248-262,315-320,400-414 and 482-496 amino acids.This study provided a theoretical basis for the screening of the dominant antigen of NS3 protein in BVDV.

To investigate the polymorphism of the promoter regions of MYOD1 and AKT3 genes and the possible molecular regulatory mechanism that affected gene expression,PCR direct sequencing was used to detect the polymorphism in the promoter regions of MYOD1 and AKT3 genes in Large White pigs.At the same time,the core promoter region,CpG island and transcription factor binding domain of MYOD1 and AKT3 genes were predicted by bioinformatics analysis.The results showed that there were 5 core promoter regions,1 CpG island region and 10 transcription factor binding domains of MYOD1 gene,and the fifth core promoter region was located in the CpG island region.A total of 6 core promoter regions were predicted for AKT3 gene,and no CpG island was found.1 SNP mutation was detected in MYOD1 gene at G-361T site by direct sequencing,but only 1 genotype was found in the experimental population,and the mutation site was located in the first core promoter region.There was 1 SNP-mutation in the promoter region of AKT3 gene T-1709C site,including TT,TC and CC genotypes,of which TT was the dominant genotype,and T was the dominant allele.Genetic polymorphism analysis indicated that the PIC value of this mutation site was 0.25 to 0.5,showing moderate polymorphism.This study preliminarily explored the polymorphism of MYOD1 and AKT3 genes promoter region and predicted the possible regulatory factors and regulatory elements in the promoter region,which would provide guidance and basis for the further study of the regulatory mechanisms of the MYOD1 and AKT3 genes for muscle growth and development and the use of mutation sites as genetic markers for molecular breeding.

This study was aimed to improve the gene structure of Gaoyou duck,and explore its regulation mechanism of double-yolk egg performance.RNA-Seq technology was used to analyze the transcriptome of ovaries in high and low double-yolk egg yield Gaoyou ducks.SNP related to the double-yolk egg performance were screened by quality control and gene annotations from RNA-Seq data.Raw reads from RNA-Seq data were selected with quality control,84 540 140-87 479 660 clean reads were screened from total 6 samples,over 70.79% of the sequencing data were covered by the annotated genome data of ducks.Based on these data,108 260-116 478 SNP were found in the exon area of the annotched genes,and the number of transition SNP was extremely significant higher than the number of transversion(P<0.01).KEGG database was used to analyze the related pathways from these selected SNP.Top 20 pathways were screened,including Ribosome signaling pathway,Carbon metabolism signaling pathway,Biosynthesis of amino acids signaling pathway,and so on.The ovaries' transcriptome of Gaoyou ducks with high and low double-yolk performance were analyzed through RNA-Seq technology,the results provided the basis for mining and annotating new genes of Gaoyou ducks,and improved the data bases for the study of its regulation mechanism of double-yolk egg performance.

This study was aimed to clone,analyze sequence and determine the tissue expression of PRLHR gene in yak.The hypothalamus,anterior pituitary,ovary,oviduct and uterus tissues of 5 female yaks and 5 female cattle were collected.The full-length cDNA sequence of PRLHR gene in yak was amplified by RT-PCR techniques,and the characteristics of PRLHR protein in yak was analyzed by bioinformatics methods.The expression of PRLHR gene in yak and cattle were detected by Real-time quantitative PCR.The results showed that the sequence of PRLHR gene in yak was 1 625 bp,which contained 1 113 bp of the CDS region,22 bp of 5'-UTR and 490 bp of 3'-UTR,encoding 370 amino acids.The CDS was high homology with Bos taurus,Bubalus bubalis,Ovis aries,Sus scrofa and Homo sapiens,indicating it was conservative in the evolutionary course.PRLHR protein in yak was an unstable hydrophobic protein with no signal peptide,but had 7 transmembrane domains.There were 13 serine phosphorylation sites,6 threonine phosphorylation sites and 4 tyrosine phosphorylation sites.It was predicted that there were 3 N-glycosylation sites and 10 O-glycosylation sites.The secondary structure of PRLHR protein in yak contained alpha helix (49.19%),random coil (31.89%),extended chain (15.68%) and beta turn (3.24%).The tertiary structure of PRLHR protein in yak had a typical domain of PrRP family in GPCRs superfamily.The results of Real-time quantitative PCR showed that the expression of PRLHR gene in oviduct of yak was significantly higher than that of other tissues (P<0.05).The expression of PRLHR gene in hypothalamus,anterior pituitary,uterus and oviduct tissues of yak was extremely significantly higher than that of cattle (P<0.01).In conclusion,cloning,sequence analysis and tissue expression of PRLHR gene in yak were successfully carried out,which provided a foundation for the further study on the regulatory role of PRLHR gene in yak reproduction.

Canine coronavirus (CCoV) is a common pathogen that causes gastrointestinal diseases in dogs,especially when co-infection with other intestinal pathogens,the mortality rate increases significantly,seriously affecting the healthy development of the canine industry.Fast,sensitive and specific diagnostic techniques play a vital role in the prevention and control of the disease.In addition to clinical diagnosis,the common CCoV laboratory diagnostic techniques mainly include virus isolation culture,electro-mirror observation and serological diagnostic techniques,however,these technologies often take a long time and a large workload,molecular diagnostic techniques mainly include ordinary RT-PCR,Real-time quantitative RT-PCR,nano PCR and other detection methods,these diagnostic techniques are greatly improved in accuracy,sensitivity,specificity and diagnostic efficiency.In recent years,with the rapid development of molecular immunodiagnostic technology and new materials development technology,new detection methods have emerged,nano antibody gold,nucleic acid probe,chip technology,and nano-biosensors and other technologies strive to be more sensitive,convenient,efficient and have the possibility of preparation kits,so as to be diagnostic concise and universal.This paper will provide a comprehensive review of the diagnostic techniques of CCoV,in order to provide reference for the development of CCoV diagnostic reagents.

This experiment was conducted to investigate the effect of dietary calcium (Ca) or phosphorus (P) deficiency on growth performance and tibia histological structure of broilers from 22 to 42 days of age based on our previous study of broilers from 1 to 21 days of age.A total of 504 one-day-old Arbor Acres male broilers were randomly assigned to 1 of 4 treatments in a completely randomized design involving a 2 (Ca levels:0.90% and 0.30%)×2 (NPP levels:0.35% and 0.18%) factorial arrangement of treatments.The 4 treatment groups were the normal control group (0.90% Ca+0.35% NPP),the P-deficient group (0.90% Ca+0.18% NPP),the Ca-deficient group (0.30% Ca+0.35% NPP) and the Ca and P-deficient group (0.30% Ca+0.18% NPP).The growth performance of broilers from 22 to 42 days of age and the tibial histological structure at 28 and 42 days of age were measured.The results showed that dietary Ca or P deficiency decreased the average daily feed intake (ADFI),average daily gain (ADG) and tibia length of broilers (P<0.05),but increased the feed to gain ratio (F/G) and the length of tibia growth plate hyperplasia area (P<0.05).Furthermore,the broilers fed the P-deficient diet had the lowest ADFI and ADG of broilers from 22 to 28 days of age and tibia length at 28 days of age compared with those fed the Ca-deficient or Ca and P-deficient diets (P<0.05).Moreover,the broilers fed the P-deficient diet had the highest mortality during days 22 to 28 compared with other treatment groups (P<0.05),and all the broilers in this group died at 28 days of age.The low Ca level increased the length of tibia growth plate hyperplasia area of broilers at 28 days of age (P<0.05),but the low P level decreased it (P<0.05).In conclusion,the growth performance and tibia histological structure of broilers from 22 to 42 days of age were the most sensitive to dietary P deficiency,followed by dietary Ca deficiency or Ca and P deficiencies;And low Ca level increased while the low P level decreased the length of tibia growth plate hyperplasia area of broilers.

