The purpose of this study was to screen and identify the predicted sequence of phospholipase C-γ1 (PLC-γ1) gene in sheep,obtain the gene sequence and analyze its bioinformatics.Four different transcripts given by GenBank (accession No.:NC_040264.1) and Ensembl (accession No.:ENSOARG00000001700.1) were compared by BLAST.Specific primers were designed in the non-homologous region.The total RNA was extracted from sheep ovary and transcribed into cDNA,to amplify the specific fragment by RT-PCR.After gel recovery,the sample was sequenced,and the gene sequence was screened and consistent with the fragment by BLAST comparison.According to the selected primers,the sheep PLC-γ1 gene was amplified by PCR with LA Taq (GC Buffer) fidelity enzyme.The gel recovery product was ligated to PUC57-T vector and transformed into Escherichia coli DH5α competent cells,and the single clone was sequenced.According to the obtained sequence,the gene and the encoded amino acid sequence were analyzed by bioinformatics technology.The results showed that the non-homologous fragment of 330 bp,was successfully amplified,and the predicted sequence of transcript X3 was consistent with it by BLAST analysis.The PLC-γ1 gene fragment of 3 870 bp in sheep was successfully amplified by sequencing.PLC-γ1 gene in sheep was closely related to Capra hircus,and had a particularly strong evolutionary relationship with Bos taurus, Equus caballus, Sus scrofa and other livestock animals.The protein molecular formula was C₆₅₈₂H₁₀₁₆₀N₁₈₁₂O₁₉₆₂S₅₈,and the molecular weight was 147.9 ku.It was a hydrophilic weak acid unstable protein,which existed in cytoplasm and had no transmembrane and signal peptide.The N- and C-terminal GC content of this gene was relatively high,reaching 80%,70% or more.It had highly conserved domains such as SH2,SH3,and C2.The ratio of α-helix,extended chain,β-turn and random coli in the secondary structure was 36.46%,16.14%,5.04% and 42.36%,respectively.There were 112 phosphorylation sites.Y428,Y509 and S514 were the key phosphorylation sites.It provided a theoretical basis for the study of PLC-γ1 protein expression in sheep,and was of great significance for studying the embryonic development of Xinjiang local breed sheep and improving the fertilization rate.

To investigate the expression of microRNAs(miRNAs) in skeletal muscles of Dezhou donkeys and further improve the regulatory mechanism of small RNA in the development of skeletal muscle of donkey.In this study,the longissimus dorsi muscle of two Dezhou donkey strains (Wutou donkey and Sanfen donkey) was selected as the research objects,by constructing small RNA sequencing library of Wutou donkey longissimus dorsi muscle (WB) and Sanfen donkey longissimus dorsi muscle (SB),bioinformatics techniques were used to carry out differential expression analysis of miRNAs between WB and SB,and predict and analyze miRNAs that might affect the meat production traits of donkey.In addition,six miRNAs with high expression were selected,and Real-time quantitative PCR was used to verify the accuracy of sequencing results.The results showed that 982 and 1 149 conserved miRNAs and 106 and 143 new miRNAs were identified in WB and SB libraries.In WB and SB comparison group (WB vs SB) based on conditions|log₂(fold_change)|≥2,P≤0.05 get 17 differentially expressed miRNAs,of which 5 were up-regulated and 12 were down-regulated.Through GO annotation,it found that differentially expressed miRNAs were significantly enriched in biological processes such as regulating cell migration and differentiation,cell division and skeletal muscle cell contraction (P<0.05).bta-miR-378d_L+1R-1_1ss22CA and bta-miR-378d_R-2 which related to skeletal muscle development in donkey was found,they target multiple genes such as NPY1R,GPR152,BEST1,KCNH8 and SPAG9.KEGG enrichment results showed that miRNAs were enriched in Wnt,MAPK,ubiquitin proteolysis system and other signaling pathways related to skeletal muscle development.Among them,a total of 207 target genes 17 differentially expressed miRNAs were involved in the Wnt signaling pathway,especially PC-5p-84483_31,hsa-miR-186-3p_R-3 and cfa-miR-329b_1ss19CT had the largest number of target genes.The results of Real-time quantitative PCR were consistent with the results of miRNAs sequencing,and it found that the expression levels of the other five miRNAs were all higher in the SB than in the WB except for eca-miR-181b.In this study,miRNAs of skeletal muscle of two strains of Dezhou donkey were analyzed for the first time,and the differences of RNA-seq between Wutou donkey and Sanfen donkey were revealed,which provided reference for elucidating the molecular regulation mechanism of skeletal muscle growth and development of Dezhou donkey.

In order to explore the structural and functions of the genes related to the adaptation of Tarim red deer(Cervus elaphus yarkandensis) to the arid environment.The sequence of thioredoxin dependent peroxidase 3 (PRDX3) gene of Tarim red deer was screened from the results of whole genome resequencing,and analyzed the expression of this gene in different tissues of Tarim red deer.The CDS sequence of PRDX3 gene of Tarim red deer was cloned and sequenced.Homology comparison,phylogenetic tree construction and bioinformatics analysis were carried out by using related software.The results showed that the expression level of PRDX3 gene in kidney was extremely significantly higher than that in lung and liver (P<0.01).The CDS region of PRDX3 gene of Tarim red deer was 660 bp in length and encoded 220 amino acids.The bioinformatics analysis using related software showed that Tarim red deer and Odocoileus virginianus (GenBank accession No.:XM_020875097.1) had the highest homology and the closest genetic distance,and with Rattus norvegicus (GenBank accession No.:NM_022540.1) had the lowest homology and the longest genetic distance.The protein molecular weight of PRDX3 in Tarim red deer was 24.42 ku and composed of 220 amino acids.It was predicted that the instability index,theoretical isoelectric point,lipid solubility index and hydrophilic average coefficient of PRDX3 protein was 25.36,5.82,85.50,and -0.05,respectively.There was O-glycosylation site and phosphorylation sites in PRDX3,but had no transmembrane region,N-glycosylation site and signal peptide,most likely located in the mitochondria.The secondary and tertiary structures of PRDX3 protein were mainly comprised of α-helix and random coil.PRDX3 protein contained the conserved domain of PRX_Typ2cys superfamily,which had relatively strong interaction with a variety of proteins.The results of this study laid a foundation for the following functional gene research of Tarim red deer.