The study was to investigate the nutritional value of various common ruminant feeds in subtropical by in vitro fermentation.Sugarcane sheath,Pennisetum purpureum Schumabcv.Red,Pennisetum purpureum Schum cv.Guiminyin,Broussonetia papyrifera,corn stover,pineapple skin,fresh bean curd residue,fresh brewer's grains,and marc of Siraitia grosvenorii were selected as fermentation substrates.Rumen fluid was collected from adult Xiangdong black goats with permanent rumen fistula.In vitro fermentation was carried out for 48 h using fully automated in vitro simulated rumen fermentation equipment.The fermentation parameters were evaluated based on total gas production (TGP),methane production,dry matter degradation rate (DMD),and volatile fatty acid (VFA) concentration.The results showed as follows:For the residues,48 h total gas production,methane production,dry matter degradation rate and VFA concentration from high to low were fresh bean curd residue,pineapple skin,fresh brewer's grains and marc of Siraitia grosvenorii.For the forages,methane production of corn stover were the highest,48 h total gas production,dry matter degradation rate and VFA concentration of Broussonetia papyrifera were the highest,and these indices of marc of Siraitia grosvenorii were the lowest.In conclusion,except marc of Siraitia grosvenorii, the other eight common feeds could be developed and utilized as high-quality feeds for ruminants according to the evaluation of their conventional nutrients,in vitro fermentation gas production and volatile fatty acids level.

The purpose of this experiment was to study the effects of grape seed meal on egg production performance,egg quality and Newcastle disease antibody level of breeder chickens during the late laying peak period.810 64-week-old Roman grey parents breeders with similar laying rate and body weight were randomly divided into 6 groups with 5 replicates in each group and 27 chickens in each replicate.The control group was fed with basic diet,while the experimental group was fed with grape seed meal instead of corn in the basic diet,which was 3%,4.5%,6%,7.5% and 9% respectively (the experimental group Ⅰ,Ⅱ,Ⅲ,Ⅳ and Ⅴ).The preliminary period is 7 days and the positive period was 30 days.The effects of different levels of grape seed meal on egg production performance,egg quality and Newcastle disease antibody level of breeder chickens at the later stage of peak laying were studied.The results showed that grape seed meal could significantly inhibit the decline of egg production performance in the late peak period of laying hens,and the average daily egg production traits in group Ⅲ were significantly higher than those in control group (P<0.01),while those in group Ⅰ and Ⅱ were significantly higher than those in control group (P<0.05).In the daily average egg production traits,the experimental group Ⅲ was significantly higher than the control group (P<0.01),and the experimental group Ⅰ and Ⅱ were significantly higher than the control group (P<0.05).In terms of laying rate traits,the experimental group Ⅰ,Ⅱ and Ⅲ increased by 12.38%,14.76% (P<0.05),18.09% (P<0.01) respectively compared with the control group.The average daily feed intake,feed-egg ratio and breaking soft egg rate of the experimental group were not significantly different from those of the control group (P>0.05).Harrington's unit,yolk weight and eggshell strength of the grape seed meal treatment group were higher than those of the control group,but there was no regular change with the increase of grape seed meal content.Grape seed meal could decrease the cholesterol content of eggs (P<0.01),and grape seed meal could increase the HI antibody level of breeder chickens (P>0.05).To sum up,replacing corn with grape seed meal in the diet of breeder chickens at the later stage of egg laying peak could improve the performance of breeder chickens and the titer of Newcastle disease antibody,improve the quality of eggs,reduce the cholesterol content in eggs,and the replacement of 6% corn was better.

The objective of this study was to investigate the nutrient composition and bacterial diversity changes of natural fermented sugarcane tops silage under different silage period.Sugarcane tops silages were taken on 1st,15th,30th,45th,60th and 90th day.Nutrient contents,pH,lactic acid,ammoniacal nitrogen,volatile fatty acids VFA and microbial diversity were analyzed.The results showed that compared with sugarcane topssilageon the first day,DM content of sugarcane tops on 15th day was decreased significantly (P<0.05).Crude protein content was decreased with the extension of silage period (P<0.05).pH was decreased with the extension of silage period and was lower than 4.0 after 15-day fermentation (P<0.05).Lactic acid content was increased(P<0.05) and reached the highest value on 30th day,the highest value was 207.17 mmol/kg.Acetate content was increased with the extension of silage period (P<0.05) and no significant changes on propionate and butyric acid.On the first day,the dominant bacteria at order level of sugarcane tops silage were Lactobacillales,Rhizobiales,Bacillales,Micrococcales,Sphingomonadales order.Lactobacillales was the most dominant order after 15-day fermentation and Enterobacteriales was the second dominant order. Lactobacillus,Staphylococcus,Sphingomonas,Methylobacteriu,Rhizobium genus were the dominant genus in sugarcane tops silage on the first day.Lactobacillus was the most important dominant genus after 15-day fermentation.It can be concluded that sugarcane tops can get a good quality under natural silage conditions without any supplemented inoculums and Lactobacillus was an important bacteria genus in the natural fermented sugarcane tops silage.

The aim was to study the effects of different feeding methods on the quality of Jiangcheng beef,and obtain the best meat quality of Jiangcheng beef.24 Jiangcheng beef aged about 2.5 years old and in good health were selected,including 9 bulls and 15 cows,which were randomly divided into 3 groups according to sex,8 in each group (3 bulls and 5 cows).The first and second groups were fed in house.On the basis of free feeding of whole plant corn silage,each cow was fed with concentrate supplement 2 kg (concentrate supplement group) and concentrate 1 kg (concentrate group) per day.The third group was fed on artificial grassland (grazing group).After 90 days of feeding,4 beefs were selected from concentrate supplement and concentrated groups,6 beefs were selected from grazing group,half male and half female,a total of 14 slaughtering experiments were carried out to determine the meat quality of Jiangcheng beef.The results showed that different feeding methods had a certain effect on the quality of Jiangcheng beef.After feeding concentrate supplement and concentrated feed,there were significant differences in water loss rate,shear force,crude fat and crude protein among concentrate supplement,concentrated and grazing groups (P<0.05),but there were no significant differences among other components (P>0.05).However,the beef quality of concentrate supplement and concentrate groups were better than that of grazing group,such as eye muscle area,backfat thickness,marbling pattern,and so on.Different feeding methods had a certain effect on the quality of Jiangcheng beef.After supplementing concentrate supplement and concentrate,the concentrate supplement and concentrate groups were better than grazing group in meat quality.The results showed that the meat quality of Jiangcheng beef could be improved by supplementing concentrate supplement and concentrate in the process of feeding,and the development of meat performance had a good prospect.The results of this study provided a theoretical basis for reasonable feeding and improving meat quality of Jiangcheng beef.

This study was conducted to investigate the effects of Chinese herbal compound fermentation powder (CHCF) on growth performance,nutrient apparent digestibility and digestive enzyme activities of weaned piglets.A total of 32 healthy 42-day-old weaned piglets (Duroc×Landrace×Large White) with an average body weight of (14.96±0.96) kg were randomly divided into 4 treatments,8 replicates per treatment,and 1 pig per replicate.The control group was fed the basal diet,and the other three groups were supplemented with chlortetracycline (CC) 75 mg/kg,CHCF 600 mg/kg,CC 75 mg/kg+CHCF 600 mg/kg.The experiment lasted for 42 days.The results showed as follow:Supplementation of CHCF had significantly increased the apparent digestibility of dry matter,organic matter and carbohydrates and the apparent digestible energy (ADE) (P<0.05),increased the apparent digestibility of essential amino acid and non-essential amino acid (Gly,Asp,Ala,Cys and Tyr),activities of lipase (duodenum,jejunum and ileum) and amylase (ileum) (P<0.05).CHCF and CC had a significant interaction on average daily gain (ADG) and ADE,the apparent digestibility of crude protein (CP) and ether extract (EE),and the apparent digestibility of Leu,Phe and Ser (P<0.05).Compared with control group,CHCF and CC group had significantly increased ADG and the apparent digestibility of CP and EE,the apparent digestibility of Phe and Ser (P<0.05),but there were no significant difference between two groups (P>0.05).In conclusion,the addition of 600 mg/kg CHCF in diets could improve the nutrient apparent digestibility,apparent digestibility of amino acid and intestinal digestive enzyme activities of weaned piglets.

To study the effect of different iron levels in diet on immune function of sheep,and explore the risk of iron overdose during sheep feeding,different doses of FeSO₄,including 500,1 000 and 1 500 mg/kg of iron,were added to the basic diet of sheep.After feeding 75 days,immune organs of sheep (spleen,thymus,portal lymph nodes,duodenal lymph nodes and palatine tonsil) were adopted.Organ structures and hemosiderin deposition were observed using traditional pathological methods.The distribution of hemosiderin and iron content in the above organs were measured.The results showed that proportion of splenic white pulp in spleen decreased,the number of lymphocytes reduced and its arrangement was loosed,accompanied by swelling and vacuolization after sheep were fed with excessive iron.Moreover,we found that thymus cortex became thinner,the medulla became thicker and fused,the number of thymus corpuscles decreased and the structure of thymus corpuscles was blurred.The cortical and medullary areas of portal lymph nodes and duodenal lymph nodes became thinner and blurred,the space between cortical lymphatic sinus and medullary sinus increased,and the structure of lymph nodules was blurred.The number of lymph nodules in parenchyma of palatine tonsil decreased,and post capillary venules appear congested.After feeding excessive iron,the iron content in immune organs increased,in which the iron content in spleen was the highest and that in palatine tonsil was the least.The iron content in spleen of low,medium and high iron excess group was extremely significantly higher than that of control group (P<0.01).The results showed that the tissue structure of sheep immune organs was damaged in varying degrees after sheep were fed with excessive iron,and the more iron added in fodder,the damage of tissue structure was more serious.The amount of iron accumulated in each immune organ was positively correlated with iron level added in the diet.Excessive iron intake seriously affects the immune function of sheep.Therefore,in the process of sheep feeding,it is necessary to strictly abide by the NRC (1985) feed standard of the United States,and the iron level in the diet should not exceed 500 mg/kg.

The aim of this experiment was to study the effects of different conjugated linoleic acid (CLA) levels on serum biochemical parameters and muscle fatty acid composition of eliminated Angus cows.The method of complete random design was adopted,forty healthy 22 months old Angus cows with body weight (417.70±30.83) kg were randomly divided into four groups with 10 cows each.The diet of control group did not add CLA,while of the experimental groups added 0.5% (0.5% CLA group),1.0% (1.0% CLA group) and 1.5% (1.5% CLA group) CLA,respectively.The experiment lasted for 105 days,which consisted of 15 days of per-feeding period and 90 days of formal period.At the end of experimental,three cows were randomly selected from each group for collect blood and slaughter,and measure serum total cholesterol (TC),triglyceride (TG),high density lipoprotein (HDL),low density lipoprotein (LDL),growth hormone (GH),insulin (INS),adiponectin (ADPN),leptin (LEP) and muscle fatty acid contents.The results showed as follows:There were no significant differences in serum TC,TG,HDL,LDL,GH,ADPN and LEP among the groups (P>0.05).The INS levels in control group and 1.0% CLA group were significantly lower than those in groups 0.5% and 1.5% CLA (P<0.05),but there was no significant difference between 0.5% CLA group and 1.5% CLA group (P>0.05).The dietary CLA supplementation had an effect on fatty acid content in muscle of elimination Angus cows.Compared with the control group,the content of C14:0 in 0.5%,1.0% and 1.5% CLA groups and the content of C16:0 in 1.0% CLA group significantly decreased (P<0.05),and the contents of C14:0 and C16:0 in 1.0% CLA groups were the lowest;The contents of C18:1n9c and monounsaturated fatty acids (MUFA) in 1.0% CLA group and C18:2n6c and polyunsaturated fatty acids (PUFA) in 1.0% CLA group and 1.5% CLA group significantly increased (P<0.05),and the content of unsaturated fatty acids in 1.0% CLA group was the highest.It could be seen that 1.0% CLA supplementation could reduce the serum INS level,whereas increase the content of MUFA and PUFA in beef,accordingly,improve beef quality of elimination Angus cows.

The purpose of this study was to analyze the effects of season and culture mode (canned milk and scattered milk) on the quality of raw milk in Hohhot,Inner Mongolia,and evaluate the quality of Hohhot canned milk according to the quality grading standard coming out in China,in order to provide reference for quality improvement and safety evaluation of raw milk in Inner Mongolia.Thirty batches of raw milk were collected from different dairy farm of Hohhot in each quarter from 2017 to 2018,a total of 240 batches in two years.According to the method of NY/T 2659-2014,NY/T 800-2004,GB 4789.2-2016 to detemine the milk fat rate,milk protein rate,somatic cell count (SCC) and colony count of raw milk.The results showed that the milk fat rate of raw milk in Hohhot from 2017 to 2018 were higher in the fourth quarter;The milk protein rate in the fourth quarter were 3.46% and 3.47%,which were significantly higher than that in the other quarters (P<0.05);The colony count in the third quarter were 2.50×10⁴ and 6.00×10⁴ CFU/mL,which were significantly higher than that in the other quarters (P<0.05);The SCC of fresh milk in 2017 was the highest in the first quarter (39.90×10⁴/mL),which were significantly higher than that in the other quarters (P<0.05),but there was no significant seasonal difference in SCC of fresh milk in 2018 (P>0.05).From 2017 to 2018,the milk fat rate of scattered milk produced by family breeding mode in Hohhot was significantly lower than that of canned milk produced by large-scale breeding mode,while SCC and colony count were significantly higher than that of canned milk (P<0.05).There were no significant difference in the milk fat rate and milk protein rate of canned milk between 2017 and 2018 (P>0.05),but SCC was significantly higher than that in 2018 (P<0.05),and the colony count was significantly lower than that in 2018 (P<0.05).To sum up,the seasons had a great influence on the milk fat rate,milk protein rate and the colony count of raw milk in Hohhot from 2017 to 2018,but had no obvious influence on SCC.In 2017 and 2018,the quality and safety level of family scattered milk were obviously lower than that of large-scale canned milk.The quality and safety level of canned milk were relatively high and reached the excellent standard.Therefore,large-scale breeding mode and high-quality milk production would become an inevitable trend.