The aim of this study was to explore the effect of different expressions of chi-miR-107-3p and rhomboid family member 2(RHBDF2) genes on the villus growth of cashmere goats in different breeds (strains) and light controlled cashmere increasing in the skin hair follicle anagen (May to July).In this experiment,the hair follicle tissues of Inner Mongolia Alashan cashmere goats,Mingai cashmere goats and Albas cashmere goats which were used the increasing cashmere yield technology by shortened photoperiod were randomly collected in July,and HE staining was used to observe the morphology differences.Real-time quantitative PCR was used to detect the ralative expression of chi-miR-107-3p and RHBDF2 genes.The results showed that the secondary hair follicles of Alashan cashmere goats were in the stage of growth and development,and the reconstruction of secondary hair follicles of Mingai cashmere goats and Albas cashmere goats (light controlled cashmere increasing) had been basically completed.The expression of chi-miR-107-3p in the skin tissue of Alashan cashmere goats was extremely significantly higher than that of Mingai cashmere goats and Albas cashmere goats (P<0.01),the expression level of RHBDF2 gene was extremely significantly lower than that of the other two breeds (P<0.01),but the expression level of RHBDF2 gene in skin tissue of Mingai cashmere goats was significantly lower than that of Albas cashmere goats (P<0.05).In conclusion,the microstructure of hair follicle in different breeds (strains) light controled cashmere goats at anagen prosperity was consistent with the results of the differential analysis of chi-miR-107-3p and RHBDF2 genes in skin tissues.The expression level of chi-miR-107-3p was high,while the expression level of RHBDF2 gene was extremely low.With the completion of secondary hair follicle reconstruction,the expression of chi-miR-107-3p decreased significantly,the expression of RHBDF2 gene increased gradually,and the expression mechanism of Inner Mongolia cashmere in different breeds (strains) was basically the same.chi-miR-107-3p and RHBDF2 genes were important regulators for the growth and development of skin hair follicles in cashmere goats.

To develop a feasible genome editing technology based on CRISPR/Cas system of Flavobacterium psychrophilum,the CRISPR/Cas system structure of Flavobacterium psychrophilum and its mechanism of action were analyzed by bioinformatics.The complete genome sequences of eight Flavobacterium psychrophilum strains were available in the GenBank database,and CRISPRCasFinder software was used to identify both clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated (Cas) proteins in the Flavobacterium psychrophilum genomes.The repeats and spacers of the CRISPR arrays were further identified using CRISPRFinder software,and the nucleotide sequence identities of cas genes were analyzed using Mega X software.Nucleotide alignment was performed with repeat sequence to search for the antirepeat portion of the trans-activating CRISPR RNA (tracrRNA),and the transcription terminator of the tracrRNA was predicted using ARNold software.The promoters of tracrRNA and crRNA precursor (pre-crRNA) were predicted using BPROM software.The nucleotide sequence identities of spacers were analyzed using Clustal X software,and CRISPRTarget software was used to predict protospacers matching the unique spacers and their protospacer adjacent motifs (PAMs).The PAM of the matching protospacers was obtained using WebLogo software.The results showed that all 8 strains had only full intact a CRISPR/Cas9 system,including a CRISPR array and three Cas proteins.The CRISPR array consisted of short repeated sequences (repeats) interspersed with short variable sequences (spacers).Spacers in all eight CRISPR arrays were 46 bp long,and the nucleotide sequence of repeat were highly similar.Spacers had a size between 29 and 31 bp,and the numbers of spacers varied from 20 to 41.All 8 CRISPR/Cas9 systems included a set of only three Cas proteins (Cas9,Cas1 and Cas2) which were highly conserved.The tracrRNA sequences shared 100% identity at the nucleotide level and found upstream of the cas9 gene.There was a stretch of 24 nucleotides where 23 nucleotides of the tracrRNA matched the repeat sequence.Each repeat carried its own minimal promoter and the pre-crRNA transcription initiated independently within each spacer.Spacer alignments among 8 different strains revealed that newly acquired spacers were integrated both at the 5'end of the CRISPR array and internally.13 of the 65 unique spacers could be mapped as protospacers on phages or plasmids.Protospacer alignments revealed an apparent PAM of 5'-GANTTTT-3'recognized by the Cas9.Taken together,the CRISPR/Cas9 system could be used to develop a feasible genome editing technology suitable for Flavobacterium psychrophilum in theory.

The purpose of this study was to explore the differentially expressed genes (DEGs) and signaling pathways related to growth and development of chicken in pituitary by transcriptome sequencing and bioinformatics analysis.Three 42-day-old healthy Tibetan chickens and White-feather broilers were selected,respectively.The pituitary transcriptome of the 2 chicken breeds was analyzed by Illumina HiSeq 2000 sequencing system.The DEGs were annotated and enriched by GO and KEGG database.The relative expression of randomly selected DEGs were verified by Real-time quantitative PCR.The results showed that 126 094 302 and 125 666 442 clean reads were obtained from Tibetan chicken and White-feather broiler,respectively.Compared with White-feather broilers,there were 392 DEGs in Tibetan chickens pituitary,of which 196 up-regulated genes and 196 down-regulated gene (P<0.05),including growth hormone gene (GH),growth hormone releasing hormone receptor gene (GHRHR) and insulin-like growth factor 2 mRNA binding protein 1 gene (IGF2BP1).GO and KEGG enrichment analysis showed that the DEGs were mainly involved in the pathways related to cell communication,such as cell adhesion molecules and neuroactive ligand-receptor interaction signaling pathways.GH,GHRHR and IGF2BP1 genes were also significantly enriched in neuroactive ligand-receptor interaction signaling pathway.The results of Real-time quantitative PCR showed that the accuracy and reliability of the transcriptome sequencing.The research revealed the key candidate genes and signal pathways for the difference in growth and development between Tibetan chicken and White-feather broiler,and provided a theoretical basis for further understanding of the regulatory mechanisms for growth and development of pituitary.

The aim of this study was to clone and bioinformatics analyze cya gene of Salmonella Typhimurium.cya gene was amplified from the Salmonella Typhimurium SL1344 strain by PCR and the sequence was cloned into pET-32a (+) vector for expression in Escherichia coli BL21 (DE3) cells and induced by addition of 1 mmol/L IPTG.At the same time,online bioinformatics software was used to analyze its bioinformatics.The results showed that cya gene from Salmonella Typhimurium was 1 185 bp.SDS-PAGE and Western blotting results showed that Cya expressed in the prokaryotic expression system was a inclusion body protein with a molecular weight of about 58 ku.Bioinformatics analysis results showed that cya gene of Salmonella Typhimurium was encoding a protein of 394 amino acids.The theoretical molecular weight of Cya protein was 44.97 ku,the molecular formular was C₂₀₂₆H₃₁₄₆N₅₆₂O₅₇₈S₁₁,and the isoelectric point(pI) was 7.01.The total number of negatively charged residues (Asp+Glu) and positively charged residues (Arg+Lys) were 43 and 42,respectively.Cya protein was a membrane protein without signal peptide and transmembrane region,containing 4 N-glycosylation sites,1 O-glycosylation sites,27 phosphorylation sites,13 linear B-cell epitopes and 11 T-cell epitopes.The secondary structure of Cya protein contained alpha helix (38.07%),extended chain (19.80%),beta turn (5.58%) and random coil (36.55%).The results laid a foundation for further study on the function of Cya protein of Salmonella Typhimurium and the establishment of an immunoassay method using Cya protein as antigen.