There are many microorganisms in the gastrointestinal tract of ruminants,such as bacteria,fungi,protozoa and archaea.Gastrointestinal microbes play an important role in the energy metabolism and central nervous system of animals.Intestinal microbes can be in direct contact with intestinal cells,producing not only metabolites that activate the endogenous central nervous system signaling mechanisms,but also independently producing or contributing to the production of many neurally active molecules.Microbial metabolites and neuroactive molecules form a complex reflex network through key pathways such as neural signaling pathways,gastrointestinal endocrine signaling pathways and immune systems.That is,gastrointestinal microorganisms and metabolites conduct signals to the central nervous system through the afferent neurons.The gastrointestinal microbes interact with the host through the main signaling pathways,affecting the body's gastrointestinal barrier,nutrient metabolism,immune response and other physiological functions and feeding behavior.The article mainly focuses on the gastrointestinal microbial species of ruminants,the "bottom-up" transmission pathway of microorganisms through the gut-brain axis,and the role of microorganisms and their metabolites in host diseases and behavior through the gut-brain axis.The possible influencing factors of gastrointestinal microbial-gut-brain axis were analyzed,and the research on the axis of ruminants is prospected in this paper.

The main active components of yeast cell wall polysaccharide are β-glucan and mannan,which compete with intestinal receptors to adsorb pathogenic bacteria and to provide energy in intestinal degradation to improve the intestinal environment of animals.They can also be used to chelate with Fe²⁺ ions to inhibit the production of hydroxyl radicals and prevent lipid peroxide chain reaction to improve antioxidant capacity.And its unique chemical sites will adsorb mycotoxins by a variety of intermolecular interactions.β-glucan and mannan enhance the specific and nonspecific immunity of the body by acting on macrophages and regulating a variety of cytokines.Currently,it can be used in aquaculture to replace chlortetracycline,mucin sulfate and other antibiotics to control the necrotizing enteritis of chickens,reduce bacterial infection in aquatic animals,and reduce the diarrhea rate of young animals such as pigs and cattle,prevent all kinds of aging diseases and promote the growth and development of animals.The results of the existing extraction methods of yeast cell wall polysaccharide were compared.The ultrasonic assisted enzyme hydrolysis rate was the highest and the effect was the best when β-glucan was extracted from yeast cell wall.The extraction of mannan by low concentration sodium hydroxide (NaOH) solution and the extraction of yeast cell wall polysaccharide by enzyme-alkali method were the best.The separation and purification techniques include deprotein,decolorization and separation of single polysaccharide.The effect of isolation and purification is compared.The best method to remove the protein from yeast cell wall polysaccharide is Sevage method.The best method of pigment removal is granular activated carbon adsorption,and the method of separation and purification of single polysaccharide by DEAE-cellulose A52 anion exchange column and Sephadex G-100 column is the best.Compared with the existing identification techniques of yeast cell wall polysaccharides,the structure identification of yeast cell wall polysaccharides includes that determination of monosaccharide composition of polysaccharides by high performance liquid chromatography,and uniformity and molecular weight of polysaccharides by high performance gel permeation chromatography.The fine structure of polysaccharide was determined by fourier transformation infrared spectrometer (FT-IR),chromatographic mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR).This review commences with the effects and applications extraction,purification and identification techniques of yeast cell wall polysaccharides.It can provide a theoretical basis for the popularization and application of yeast cell wall polysaccharides as an antibiotic substitute in animal husbandry production and offer a new thinking direction for the research and development of antibiotic substitutes.

This study was aimed to investigate the genetic differences and genetic diversity in Luchuan pig populations,provide reference for germplasm improvement and innovation.A total of 29 samples of Luchuan pigs and some samples of Guangxi local pigs were used as experimental materials,three bands of clear and reproducible ISSR primers were used,and DNA fragments obtained by PCR amplification and electrophoresis were used to carry out cluster analysis and clustering trees were constructed to analyze the genetic differences in Luchuan pig populations.The results showed that a total of 182 bands were found on the ISSR molecular markers,84 of which were polymorphic.The total DNA fragment bands amplified by three ISSR primers were different from the polymorphic DNA fragments,which indicated that the selected three ISSR molecular markers were abundant in Luchuan pigs populations.The genetic similarity coefficient in Luchuan pig populations rangeed from 0.43 to 1.00,which indicated that the genetic differences in populations were larger,and the genetic background was rich.UPGMA cluster analysis results showed that Luchuan pigs and Chang Lu binary pigs,Bama Xiang pigs and Huanjiang Xiang pigs could be grouped together,and Da Lu binary pigs,ternary pigs and Longbao pigs were grouped together.This study confirmed that the selected three ISSR primers could be used as effective genetic markers for analyzing genetic difference in Luchuan pig populations,enriched the current ISSR molecular marker resources of Luchuan pigs,and also indicated the rich genetic diversity in Luchuan pig populations.

The aim of this study was to investigate the expression and methylation levels of the uncoupling protein 3 (UCP3) in subcutaneous adipose tissue of Bama and Tibetan pigs.The expression of UCP3 gene at mRNA level was detected by Real-time quantitative PCR method.CpG islands were predicted for the sequence in pig UCP3 gene promoter region (-3 580 to +920 bp) using online software MethPrimer.The difference methylation level of UCP3 gene in Bama and Tibetan pigs was detected by bisulfite sequencing PCR (BSP).The results showed that the expression of UCP3 gene in subcutaneous adipose tissue of Bama pigs was significantly higher than that of Tibetan pigs (P<0.05).Three CpG methylation islands were predicted in the promoter region of UCP3 gene,which were recorded as CpG island1 (-3 171 to -2 928 bp),CpG island2 (-154 to -2 bp) and CpG island3 (+648 to +806 bp).The methylation levels of CpG island1 and CpG island3 were less different in Bama and Tibetan pigs,while the methylation level of CpG island2 (42.61%) was significantly higher in Tibetan pigs than that in Bama pigs (24.49%).A black and white dot maps were built based on the CpG island2 methylation of Bama and Tibetan pigs,the methylation frequency of CpG sites 4,8,9,10,11,12 and 15 in Tibetan pigs were 28.26%,17.39%,26.09%,26.09%,26.09%,23.91% and 34.78% higher than that in Bama pigs,respectively.In addition,three transcription factor binding sites (SP2,PPARγ and EGR1) were predicted at CpG island2.The results indicated that the differential expression of UCP3 gene in subcutaneous adipose tissue of Bama and Tibetan pigs might be caused by the different methylation levels of CpG island2.The higher DNA methylation of adipose tissue in Tibetan pigs might inhibit the expression of UCP3 gene at mRNA level by inhibiting the binding of transcription factors to promoter regulatory regions.

In order to reveal the effect of discoidin domain receptor 1 (DDR1) gene on the lactation performance of buffalo,in this study,the eukaryotic expression vector of buffalo DDR1 gene was constructed,and its optimal transfection time was groped.The effects of DDR1 gene expression on buffalo mammary epithelial cells were also analyzed.Results of agarose gel electrophoresis showed that the fragment size of vetor was 8.8 kb,it was the same as the target carrier fragment size,and the sequencing results showed that the match rate with the target fragment sequence was 100%.Results of cell transfection test showed that the optimal transfection time of DDR1 gene over expression was 48 h.Results of cell proliferation test showed that there was no significant difference in the relative fluorescence value between the DDR1 gene over expression group and the control group (P>0.05).Apoptosis test results showed that the late apoptosis rate was not affected (19.87% VS 17.49%) by the DDR1 gene over expression (P>0.05),but the early apoptosis rate of buffalo mammary epithelial cells (6.48% VS 1.35%) was extremely significantly increased (P<0.01).Moreover,DDR1 gene over expression increased the expression of apoptosis-inhibiting genes BCL-2 and XIAP in buffalo mammary epithelial cells,and reduced the expression of apoptosis-promoting gene P53.In addition,over expression of the DDR1 gene significantly improved the migration rate of buffalo mammary epithelial cells (66.26% VS 58.76%,P<0.05).In conclusion,the research successfully constructed the eukaryotic expression vector of buffalo DDR1 gene and proved that over expression of DDR1 gene promoted the early apoptosis and migration of buffalo mammary epithelial cells,and laid a foundation for the future study of the development and lactation performance of buffalo mammary gland.

This study was aimed to investigate the effect of supernumerary teat (SNT) on the milk performance of dairy cows and the relationship between mammogenesis gene LGR5 and supernumerary teat.Dairy herd improvement (DHI) data of 325 supernumerary teat and 737 non-supernumerary teat dairy cows in the same born year were collected to compare the differences in milk performance.The differential expression of LGR5 gene between supernumerary teat and non-supernumerary teat dairy cows was performed by Real-time quantitative PCR.The genetic mutation of LGR5 gene was detected by direct sequencing method.The results showed that the daily milk yield,peak milk yield and lactation persistence of non-supernumerary teat dairy cows were better than that of supernumerary teat dairy cows,but did not reach the statistical significant level.The expression of LGR5 gene in supernumerary teat dairy cows was significantly higher than that in non-supernumerary teat dairy cows (P<0.05).3 SNPs (rs42849472,rs42849471 and rs42849470) were identified in LGR5 gene intron 1.The frequency of AA genotype of 3 SNPs in supernumerary teat dairy cows were extremely significantly higher than that in non-supernumerary teat dairy cows (P<0.01).The results suggested that LGR5 gene might be involved in the occurrence of supernumerary teat in dairy cows,and could be used as an effective candidate gene.

To investigate the fat deposition mechanism of Hulun Buir sheep,twenty one Hulun Buir ewes at five months old were divided into two groups,Baerhu group(semi-ellipsoid tail,n=11) and short-tailed group(small-peach tail,n=10),and fed under the same management conditions.The carcass weight,body size and tail type indexes of two groups were measured.Adipose tissues from 8 body locations were used to analyze adipocyte morphology and triglyceride(TG) content.Moreover,the adipose metabolism related genes were detected by Real-time quantitative PCR.The result showed that two populations were significantly or extremely significantly differed in length,width and weight of tail as well as carcass weight and body length (P<0.05;P<0.01).Although the morphology of adipocytes in the same part of two populations were basically the same,the area and volume of adipocytes in the tail of Baerhu population were larger than that of short-tailed ewes (P<0.01).And TG content in adipose tissues including subcutaneous,omentum and tail tissues were significantly or extremely significantly different between two sheep groups (P<0.05;P<0.01).The mRNA levels of lipid-metabolism related genes,including ATGL,PPARγ,C/EBPα and SREBP,were higher in Baerhu population than that in short-tailed group (P<0.05; P<0.01).For the Baerhu ewes,the expression of ATGL and SREBP was negatively or positively associated with the TG content (r=-0.965,0.972),respectively.In summary,it was preliminarily revealed that it had significant differences of adipose deposition ability between two populations.The study was helpful for further exploring the molecular mechanism of fat tail formation in sheep and providing a theoretical basis.

This experiment was conducted to investigate the effects of different slaughter weights on slaughter performance,carcass traits and meat quality of Sushan pigs,so as to determine the optimal slaughter weights of Sushan pigs.60 Sushan pigs,with 30 kg of average body weight and similar body conditions,were selected and randomly assigned to three groups (A,B and C),and there were 20 pigs in each group.When the body weight reached about 90,100 and 110 kg respectively,three boars and three sows in each group were slaughtered.The results showed:With the increase of slaughter weights of Sushan pigs,there were significant differences in slaughter performance among groups.The lean meat rate of group A was 63.18%,which was significantly higher than that of groups B and C (P<0.05).The fat content,the fat thickness of shoulder thickness,and the backfat thickness of reciprocal 3-4 rib of group C were significantly higher than those of group A (P<0.05).The slaughter rate,skin rate and bone ratio had no significant difference in three treatment groups.There was no significant difference in water content,crude protein and crude fat of longissimus dorsi among the three groups (P>0.05),but the crude ash content of group A was significantly higher than that of groups B and C (P<0.05).The L value of group B was 43.50,significantly higher than that of group C (P<0.05),the pH_{1 h} and pH_{24 h} values of group C were 6.40 and 5.83 respectively,it was higher than groups A and B (P<0.05 or P<0.01).With the increase of slaughter weight,saturated fatty acids and monounsaturated fatty acids showed an upward trend,polyunsaturated fatty acids showed a downward trend,and saturated fatty acids in group C were significantly higher than those in group A (P<0.05).Total amino acids,essential amino acids and flavor amino acids in group A were higher than those in group B and C,but the difference was not significant (P>0.05).Therefore,considering the results of this experiment,the best slaughter weights of Sushan pigs were about 90 kg.

The aim of this study was to investigate the effect of sesamin (SES) added to in vitro culture (IVC) on the development of early mice embryos.The fertilized eggs of 6-week-old female Kunming mice were randomly divided into control group and different concentrations (10,50,100 μmol/L) SES groups.The apoptosis rate and total cell number in blastocysts were detected by Fluorescein-dUTP and Hoechst 33342 immunofluorescence staining,respectively.DCFH-DA was used to detect the levels of reactive oxygen species (ROS) in early embryos;The levels of glutathione (GSH) in early embryos were detected by CMF₂HC;Early embryonic mitochondrial membrane potential intensity was measured using JC-1.The results showed that the segregation rate and blastocyst rate of SES groups were higher than those in control group,but there was no significant difference (P>0.05).The number of cells in 50 μmol/L SES group was significantly higher than that in control group (P<0.05),and the apoptosis rate was significantly lower than that in control group (P<0.05).Compared with control group,the ROS level in the 50 μmol/L SES group was significantly lower (P<0.05),and the GSH level and mitochondrial membrane potential level were significantly increased (P<0.05).The results showed that the addition of SES in IVC could increase the number of cells in blastocyst,decrease the apoptotic rate and the ROS level,increase the GSH level,improve the mitochondrial function of early embryos,reduce the damage of oxidative stress of embryos,and improve the quality of early embryonic development in mice.