This study was aimed to knockdown the embryonic development related genes SUV39H1/SUV39H2 in porcine embryonic fibroblasts (PEFs) by CRISPR/Cas13d system,so as to establish a CRISPR/Cas13d mediated gene knockdown system in pigs.According to the coding sequences of SUV39H1/SUV39H2 genes,3 sgRNAs were designed and synthesized in the form of single stranded oligonucleotides.After annealing,they were connected with the sgRNA expression vector linearized by BSPQ Ⅰ.The sgRNA-expressing vectors of SUV39H1 and SUV39H2 genes sgRNA were constructed and sequenced by Sanger software.The expression vector of Cas13d and sgRNA vector targeting SUV39H1/SUV39H2 genes were transfected into PEFs at the ratio of 1∶1,1∶2,1∶4,2∶1 and 4∶1.After 48 h,the cells were collected,and the transfection efficiency was detected by flow cytometry.The knockdown efficiency was detected by semi-quantitative PCR and Real-time quantitative PCR.Semi-quantitative PCR and immunofluorescence were used to detect the level of target gene transcripts and histone H3K9me3 after SUV39H1/SUV39H2 genes knockdown.Sequencing results showed that 3 sgRNAs for each of two genes were successfully inserted into the vector.Flow cytometry results showed that the transfection efficiency was about 70%.Semi-quantitative PCR results showed that 3 sgRNAs extremely significantly reduced the expression of SUV39H1/SUV39H2 genes compared with control group (P<0.01).SUV39H1-sgRNA-2 and SUV39H2-sgRNA-1 could reduce the expression of SUV39H1 and SUV39H2 by 50%.The survival rate of Cas13d∶sgRNA in 1∶1,1∶2 and 1∶4 groups were higher than that of 2∶1 and 4∶1 groups.The knockdown efficiency of Cas13d∶sgRNA in 1∶2,1∶4,2∶1 and 4∶1 groups were significantly higher than that of 1∶1 group (P<0.05),and the knockdown efficiency of Cas13d∶sgRNA in 1∶2 group was the highest (70%).Semi-quantitative PCR results showed that transfection of SUV39H1-sgRNA-2 and SUV39H2-sgRNA-1 extremely significantly reduced the expression of SUV39H1 and SUV39H2,and the expression level was 25%-30% of control group (P<0.01).The results of immunofluorescence showed that the level of histone H3K9me3 decreased significantly after SUV39H1 and SUV39H2 genes knockdown (P<0.05).Therefore,in this study,CRISPR/Cas13d system was used to knockdown SUV39H1 and SUV39H2 successfully,and downregulate H3K9me3 level catalyzed by them.

The purpose of this study was to explore the gene function of ephrin A5(EFNA5) in Chinese Red steppe,and to detect the difference of EFNA5 gene expression in different tissues of Chinese Red steppe by bioinformatics analyzed.Primers were designed according to the predicted sequence of EFNA5 gene in GenBank (accession No.:NM_001076432.1).Verification of complete coding sequence of EFNA5 gene was obtained by PCR and sequencing.Using analysis software to carry out sequence similarity alignment and construct phylogenetic tree.The corresponding amino acid sequence was obtained.The physical and chemical property and subcellular structure was analysed by online prediction software.Protein hydrophilicity,hydrophobicity and phosphorylation sites were analysed.Protein secondary structure was predicted and protein tertiary structure model was constructed.EFNA5 gene expression in tissues of Chinese Red steppe was detected by Real-time quantitative PCR.The results showed that the EFNA5 gene of Chinese Red steppe had the highest similartiy with common cattle and American bison (100% and 99.8%),and with sheep and human were 95.6% and 97.2% respectively,and lower similartiy with dolphins,chickens,horses,rhesus monkeys,mice and pigs (83.6%,85.8%,84.5%,84.9%,84.1% and 82.8%,respectively).The CDS of EFNA5 gene was 687 bp,encoding 228 amino acids.The molecular formula of the protein was C₁₁₈₃H₁₇₉₁N₃₁₅O₃₃₉S₁₃,the molecular weight was 26.266 ku,the theoretical isoelectric point was 5.97.The protein instability index was 49.66,and the total average hydrophilicity was －0.313. Phosphorylation sites prediction analysis showed that there were 27 phosphorylation sites in EFNA5 protein.The results of subcellular localization showed that EFNA5 protein was mainly located in cytoplasm (26.1%),secretory system vesicles (27.1%),nucleus (13.0%),mitochondria (13.0%),plasma membrane (8.7%),endoplasmic reticulum (4.3%),golgi apparatus (4.3%),cytoskeleton (4.3%) and vacuole (4.3%).The main forms of secondary structure were α-helix (18.9%),β-turn (30.2%),β-fold (26.4%) and irregular crimp (24.5%),which were consistent with the prediction of tertiary structure.The expression of EFNA5 gene was the highest in kidney and stomach,and the least in lung.There was significant difference in different tissues of Chinese Red steppe (P<0.05).This study could provide reference for further investigation of the effects of EFNA5 gene on lipid metabolism in livestock and the screening of quality candidate genes in Red steppe.

The purpose of the experiment was to compare the differences of rumen tissue structure,especially the changes of ultrastructure of ruminal papillae in Hu lambs at different weaning age,so as to provide the basis for determining the suitable weaning age of Hu lambs.Twenty-four newborns of Hu sheep with birth weight of 2.80 kg±0.08 kg were randomly divided into three groups,8 lambs in each group,weaning at 30 days old (EEW-30),45 days old (EW-45) and 60 days old (NW-60),respectively.Four lambs in each group were slaughtered at corresponding weaning time and at the age of 75 days,respectively.The body weight and rumen weight were measured,and the ruminal relative weight was calculated.The rumen was sampled for morphological measurement.The results showed that:①The body weight of lambs at weaning time in EEW-30 group was extremely lower than that in NW-60 group (P<0.01),but there was no significant difference between EW-45 and NW-60 groups (P>0.05),and neither was any significant difference of the body weight among 3 groups at 75 days old (P>0.05).The change of the ruminal relative weight in 3 groups was in accord with that of the body weight.②Under the light microscope,the tunica mucosa thickness of rumen in EEW-30 group was significantly lower than NW-60 group (P<0.01),and there was no significant difference between EW-45 group and NW-60 group (P>0.05) at weaning age.At 75 days,the tunica mucosa thickness of rumen in EW-45 group was significantly higher than that in NW-60 group (P<0.05).At weaning age,the rumen papillae of EEW-30 group was extremely significant shorter than that in NW-60 group (P<0.01),and EW-45 group was also significantly shorter than NW-60 group (P<0.05).However,there was no significant difference in the width and area of rumen papilla at different weaning time and at day 75 (P>0.05).③Under scanning electronic microscope,the number of ruminal micro papillae structure per μm² at weaning age in EEW-30 group was extremely significantly lower than that in NW-60 group (P<0.01),but there was no significant difference between EW-45 group and NW-60 group (P>0.05).At the age of 75 days,there was no significant difference in the number of ruminal micro-papillae structure per μm² among the three groups (P>0.05).The results showed that there was significant differences in ruminal morphology between EEW-30 and NW-60 groups at weaning age,while EW-45 and NW-60 groups were more similar,and the change trend of ultra-microstructure of ruminal micro-papillae was the same.It was suggested that weaning at 45 days of age was more appropriate.