The aim of this study was to explore the crossbreeding effect of Luxi cattle,so as to promote genetic improvement of Luxi cattle in production performance.111 healthy steers were selected and divided into four groups:Group A included 29 first filial generation (F1) of Limousin cattle×Luxi cattle;Group B included 26 F1 of Simmental cattle×Luxi cattle;Group C included 27 F1 of Japanese Black cattle×Luxi cattle;Group D was the control group including 29 Luxi cattle.Fattening and slaughtering test was in the same feed farm and management condition,and the fattening effects were comprehensively analyzed.The results showed that Limousin cattle,Simmental cattle and Japanese Black cattle could carry out better crossbreeding improvement on Luxi cattle.The Daily weight gain of Simmental cattle×Luxi cattle was significantly higher than that of other groups (P<0.01),and the dressing percentage,carcass weight and slaughter rate of Simmental cattle×Luxi cattle were significantly higher than that of other 3 groups (P<0.05).The marbling score was the best in group C.There was no significant differences of daily mean intake and daily feed cost among four groups (P>0.05).In conclusion,Simmental cattle×Luxi cattle showed the stronger meat production,and Japanese Black cattle×Luxi cattle was the best in marbling.

In order to study the effects of acute cold stress on the cold resistance,production performance and disease resistance of Altay sheep by affecting the change of heat shock protein content.This experiment was set in the normal temperature group (15 ℃±2 ℃) and the cold stress group (-25 ℃±2 ℃),each group of 5,respectively.The heart,liver,spleen and kidney tissues were collected from the normal temperature group and the acute cold stress group after 24 h.The changes of HSP60,HSP70 and HSP90 mRNA contents were detected by Real-time quantitative PCR and homology analysis of HSP60, HSP70 and HSP90 genes.The results showed that the homology of HSP70 and HSP90 genes with the existing Ovis aries sequences were higher than 99%,and the HSP60 gene had more than 99% homology with the existing Bos taurus sequence.After cold stimulation,the expressions of HSP60 gene in heart,liver,spleen and kidney were higher than those in normal temperature group,and the expression of HSP60 gene in kidney was extremely significant different from that in control group (P<0.01).The expressions of HSP70 gene in heart,liver,spleen and kidney were higher than those in normal temperature group,and the expressions of HSP70 gene in liver and kidney were extremely significant different (P<0.01).The expressions of HSP90 gene in heart,liver,spleen and kidney were higher than those in normal temperature group,and the expression was significantly different in liver (P<0.05),but the expressions difference in spleen and kidney were extremely significant (P<0.01).It demonstrated that acute cold stress greatly stimulates the individual's heat production function,and changed its energy metabolism through the regulation of heat-producing related genes in the pathway,thus improving the survival rate of cells,enhancing the body's tolerance to environmental stress,and better adapting to the changes of environmental temperature.The results of this experiment provided a theoretical basis for further in-depth study of the effects of acute cold stress on Altay sheep.

This study was aimed to explore the polymorphisms of BMP15 gene exon 1 and its association with egg quality in Sizhou chicken.The direct sequencing of PCR products was adopted to detect the polymorphism of BMP15 gene exon 1 in Sizhou chicken.After direct sequencing,sequence adjusting were conducted using DNAStar software.Data of genotype frequency was counted by Excel 2007 and analyzed by χ² test.The association of genotype and egg quality were analyzed with SPSS 18.0.The results showed that BMP15 gene exon 1 in Sizhou chicken had four SNPs:397C/T,473A/G,603C/T and 612C/T,which were not in Hardy-Weinberg equilibrium (P<0.05);The polymorphic information content (PIC) of 397C/T and 603C/T loci were 0.3488 and 0.3299,respectively,belonging to moderate polymorphism (0.25 < PIC < 0.5).The PIC of 473A/G and 612C/T loci were 0.1527 and 0.2075,respectively,belonging to low polymorphism (PIC<0.25).The results of correlation analysis showed that the 397C/T locus significantly affected the protein height,Hastelloy unit and egg yolk weight of Sizhou chicken (P<0.05);There were no significant difference of 473A/G and 603C/T loci with egg quality in Sizhou chicken (P>0.05);The 612C/T locus had a significant influence on egg length of Sizhou chicken,respectively (P<0.05).This results suggested that BMP15 gene exon 1 had a larger genetic effect on the protein height and egg length in Sizhou chicken.BMP15 gene might be one of the genes affecting the egg quality in Sizhou chicken,and could be used as a molecular marker for egg quality traits such as protein height and egg length and diameter in Sizhou chicken.

The study was aimed to express the EP402R gene of African swine fever virus (ASFV) Georgia 2007/1 strain via prokaryotic expression system,obtain the recombinant CD2v protein,and prepare polyclonal antibodies against the purified recombinant CD2v protein.After codon optimization,ASFV EP402R full-length gene was linked into pET-28a(+) expression vector to construct prokaryotic recombinant expression plasmid.After induction by 1 mmol/L IPTG at 16 ℃ for 12 h,the recombinant protein was identified by SDS-PAGE and Western blotting.The purified recombinant CD2v protein was used as immunogen to prepare mouse anti-CD2v polyclonal antibodies.The antibody titer was measured by indirect ELISA and the specificity was further analyzed by indirect immunofluorescence assay (IFA) and Western blotting.The results showed that ASFV EP402R gene was successfully cloned into pET-28a(+),and pET-28a-EP402R was obtained.The recombinant plasmid was transformed into E.coli BL21(DE3) for expression,the recombinant protein was expressed mainly in the form of inclusion bodies,with molecular mass at about 47 ku,while some of the recombinant protein could also exist in a soluble form.Western blotting results showed that the purified protein had good immunoreactivity.The indirect ELISA result showed that the polyclonal antibodies had a high titer of 1:512 000,IFA and Western blotting results indicated that it could specifically recognize recombinant CD2v protein.These results confirmed the recombinant CD2v protein expressed via prokaryotic system had good immunogenicity,and the prepared polyclonal antibodies had high titer and specificity.This research provided technical support for further study of ASFV EP402R biological function,as well as its gene-deletion based vaccine development.

In order to establish an indirect ELISA method for detection of Brucella antibody in serum,the wzt gene fragment was amplified from the strain of Brucella melitensis QY1 by PCR and ligated into the pET-30a vector to construct the plasmid pET-30a-wzt.The correct plasmid was transferred to E.coli BL21(DE3) competent cells were expressed by prokaryotic expression system,and the expressed products were analyzed by SDS-PAGE and Western blotting,and then the wzt recombinant protein was purified.The indirect ELISA assay for Brucella was established using wzt recombinant protein as the detection antigen and gradually optimizing the conditions.The results showed that the prokaryotic expression vector of pET-30a-wzt was successfully constructed and expressed in BL21(DE3) host bacteria.SDS-PAGE and Western blotting results showed that the recombinant protein was about 35 ku,which had good reactogenicity.The optimal coating concentration was determined to be 15 μg/mL,the optimal dilution of serum was 1:80,and the optimal dilution of the enzyme-labeled antibody was 1:5 000.The critical value was determined by detecting 24 negative samples,the sample was positive when the D_{450 nm} value was ≥ 0.30,and the sample was negative when the D_{450 nm} value was <0.30.The specificity test results showed that the method did not cross-react;The intra-and inter-assay coefficients of variation were both <10%;120 samples of serum were used in this method,the test was carried out and verified by the Rose-Bengal agglutination test,and the coincidence rate was 96%.This results indicated that the indirect ELISA method established in this experiment provided a reliable technical means for the detection of brucellosis.