Organoids are derived from self-organizing and self-renewing stem cells,they are cell clusters formed after 3D culture in vitro using the self-organizing properties of stem cells,they are closely related to the source organs and reproduce the 3D cell structure of the source organs.In addition,they provide a new model for exploring the pathogenesis of the source organ.The organoid system is derived from cells cultured in vitro by adding extracellular matrix (ECM) substrates,small molecules,and growth factors to medium and regulated by autocrine,paracrine or adjacent secretory signals.The interaction of these factors creates a dynamic environment that guides the self-renewal,differentiation and the self-assembly of stem cells in organoids.The organoids which derived the reprogramming method by induced pluripotent stem cell (iPSC) combined with 3D organoid,establishes contact between animal models and human clinical trials.It is a complement to study cell experiments in vivo.Compared with traditional cell culture methods,organoids provide the new models for studying developmental biology and pathophysiology of the source organ in vitro.It is also important in the field of regenerative medicine and personalized medicine.In the future,maybe it will be used to research the mechanism of action of nutrient,drugs,poisons and toxins,as well as used in the fields of drug screening and regenerative medicine.The development of organoid technology has enhanced people’s understanding of the physiological and biochemical functions of organs and tissues.In this article,the author introduces the culture and application of organoids such as intestine,brain,lung,liver,uterus,ovary,etc.which provides reference for the scientific research and application of organoids.

The purpose of this study was to investigate the effects of transportation on muscle injury and oxidative stress in Ili horses.Five 24-month old Ili mares with similar body weight were selected.The transportation test was carried out after 7 days of environmental adaptation,no food or water was allowed during transportation.The total transportation distance was about 400 km and the transportation time was 8 hours.The blood samples were collected from horse jugular vein at 0 (T1),4 (T2),8 (T3) of transportation and 12 h (T4) after unloading.The serum samples were determined for related physiological and chemical indicators.The results showed that compared with T1,the muscle damage biochemical markers myoglobin (MYO) and cardiac troponin T (cTnT) increased significantly at T2(P<0.05),and increased extremely sinificantly at T3 and T4 (P<0.01).Lactic dehydrogenase(LDH) increased significantly at T3(P<0.05);The biochemical markers of inflammation C-reactive protein (CRP),interleukin-6 (IL-6) increased extremely significantly at T2,T3 and T4 (P<0.01).Cortisol(COR) increased extremely significantly at T2 and T3 (P<0.01),and increased significantly at T4 (P<0.05).Total antioxidant capacity (T-AOC),glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) decreased extremely significantly at T2,T3 and T4 (P<0.01).Catalase (CAT) decreased significantly at T3 and T4 (P<0.05).Reactive oxygen species (ROS) increased significantly at T2 and T4 (P<0.05),and increased extremely significantly at T3 (P<0.01).Malondialdehyde (MDA) increased significantly at T3 and T4 (P<0.01).Therefore,transportation could cause muscle damage and oxidative stress in Ili horses,and the adverse effects could not be eliminated within 12 h.

In recent years,the Semen Cuscutae was often used in the treatment of reproductive endocrine diseases in clinic,and the effect is remarkable.Semen Cuscutae is a kind of convolvulaceae plant,which has the advantages of small side effects,no drug resistance,rich in a variety of nutrients,low cost and so on.It is a pure natural medicinal plant.The total flavonoids of Semen Cuscutae is the most effective component in Semen Cuscutae,which has the functions of preventing diarrhea,nourishing liver and kidney,improve sperm quality,enhance male function and pacifying fetus,etc.It is not only regulates reproductive endocrine activities of male and female animals,but also has pharmacological effects on immune,cardiovascular and cerebrovascular systems.Reproductive endocrine activities in animals are mainly regulated by the hypothalamus-pituitary-gonadal axis,including the secretion of reproductive hormones,spermatogenesis and follicular development,which play an important role in animal reproduction.Most of the animal reproductive endocrine diseases are linked to the hypothalamus-pituitary gonadal axis adjustment by the developmental state of the reproductive hormones and reproductive organs,reproductive hormones can cause hypothalamus-pituitary-gonadal axis functional disorders by direct effect and indirect effect.Given that hormonal drugs are currently prohibited in economic animals,there is an urgent need to study the effects and mechanisms of safe,effective and low-toxic chinese herbal medicines to lay the foundation for their wide application.In this paper,the main pharmacological effects of total flavonoids of Semen Cuscutae were described,and the regulatory effects of total flavonoids of Semen Cuscutae on different levels of endocrine activity of hypothalamus-pituitary-gonad axis were described in detail.The purpose of this study is to provide theoretical reference for further study on the target and mechanism of total flavonoids of Semen Cuscutae on the regulation of hypothalamus-pituitary-gonad axis.

The skeletal system is crucial to the life activities of the organism,it provides the hematopoietic micro-environment,mechanical support,organ protection and other functions,as well as a reservoir of calcium and other minerals.The phenotypic diversity of chicken tailbone is a good resource for study of skeletal phylogeny and mechanism.To understand the molecular mechanism of skeletal developmental termination in the Rumpless chicken,this paper summarized the development of skeletal,the skeletal research in Rumpless chicken,and focus on the key signaling pathways of Notch and Wnt that influence the characteristics of rumpless and research progress of the Rumpless chicken.Target genes of Notch and Wnt signaling pathways and their interactions can form the characteristic of periodic expression,which is a molecular pathway regulating body segment formation and tailbone extension.However,the precise regulation of the interaction between the receptor and ligand and the core elements in the signaling pathway are still unclear,and more functional genomic and proteomic technology are needed for further study.

In order to explore the effect of feeding modes on fatty acid composition in muscle and adipose tissue of Tan sheep lambs,24 healthy 2-month-old Tan sheep lambs with similar weight (14.16 kg±0.58 kg) were randomly divided into house feeding group and grazing group.12 of each group were slaughtered at 4 months of age,and the longissimus dorsi muscle,biceps femoris,diaphragm,subcutaneous fat,perirenal fat and tail fat were collected for the determination of fatty acid contents by gas chromatograph.The results showed that:①In saturated fatty acids of muscle tissue,compared with the grazing group,the contents of C4∶0 and C16∶0 in biceps femoris of Tan sheep lambs in the house feeding group were significantly increased (P<0.05),and C8∶0,C12∶0,C14∶0 in the diaphragm content were significantly reduced (P<0.05).In unsaturated fatty acids of muscle tissue,compared with the grazing group,the contents of C16∶1,C20∶4n6 in longissimus dorsi muscle of Tan sheep lambs in the house feeding group were significantly reduced (P<0.05),C18∶3n6,C18∶3n3 content significantly increased (P<0.05),C20∶5n3,n-3 PUFA extremely significantly increased (P<0.01).Compared with the grazing group,the content of C20∶4n6 in biceps femoris of Tan sheep lambs in the house was significantly reduced (P<0.05),and the contents of C17∶1,C18∶3n3,C20∶5n3 and n-3 PUFA were extremely significantly increased (P<0.01).Compared with the grazing group,the content of C16∶1 in diaphragm muscle of the house feeding group was significantly reduced (P<0.05).② Among the saturated fatty acids in adipose tissue,compared with the grazing group,the contents of C11∶0 and C15∶0 in subcutaneous fat of the house feeding group were significantly reduced (P<0.05),and the content of C13∶0 was extremely significantly reduced (P<0.01).Compared with the grazing group,the contents of C10∶0 and C14∶0 in perinephric fat of Tan sheep lambs fed in the house were significantly reduced (P<0.05),and the contents of C4∶0 and C18∶0 were extremely significantly increased (P<0.01).Compared with the grazing group,the content of C11∶0 in tail fat of Tan sheep lambs fed in the shed significantly decreased (P<0.05).Among the unsaturated fatty acids in adipose tissue,the content of C18∶2n6t in the subcutaneous fat of Tan sheep lambs fed in the house was extremely significantly increased (P<0.01),and the contents of C14∶1,C16∶1 and C18∶3n6 were extremely significantly reduced (P<0.01).Compared with the grazing group,the contents of C18∶1n9t,C18∶3n3 and n-3 PUFA in perinephric fat of Tan sheep lambs fed in the house were significantly increased (P<0.05),and the content of C14∶1 was significantly decreased (P<0.05),the contents of C17∶1,C20∶2 and C18∶2n6t increased significantly (P<0.01).Compared with the grazing group,the contents of C14∶1 and C16∶1 in tail fat of Tan sheep lambs in the shed were significantly reduced (P<0.05),and the content of C18∶2n6t was extremely significantly increased (P<0.01).In conclusion,fatty acid contents in muscle and adipose tissue of Tan sheep lambs were affected by different feeding modes.The content of saturated fatty acid in biceps femoris muscle and unsaturated fatty acid in perirenal fat of Tan sheep lambs fed in the house were significantly higher than that of grazing Tan sheep lambs.

To study the difference of blood physiological and biochemical indexes of Jilin sika deer in different growth stages,and provide basis and reference for health testing,disease control and sika deer breeding,a total of 358 blood samples from 93 lactating deer (37 males and 56 females),135 growthing deer (40 males and 95 females) and 130 adult deer (42 males and 88 females) in Zuojia,Shuangyang and Dongfeng areas were collected.13 blood physiological indexes and 19 biochemical indexes were detected.The results showed that there was no significant difference in physiological and biochemical indexes between lactating male deers and lactating female deers,except erythrocyte system,blood glucose and creatine kinase (P<0.05).There were significant differences in hematocrit,mean volume of red blood cells,mean hemoglobin content,mean hemoglobin concentration,alkaline phosphatase and lactate dehydrogenase between males and females in growthing deer and adult deer (P<0.01).Among the male deer at different growth stage,there were significant differences in aspartic acid aminotransferase,prealbumin,total bile acid and blood glucose (P<0.05),extremely significant differences in erythrocyte system indexes,creatine kinase,calcium,inorganic phosphorus and creatinine (P<0.01) and no significant difference in albumin and amylase (P>0.05).Among the female deer at different growth stages,total lymphocyte,the total number of red blood cells,neutrophils were significantly different (P<0.05),the total number of white blood cells,red blood cell system indicators,aspartate amino transferase,alanine aminotransferase,creatine kinase indexes of inorganic phosphorus,calcium were extremely significantly different (P<0.01),and no significant difference in glutamyl transpeptidase,total bile acid,albumin (P>0.05).It was indicated that the physiological and biochemical indexes in sika deer were irregular and significantly different at different growth stages and sex,and some of the differences might be related to the biological characteristics of sika deer such as antler and sex difference.The reference range of physiological and biochemical indexes obtained in difference gender sika deer at different growth stages provided a certain theoretical basis for seed selection and breeding,disease diagnosis,development and utilization of resources of sika deer,and also provided reference basis for protection and health assessment of wild Northeast sika deer resources.

This experiment was conducted to study effects of long-term Lactobacillus reuteri 1 (LR1) on the expression of enzymes involved in protein digestion in pancreas and stomach,antioxidant capacity and amino acid profile of meat in pigs.A total of 144 cross-bred (Duroc×Landrace×Yorkshire) weaned piglets (average body weight 6.49 kg±0.04 kg) at 21 days were randomly assigned to 3 groups.The diet in control groups was basal diet,the diet in antibiotic group was basal diet supplemented with antibiotics (100 mg/kg olaquindox plus 75 mg/kg aureomycin for weaned piglets,and 75 mg/kg aureomycin for growing and finishing pigs),and the diet in LR1 group was basal diet supplemented with 5×10¹⁰ CFU/kg LR1.The experiment lasted for 175 days.The results showed as follows:① Antibiotics decreased the expression of elastase in pancreas compared to control (P<0.05),LR1 increased pepsinogen A in antrum,cardia and fundus of stomach in pigs compared to control and antibiotics (P<0.05).② Compared to control group,antibiotics increased activities of total axtioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) in longissimus dorsis muscle (LDM) (P<0.05).LR1 did not affect malondialdehyde (MDA) content,activities of T-AOC、SOD and glutathione peroxidase (GSH-Px)in LDM of pigs (P>0.05).③ Both antibiotics and LR1 did not affect significantly total amino acids profile following hydrolysis (P>0.05).Antibiotics decreased free methionine (bitter amino acids),total content of sweet amino acids and free amino acids compared to chilled fresh pork at 24 h in contron group (P<0.05),and LR1 decreased methionine (P<0.05).Antibiotics and LR1 decreased the content of sweet,bitter,essential amino acids and total free amino acids (P<0.05),additionally,antibiotics decreased the content of flavor amino acids of chilled fresh pork at 48 h (P<0.05).In conclusion,long-term LR1 improved digestive function of protein in stomach and flavor of chilled fresh pork at 24 h of pigs.Long-term antibiotics decreased digestive function of pancreas,improved antioxidant capacity of muscle,but decreased flavor of chilled fresh pork at 24 h and 48 h of pigs.

The aim of this study was to investigate the effects of walnut green husk and its extract extracted with 25% ethanol on intestinal morphology,mucosal antioxidant activity and microbial diversity of Yellow-feather broilers.144 1-day-old male Yellow-feather broilers were randomly divided into 3 groups with 12 replicates in each group and 4 chickens in each replicate.The control group was fed with basic diet,and the experimental groups Ⅰ and Ⅱ were fed the basic diet with 1% walnut green husk powder or the extract until 70 days old.At the end of the experiment,one chicken was randomly selected and sacrificed from each replicate,small intestine was taken,and villi height and crypt depth of duodenum,jejunum and ileum were measured.The activities of total superoxide dismutase (T-SOD),copper zinc superoxide dismutase (CuZn-SOD),manganese superoxide dismutase (Mn-SOD),total antioxidant capacity (T-AOC),catalase (CAT) and glutathione reductase (GR) in duodenal mucosa were measured.All ileal contents of each chicken were collected and 16S rDNA high-throughput sequencing technology was used to analyze the differences of intestinal microbial diversity and relative abundance.The results showed that,compared with the control group,adding walnut green husk and its extract had no significant effect on villus height,crypt depth and villus height/crypt depth ratio of duodenum,jejunum and ileum (P>0.05).The activities of T-SOD,CuZn-SOD,Mn-SOD,CAT and T-AOC of duodenal mucosa were not significantly affected (P>0.05).After adding walnut green husk and its extract,the number of observed microbial species in ileum increased significantly (P=0.05),the index of microbial diversity and species richness somewhat increased (P>0.05).Firmicutes was the dominant phylum in ileum of broiler in control group,more than 99% of the total bacteria.The addition of walnut green husk and its extract somewhat decreased the relative abundance of Firmicutes,and increased that of Bacteroidetes,but the differences were not significant (P>0.05).Lactobacillus,the dominant genus in the ileum of Yellow-feather broilers in control group,accounting for 71% on average,somewhat decreased by walnut green husk and the extract (P>0.05).Lactobacillus aviarius,Lactobacillus salivarius, Lactobacillus agilis,Lactobacillus ingluviei and Lactoba cillus reuteri were the dominant species in the ileum of Yellow-feather broilers in control group.The abundance of Lactobacillus aviarius was significantly increased after adding walnut green husk and the extract (P<0.05),and the Lactobacillus agilis was significantly increased after adding the extract (P<0.05).It could be concluded that adding 1% walnut green husk or its extract to broiler diet did not affect intestinal morphology and mucosal antioxidant capacity,but intestinal microbial diversity and species richness increased,and the abundance of beneficial bacteria such as Lactobacillus aviarius and Lactobacillus agilis increased.