To assess the genetic diversity of ORF5 gene of porcine reproductive and respiratory syndrome virus (PRRSV) in Zhejiang province from 2014 to 2016,ORF5 gene was amplified and sequenced in the PRRSV positive samples which were collected from different places of Zhejiang province.The homologous analysis and genetic analysis were conducted by DNAStar and Mega 5.1 softwares.Homologous analysis showed that ORF5 gene of 54 PRRSV strains shared 83.1% to 100% nucleotide homology and 80.6% to 100% amino acid homology.ORF5 gene of 54 PRRSV strains shared 83.8% to 90.4% nucleotide homology and 81.6% to 90.0% amino acid homology with classical strain VR2332.ORF5 gene of 54 PRRSV strains shared 83.5% to 99.5% nucleotide homology and 83.1% to 99.0% amino acid homology with virulent strain JXA1.The phylogenetic tree based on sequences of ORF5 gene suggested the 54 strains could be classified in subgenotypes Ⅰ,Ⅲ and Ⅳ,among them,subgenotypes Ⅲ and Ⅳ were new strains in Zhejiang province in recent years.Compared with antigen epitopes,54 PRRSV strains had higher variation in the signal epitope and non-neutralizing antigen epitopes,and had little variation in neutralizing epitope.The results indicated that four subtypes of PRRSV existed in Zhejiang province,but mainly prevailing the subgenotype Ⅰ PRRSV strain.

This study was aimed to understand the prevalence of orf virus (ORFV) in Chongqing.18 samples of clinical suspected orf disease materials were collected from sheep farms in Dazu,Chongqing,etc,and DNA of the samples was extracted,which was identified as positive by PCR.Positive disease materials were routinely treated,inoculated with lamb testicular cells (LT),and blind transmitted to the F5 generation.Toxic LT cell lesion CPE was observed,and F5 generation cell cultures were systematically identified by indirect immunofluorescence test (IFA),PCR amplification,sequencing,homology alignment,genetic evolution analysis and TCID₅₀ determination.The results showed that the nucleic acid positive rate of ORFV detected by PCR was 61.1% (11/18).After LT cells were inoculated with Dazu strain for 36 h,the long-spindle cells became round,fused,reticulated and shriveled,and most of the cells fell off after 72 h.IFA observed the specific green fluorescence in cytoplasm,and the fluorescence was distributed around the nucleus.Specific target bands of F1 to F5 cell cultures were amplified by PCR with a size of 1 137 bp.The sequencing results were compared with 19 strains of ORFV B2L genes in GenBank,and the nucleotide homologies were 97.9% to 100%,which was up to 100% of the homology with the SA00 isolates from the United States,and the genetic evolution analysis showed that they were in the same evolutionary branch,with close relationship.The TCID₅₀ value of F5 generation cell culture was 10^-5.68/0.1 mL,which could be stable proliferation in cells.In conclusion,this study successfully isolated and obtained an ORFV strain,provided biological materials for the follow-up ORFV research,and laid a foundation for the prevention and control of stomatitis in Chongqing.

In order to study the effect of nasal drip immunization on tracheal function of chickens,tracheal cilia swing rate,tracheal morphology and mucus secretion after nasal drip immunization with Newcastle disease vaccine in SPF chickens were studied.Eighty 7-day-old SPF chickens were randomly divided into control group and test group,40 chickens in each group.The test group received nasal immunization with attenuated Newcastle disease vaccine and the control group received nasal saline.At 0,2,4 and 6 days post immunization,10 chickens were killed randomly in each group.Tracheal cilia swing rate was measured by red blood cell tracer method.Paraffin tissue sections were collected from trachea for HE and Alcian Blue pH 2.5 and pH 1.0 staining.The results showed that the cilia swing rate of SPF chickens in test group was higher than that in control group at 0 and 2 d post immunization,but the difference was not significant (P>0.05).After nasal drip immunization,the trachea structure of SPF chickens was intact without obvious damage.After nasal drip immunization,the distribution volume of acid mucus in trachea of SPF chickens in test group was significantly higher than that of control group at 0 and 2 d post immunization (P<0.05),then decreased significantly,and increased at 6 d post immunization,but the difference was not significant (P>0.05).The distribution volume of sulfated mucus increased significantly at 0 and 2 d post immunization (P<0.05),and then decreased in accordance with the control group.The results showed that the tracheal mucus secretion of SPF chickens increased in two days after nasal drip immunization,and then returned to normal level.There was no significant effect of immunization on tracheal morphology and cilia swing rate.

The aim of this study was to investigate the pathogenicity and drug resistance of 7 Streptococcus suis type 3 (SS 3) strains isolated from piggery in Tianjin.The pathogenicity of bacteria with doses of 1.0×10⁷,1.0×10⁸ and 1.0×10⁹ CFU/mice was studied.The virulence genes mrp,ef,sly,gdh,gapdh,fbps and orf2 of 7 strains of SS 3 strains were detected by PCR.Drug susceptibility tests of the 7 strains of SS 3 were also carried out.The results of pathogenicity test showed that 6 and 3 strains of SS 3 could cause 100.0% (5/5) morbidity and death in mice at inoculation dose of 1.0×10⁹ and 1.0×10⁸ CFU/mice,respectively.One strain of SS 3 could still cause 80.0% (4/5) morbidity and death in mice at inoculation dose of 1.0×10⁷ CFU/mice.The order of pathogenicity of the 7 strains of SS3 strains in mice was R15056 > R15042=S15030 > Y12024=Y09011 > Y13125 > Y13164.The results of virulence gene test showed that there were three virulence genotypes:R12024,Y13164,R15042 and S15030 were positive for gdh,gapdh,fbps and orf2 virulence genes;Y13125 and R15056 were positive for sly,gdh,gapdh,fbps and orf2 virulence genes,and Y09011 was positive for gdh,fbps and orf2 virulence genes.Drug susceptibility test showed that 7 SS 3 strains were relatively sensitive to cefquinoxime,followed by amoxicillin,ciprofloxacin and sulfamethoxacil sodium.However,they showed 100.0% resistance to doxycycline,and all strains showed more than 3-fold resistance.This study laid a foundation for the further development of epidemiological characteristics and pathogenic mechanism of SS 3 strains in Tianjin,and could provide theoretical guidance for the comprehensive prevention and control of SS 3 strains in Tianjin.

The aim of this study was to analyze the drug resistance of porcine Escherichia coli in some areas of Guangxi and drug resistance genes contained in some strains,and provide a reference for the prevention and treatment of animal Escherichia coli diseases.The experiment used Macconkey agar medium and Eosin-Methylene Blue agar medium preliminary identification,microscopic microscopy,molecular biology 16S rRNA identification,drug sensitivity double broth microdilution method and other methods to isolate and identify Escherichia coli,combined with PCR-related techniques were used to detect more than 30 drug resistance genes in some strains.The results showed that 65 strains of porcine Escherichia coli were isolated from pig-derived materials collected from a certain area of Guangxi,all of which were multi-drug resistant Escherichia coli and concentrated in six-drug resistance and seven-drug resistance.According to the investigation of the antibiotic-resistant drugs,it was found to be highly resistant to β-lactams,sulfonamides,macrolides,and the drug resistance rates were 98.46%,90.77% and 84.62%,respectively.Further analysis results found that it was mainly resistant to antibiotics such as ceftiofur sodium,amoxicillin,sulfamonomethoxine,azithromycin,and sensitive to meropenem,amikacin and colistin.The resistance genes contained in some strains were found to contain tetracycline (tetA and tetM),β-lactams (CTX-m-u) and sulfonamide (Sul2) resistance genes.In summary,it was found that the drug resistance of porcine Escherichia coli in Guangxi was serious,and there were relatively many drug resistance genes.Therefore,in the actual production,it was necessary to use related measures such as combination therapy for disease treatment to achieve rapid and effective treatment of diseases.