The experiment was conducted to investigate the effects of peNDF in the diet on ruminating,digestibility,production performance,and rumen fermentation of Holstein bulls,to provide a basis for the formulation of TMR with suitable content of peNDF in fattening bulls.30 healthy Chinese holstein bulls (no castrated) at 11 months of age and weighted 345.57 kg±23.53 kg had been selected and randomly divided into 3 groups,groupⅠ,Ⅱ,and Ⅲ,feeding peNDF_8.00 at the content of 12.40%,13.75%,and 14.85%,respectively.The results showed that the diet intake frequency of group Ⅰwas significantly higher than group Ⅱ and Ⅲ (P<0.05),while the regurgitation time and the regurgitation time per kg DM and NDF of group Ⅱ and Ⅲ was significantly higher than group Ⅰ (P<0.05).There was no significant difference in DMI,the peNDF intake,ADG,and F/G,as well as the apparent digestibility of DM,CP,NDF,ADF,Ca,and P among 3 groups (P>0.05).Group Ⅲ had the significantly higher rumen pH but the significant lower propionic acid content than the others.The acetic acid concentration,ration of acetic acid to propionic acid (A/P),and the NH₃-N concentration of group Ⅱ and Ⅲ in rumen were significantly higher than group Ⅰ (P<0.05).In the present experiment conditions,3 different peNDF_8.00 content of the diet did not affect the product performance and the apparent digestibilities of nutrients of Holstein bulls,meanwhile,feeding by the peNDF_8.00 content of 13.75% and 14.85%,the fattening Holstein bulls could have a better rumen fermentation performance with the increasing rumination time,the acetic acid concentration,A/P,and the NH₃-N concentration.

The purpose of this study was to investigate the effects of adding different levels of DHA-rich microalgae to the diet on the growth performance,body measurement index,slaughter performance,and serum antioxidant indices of Qaidamford cattle.A total of 24 healthy 24-month-old Qaidamford bulls were randomly assigned to three groups with 8 cattle per group.GroupⅠ was taken as a control group and fed with basal diets.Groups Ⅱ and Ⅲ were supplemented with 100 and 200 g/d DHA-rich microalgae powder in the basic diet,respectively.The pre-experimental period lasted for 10 days,and the experimental period lasted for 42 days.Growth performance,body size indicators,slaughter performance related indicators,and total antioxidant capacity (T-AOC), superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content in serum were determined.The results showed that,①Compared with the control group,the average daily gain of groupⅡ was significantly higher (P<0.05),the feed-to-weight ratio were significantly lower of groups Ⅱ and Ⅲ (P<0.05).The body size and slaughter performance had no significantly change among three groups (P>0.05) ②Serum T-AOC and SOD activitie were significantly increased (P<0.05),and the content of MDA (P<0.05) was significantly reduced by adding DHA-rich microalgae to the diet.The GSH-Px activity of group Ⅲ was significantly higher than that of the control group (P<0.05).In conclusion,adding 100 g/d of DHA-rich microalgae to the diet significantly improved the feed conversion rate and serum antioxidant capacity of Qaidamford cattle under the experimental conditions.

In order to explore the genetic effects of solute carrier family 4 member 9 (SLC4A9) expression and polymorphism on the eggshell quality in duck,the expression levels of SLC4A9 gene in different tissues of Sansui ducks were detected by Real-time quantitative PCR,the SNP of SLC4A9 gene in Sansui ducks were directly sequenced by PCR products,and the genetic effect of SNP on the eggshell quality in Sansui ducks was analyzed.The results showed that the SLC4A9 gene mRNA was expressed to varying degrees in 10 tissues of Sansui ducks,with highly specific expression in heart and pancreas,moderate expression in kidney,and low expression in others.A total of 10 SNPs were detected among which three were located on exons and all were missense mutations(g.6803 C>T,g.7065 C>T and g.7089 C>G),and seven were located in introns (g.7162 C>G,g.11044 G>A,g.11090 T>C,g.11234 C>T,g.11261 C>T,g.11349 C>T and g.11403 G>A).Among them,g.6803 C>T mutation led to alanine (GCG) turning into valine (GTG),g.7065 C>T mutation caused alanine (GCC) to valine (GTC),g.7089 C>G mutation caused alanine (GCA) to glycine (GGA).χ² test found that g.6803 C>T,g.11090 T>C and g.11349 C>T deviated from Hardy-Weinberg equilibrium (P<0.05),and the genotype distribution of the other SNPs were in accordance with Hardy-Weinberg equilibrium (P>0.05).Correlation analysis results showed that the eggshell strength of CC and CT genotypes at g.11349 C>T was significantly higher than that of TT genotype (P<0.05).In conclusion,10 SNPs of SLC4A9 gene all affected the eggshell quality of Sansui ducks,and g.11349 C>T reached a significant level,which could provide reference for the improvement of eggshell quality and the selection of genetic markers in ducks.

The purpose of the experiment was to study the difference of stride index and stride frequency index between different performance of young Yili horses,and explore the correlation between the two indexes and performance,so as to improve the scientificity and accuracy of individual selection and performance measurement of Yili horses.In total,72 Yili horses aged 2 years were enrolled in this study.The stride characteristics and body size index were measured to calculate the stride length index and stride frequency index.The differences of stride length index and stride frequency index between different distance and performance were analysed,and made partial correlation analysis between each index and racing time,and probed into the correlation between each index and performance.The results showed that in the 1 600 m race,withers height stride frequency index and body length stride frequency index of the elite group were extremely significantly higher than these variables of the ordinary group (P<0.01) and significant negative correlate with racing time (P<0.01).The withers height stride length index,body length stride length index,withers height overlap stride length index and body length overlap stride length index in the elite group were extremely significantly higher than these variables in the ordinary group (P<0.01),and were significant negative correlated with racing time (P<0.01).In the 3 600 m race,the withers height stride frequency index and body length stride frequency index of the elite group were extremely significantly higher than these variables of ordinary group (P<0.01),and body length stride frequency index were significant negative correlated with the racing time (P<0.01),withers height overlap stride length index and body length overlap stride length index of the elite group were significantly higher than that of the ordinary group (P<0.05),and negatively correlated with the racing time (P<0.05).The withers height stride frequency index and body length frequency index were filtrated as primary indices for young Yili horses,and could be widely used in breeding and training to improve the efficiency of performance evaluation.

The combination of cryopreservation of semen and artificial insemination technology plays an important role in the improvement of cattle breeds and the protection of excellent germplasm resources.Despite the motility of frozen bovine sperm is more than 60%,the damage and deficiency of frozen sperm are common,especially the fertilization ability is significantly lower than that of fresh sperm.This article introduced the main damage caused by freezing thawing process to bovine sperm,including the morphological integrity,vitality and genetic material changes of sperm.The main reasons of sperm damage caused by freezing were discussed,including the formation of ice crystals and oxidative stress in cells,and the possible mechanism.The common methods to improve the quality of frozen sperm,such as adding cryoprotectants,optimizing freezing procedures and adding antioxidants,were reviewed in detail.The main issue of sperm cryopreservation was discussed,and this article would provide a theoretical basis for enhanced cryopreservation of livestock semen.

In order to provide basic data for genetic selection,breeding and fine housing of Beijing-You chickens,the females from pure line of Beijing-You chickens were used to identify and compare the feather development,growth and reproductive performance of early and late feathering hens.According to the relative length characteristics of the primary feathers and the coverts,534 newborn female chicks were identified and divided into early and late feathering subgroups.For which,the early feathering included K1 (primary feather is at least 5 mm longer than the covert feather) and K2 (primary feather is 2-5 mm longer than the covert feather).The late feathering included M1 (the primary feather length is equal to or maximumly 2 mm longer than the covert feather),M2 (the primary feather is shorter than the covert feather) and M3 (the primary feather is not growing out).PCR method was used to verify the female chicks at 6 week of age.Before 7 d of age,the length of primary feathers and coverts were measured every other day,and every other week from 7 to 42 d.Body weight was measured every week from 1 to 8 weeks,and every other week from 9 to 18 weeks.The egg production performance including age at the first egg,egg number until 43 and 52 weeks of age,clutch traits were calculated according to the individual egg production records.The results showed that identification of early and late feathering chickens using phenotype characteristics and PCR method was consistent.The early and late feathering accounted for 25% and 75% of Beijing-You chickens,respectively.The majority of late feathering hens were M2 type,and there were a few M1 and M3 type.The primary feather length of early feathering chickens was higher than that of late feathering chickens within 21-day old (P<0.05).At 28 d,there was no significant difference between M1 and M2(P>0.05),but M3 was still shorter than that of early feathering chickens (P<0.05).Compared with late feathering,the body weight of early feathering Beijing-You chickens was heavier after 70 d of age (P<0.05),and the age at the first egg was significantly earlier (P<0.05),but the egg number of 43 weeks was lower (P<0.05).Except egg shape index of early feathering hens was higher than that of late feathering ones (P<0.05),there was no significant difference in other egg quality traits.In all,the results in this study indicated that the growth and egg production performance were different in early and late feathered Beijing-You females,and there should be differences in breeding direction and production management,including performing selection of early sex maturity,which was the age of the first egg in the late feathered line and adjusting the dietary energy and protein level of the early feathered line.

The purpose of this study was to investigate the association of the polymorphisms of myosin heavy chain 3 (MYH3) and myosin heavy chain 8 (MYH8) gene with growth traits in Yanbian Yellow cattle.In this study,the genetic variation of MYH3 and MYH8 genes were analyzed by DNA direct sequencing method and high resolution melting curve in 120 heads of Yanbian Yellow cattle,and the correlation analysis was conducted based on the growth character data of Yanbian Yellow cattle.The results showed that a T>G mutation at locus 29602748 of the MYH3 gene was detected,containing two alleles:T and G,and three genotypes:TG,TT and GG.One C>T mutation at locus 29406042 of MYH8 gene was detected,in which there were two alleles:C and T,and three genotypes:CT,CC and TT.Correlation analysis showed that the T>G mutation at locus 29602748 of MYH3 gene significantly affected the height at hip cross and hip width of Yanbian Yellow cattle at 24 months age,that the height at hip cross of GG genotype was significantly higher than TT genotype (P<0.05),and the hip width was significantly higher than TT and TG genotypes (P<0.05).The C>T mutation at locus 29406042 of MYH8 gene significantly affected the chest circumference and body weight of Yanbian Yellow cattle at 24 months age,indicating that the body weight of CT and TT genotypes were significantly higher than CC genotype (P<0.05).Moreover,The chest circumference of TT genotype was significantly higher than CC genotype (P<0.05).In summary,the results of this study indicated that the polymorphisms of MYH3 and MYH8 genes were related to some growth traits of Yanbian Yellow cattle and could be used as candidate genes for marker-assisted selection in modern beef cattle breeding.

The purpose of this study was to analyze the changes of intestinal microflora in ducks infected with Riemerella anatipestifer (RA) in different periods,and to explore the effect of Riemerella anatipestifer on intestinal microflora and the possible pathogenic mechanism.Using universal primers of 16S V4-V5 region of bacteria,the DNA of duck rectal contents in Riemerella anatipestifer uninfected group (Riemerella anatipestifer infected 0 day group) and Riemerella anatipestifer infected 1,2,3,5,9 and 14 days were amplified.The amplified products were sequenced on Ion S5^TMXL sequencing platform after DNA library construction.The total number of Raw Reads was 4 710 688,with an average of 84 119 sequences per sample.The total number of OTUs annotated to the database was 4 528.Alpha diversity analysis showed that Shannon and Simpson indexes had no significant difference between Riemerella anatipestifer infected groups and uninfected group (P>0.05).The indexes of Chao1,Ace,Observed-species and PD-whole-tree decreased gradually from 0 to 5 d after infection,and increased gradually from 5 to 14 d after infection,especially at 3 and 5 d after infection,which were significantly lower than those in uninfected group (P<0.05).Beta diversity analysis showed that there were significant differences between the uninfected group and the infected groups at 6 time points (P<0.05).The species difference between the uninfected group and 3 d after infection was the largest,followed by 2,5,9,1 and 14 d after infection.The cluster heat map of species abundance showed that the microbial genera with relatively high species abundance were different in the uninfected group and the infected groups at different time points.At the phylum level,Firmicutes,unidentified-Bacteria,Bacteroidetes,Fusobacteria and Proteobacteria were the main pathogens in Riemerella anatipestifer infection groups.At the genus level,Bacteroides was the dominant genus in all infected and uninfected groups.The number of Campylobacter and Fusobacterium in infected groups increased significantly.In this experiment,the intestinal microflora data of Riemerella anatipestifer uninfected group and different infection time groups were obtained.The differences between the infected group and uninfected group were analyzed and compared.Some characteristic bacteria were found,which could provide theoretical basis for the pathogenicity research of Riemerella anatipestifer.