In order to understand the drug resistance of Escherichia coli (E.coli) in sheep in Naqu city,Tibet,and to guide clinical rational drug use,92 fresh non-polluting diarrhea samples were collected from sheep in Naqu city,Tibet,and were separated by colour medium of E.coli,examined by Gram staining microscopy,biochemical identification,molecular biological identification and biochemical identification of diarrhogenic E.coli,drug sensitivity test and drug resistance gene detection.The results showed that the isolates were blue colony and pink brevibacterium on the colour medium of E.coli.26 strains of E.coli from sheep were isolated by biochemical identification and PCR detection of 23S rRNA,the isolation rate was 28.3%,25 strains of which accorded with the biochemical characteristics of diarrhogenic E.coli,and the isolation rate of diarrhogenic E.coli was 27.2%.Drug susceptibility test showed that 25 strains of sheep-derived E.coli were resistant to ampicillin with a resistance rate of 24.0%,followed by carboxybenicillin and kanamycin with a resistance rate of 8%,piperacillin,cefuroxime,gentamicin,tetracycline and minocycline with a resistance rate of 4%.Drugs such as norfloxacin,oxygen,floxacin and ciprofloxacin were very sensitive and could be used as clinical drugs.The detection rate of bla_TEM gene was 100% in 5 drug-resistant genes,which indicated that all isolates contained corresponding drug-resistant genes.The above results showed that there was resistance to many drugs in E.coli isolated from sheep in Naqu,Tibet,suggesting that rational drug use and combined drug use should be emphasized in clinical practice to slow down the emergence of drug resistance of E.coli.

The study was aimed to observe the effects and preliminary mechanism of Spirulina kinase (SPK) on atherosclerosis (AS) model rats.60 SD rats were randomly divided into normal control group,model group,positive control group (simvastatin,0.005 g/kg),SPK low-dose,medium-dose and high-dose groups(80,160 and 320 U/kg).Except for normal control group,other groups were fed with high fat feed to establish the atherosclerosis model and were given relevant medicine intragastrically once a day for 12 weeks.The artery blood was collected and the content of total cholesterol (TC),high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol (LDL-C) levels,contents of MDA,NO and SOD activity were measured,the atherosclerosis index (AI) was caculated.The change of thoracic aorta were observed by hematoxylin and eosin (HE) staining under microscope.The results showed that:Compared with normal control group,AI and MDA content were significantly increased (P<0.05),SOD activity and NO level were significantly decreased(P<0.05) in model group;Compared with the model group,MDA content was significantly decreased (P<0.05),while SOD activity and NO level were significantly increased (P<0.05) in SPK high-dose group.Pathological results showed that the model group had obvious atherosclerotic plaque formation.The formation of atherosclerotic plaque in SPK groups was significantly reduced,especially in the middle and high dose groups,there was no obvious difference comparing to the normal control group.In conclusion,SPK had obvious effect of preventing AS.Its mechanism might be related to the decrease of MDA and the increase of SOD activity and NO level.

Qinhong perfusate is a newly developed Chinese medicine compound preparation for the treatment of dairy cow mastitis.In order to ensure the safety of clinical medication and the stability of production technology,the quality standard of Qinhong perfusate was studied and established.Scutellaria baicalensis,honeysuckle and safflower were identified by TLC,referring to the inspection methods of perfusate in Chinese Veterinary pharmacopoeia.The relative density,pH,sterility,related substances and contents of Qinhong perfusate were examined,and baicalin was quantitatively detected by high performance liquid chromatography.The results showed that the samples of Qinhong perfusate showed the same color spots at the corresponding positions with the control medicinal materials and the control products.The TCL method for identification of Scutellaria baicalensis,honeysuckle and safflower had good specificity,reproducibility and durability.The relative density,pH,sterility,related substances and loading amount of Qinhong perfusate all conformed to the relevant regulations,and the relative density of Qinhong perfusate was not less than 1.0 (30 ℃) and the pH was 5.0-7.0.The precision,stability,repeatability,linearity,specificity and durability of the HPLC method for determination of baicalin content in Qinhong perfusate were good.The linear relationship between baicalin content and peak area was good in the range of 0-9.20 μg(R²=0.9999).The average recovery was 100.04%(RSD=1.34%,n=9).The established quality standard was accurate,feasible and reproducible.It could be used for the quality control of Qinhong perfusate and laid a foundation for its large-scale production and clinical application.

The purpose of this study was to evaluate the acute oral toxicity of florfenicol premix to Wistar rats.The median lethal dose (LD₅₀) was measured using the modified Up-and-Down Procedure (UDP) revised by OECD.The adverse effects of florfenicol premix on the biological system and main organs of Wistar rats were determined by weight gain,organ coefficient,haematology,clinical chemical examination and histopathological examination.According to the LD₅₀>5 000 mg/kg of florfenicol,the limit test of UDP was selected,and the toxic reaction and death were recorded by using a fixed number (5) of animals with a dose of 2 000 mg/kg for 14 days.LD₅₀ was calculated by AOT425StatPgm program,and the other 3 Wistar rats were given the same dose of normal saline as control.The results showed that none of the five rats died,LD₅₀>2 000 mg/kg.During the test,there were no signs of toxic reaction in the treatment group.Compared with the control group,the haematological parameters in the treatment group had no significant change,but in the clinical chemical examination,the level of ALT in the treatment group was significantly higher than that in the control group (P<0.05),suggesting the toxic damage of the pharmaceutical preparation to the liver.The results of histopathological examination showed that the treatment group had no toxic damage to the main organs,such as heart,liver,spleen,lung,kidney and duodenum,and the toxic target organs could not be determined for the time being.The results showed that the use of FF premix in the safe dose range was safe and reliable,and more toxicity information still needed to be determined by long-term toxicity test.

Probiotics play an important role in the intestinal tract of healthy teleost.They not only inhibit pathogenic microorganisms,but also stimulate and enhance the intestinal mucosal immune system and play an important role in intestinal immunity.In recent years,mucosal immunity of teleost has become a hot research topic because of its diversity and unclear definition.The direct contact between teleost and aquatic environment makes the surface of the intestinal tract to various pathogens.Immunoregulation is an effective preventive measure in teleost,and probiotics can enhance the intrinsic immunocompetent cells and factors on the surface of intestinal mucosa to play a role in the pathogen.Probiotics enter fish mainly by oral administration,and intestinal tract as its main target organ produces specific immune response to fish.Therefore,the study of probiotics affecting intestinal mucosal immune system deserves attention.Compared with mammals,teleosts have more diffuse intestinal lymphatic system.The immune cells necessary for the local immune response are abundant in the intestinal mucosa,and the local immune response can be monitored in the intestinal tract of the immunized fish.In this paper,the intestinal mucosal immune system of teleost and the effects of probiotics on intestinal mucosal immunity of teleost in recent years were reviewed,and the further research on probiotics of teleost was prospected,in order to provide reference for the follow-up study on the interaction between probiotics and teleost.