In order to understand the biological characteristics of Lactobacillus phage,and provide experimental data and reference for the prevention and control of phage pollution during fermentation production,in this study,Lactobacillus phages were isolated from dairy products and kimchi samples by spot test and double-layer agar plate method,L.casei ATCC 393,L.pentosus KLDS 1.0413 and L.brevis ATCC 367 were used as indicator bacteria.The morphology of the phage was observed by transmission electron microscopy,and the packaging mechanism of the phage was analyzed by the restriction enzyme activity of the phage genome,the host range and one-step growth curve were measured,the structural proteins of the phage were analyzed by SDS-PAGE to analyse the biological characteristics of the obtained phage.The results showed that three virulent Lactobacillus phages were isolated and named as Lc,Lpen and Lbre,respectively.The results of electron microscopy showed that phage Lc and Lpen were consisted of polyhedral heads and non-contractive tails,while phage Lbre was consisted of polyhedral heads and contractile tails.According to morphology analysis,Lc and Lpen belonged to the Siphoviridae family,B1 category,and Lbre belonged to the Myoviridae family,A1 category.The genomes of phages were extracted and subjected to restriction endonuclease.The results of nucleic acid electrophoresis showed that the genomes of phage Lc and Lpen both had heterogeneous blunt ends,and the packaging mechanism was full-head packaging,namely pac-type,while Lbre contained stickiness end,the packaging mechanism belonged to cos-type.The phage Lc and Lbre were host-specific,and the host spectrum of phage Lpen was broader.The one-step growth curve showed that the latent period of Lc,Lpen and Lbre were 60,45 and 150 min respectively,the burst period were 45,90 and 105 min respectively,and the burst size were 47,24 and 30 PFU/cell respectively.SDS-PAGE testing indicated that phage Lc had 7 structural proteins,while phage Lpen,Lbre had 5 structural proteins,their protein molecular weight were about 50,55 and 50 ku,respectively.In conclusion,three virulent Lactobacillus phages were isolated and their biological characteristics were indentified,which provided important experimental data and theoretical basis for understanding the prevention and control of virulent Lactobacillus phages contamination.

To explore the genetic variation and phylogenetic evolution of Haemaphysalis flava (H. flava) collected from different geographical areas in China,a total of 20 H. flava from Hunan and Henan provinces were used in this study,their internal transcribed spacer Ⅱ (ITS-2) and NADH dehyd ogenase subunit Ⅰ (nad1) sequences were amplified by PCR,and the genetic variations in the obtained sequences were analyzed.Moreover,the phylogenetic relationship of H. flava from two provinces was analyzed,and two phylogenetic trees were established using the obtained ITS-2 and nad1 genes sequence.The results showed that the intra-specific sequence variations of ITS-2 and nad1 genes within H. flava from Hunan and Henan provinces were 0 to 1.9% and 0 to 0.4%,respectively.The intra-specific sequence variations of ITS-2 and nad1 genes within H. flava from Hunan province were 0 to 1.4% and 0 to 0.4%,respectively.The intra-specific sequence variations of ITS-2 and nad1 genes within H. flava from Henan province were 0 to 1.2% and 0 to 0.4%,respectively.The inter-specific sequence variations of ITS-2 and nad1 genes of H. flava were 37.0% to 52.0% and 13.6% to 24.8%,respectively.The phylogenetic analysis showed that all samples are on a clades in two phylogenetic trees established by ITS-2 and nad1 genes sequence.The results indicated that there was genetic variation between H. flava from Hunan and Henan provinces,but the genetic variation and genetic diversity of H. flava from Henan province were higher than that from Hunan province.The phylogenetic relationship of H. flava from Hunan and Henan provinces was closed.The results suggested that there was gene exchange within H. flava from Hunan and Henan provinces,and no genetic differentiation between all samples.

In order to understand the bovine infection with D’Aguilar virus (DAGV),BHK21 cells were used to isolate the virus from the blood samples of healthy cattle collected in Jinghong city,Yunnan province in 2019.The samples with cytopathy were identified by morphology,genomic banding and molecular biology.The sequences of S2,S3 and S7 genes were determined and compared.The results showed that five samples of CPE were found.The complete virus particles were observed by electron microscope,it was about 50 nm in diameter.The results of agarose gel electrophoresis showed that the genome of 5 newly isolated viruses was 10 segments,and the banding pattern of the virus genome was characterized by 3-3-4 electrophoresis.The banding pattern of the viruses were similar to that of the DAGV V106/YN/2014 strain isolated in Yunnan province in 2014.The nucleotide and amino acid sequences of S2,S3 and S7 genes of the five strains were 100%,the S3 and S7 genes had the highest homology with PALV isolated in China,and the S2 gene had the highest homology with DAGV isolated from Japan.Genetic analysis of S2,S3 and S7 genes showed that the similarity of the five newly isolated strains were 100%.The S7 and S3 genes were in the same branch with some known PALV strains isolated from China,indicating that these five strains were PALV.The S2 gene of five strains was located in the same branch as some DAGV strains isolated from Japan.It was further confirmed that these five strains were PALV DAGV.In this study,five strains of DAGV were successfully isolated,and the genetic evolution analysis of gene fragments were carried out to provide the basis for further epidemiological study of DAGV.

The aim of this study was to investigate the effects of PGD₂/DP1 receptor pathway on the expression of inflammatory mediators HMGB-1 and PAFR,and the degree of tissue injury in the endometrium of dairy cows infected with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).The endometrial tissues of cows were treated with 1×10^-6 mol/L DP₁ receptor agonists (BW-245C and 15d-PGJ2) and infected with E. coli and S. aureus at a concentration of 1×10⁶ CFU/mL.The expression of HMGB-1 and PAFR was detected by Real-time quantitative PCR,immunohistochemistry,and the tissue injury was evaluated by HE staining.The results showed that the expression of HMGB-1 and PAFR of endometrium tissue in dairy cows was significantly higher than that in blank control group after E. coli and S. aureus infection at a concentration of 1×10⁶ CFU/mL (P<0.05),while the expression of HMGB-1 and PAFR in bovine endometrium was significantly inhibited by DP₁ receptor agonists (P<0.05).HE staining showed that the endometrial epithelial cells and glandular epithelial cells was completely exfoliated,necrotic and disintegrated after E.coli and S.aureus infection.While the DP₁ receptor agonists treated bovine endometrium,it significantly reduced the degree of injury of bovine endometrium (P<0.05).The results of immunohistochemical staining were consistent with those of the above two methods.The results showed that PGD₂ could inhibit the expression of HMGB-1 and PAFR and reduce the degree of tissue damage in the bovine endometrium infected with E.coli and S. aureus,which might be mediated by DP₁ receptor.

There are many kinds of zoonotic parasites with a wide range of hosts and serious damage.Schistosomiasis,echinococcosis,cysticercosis,trichinosis and toxoplasmosis are common parasitic zoonoses.Humans and livestock suffer from parasitic diseases,which have a significant impact on public health and livestock farming.Controlling the source of infection,cutting off the route of transmission and protecting susceptible groups are the comprehensive prevention and control measures to control the epidemic of parasitic zoonoses.In particular,vaccinations is one of the ideal and effective ways to cut off parasites’ life cycle,and control and even eliminate parasitic zoonoses.It is the premise and key to develop a vaccine to select an effective antigen screening method to excavate the potential candidate molecules for vaccine.With the development of antigen screening technology,more new antigens and protective peptides have been discovered.Existing antigen screening methods mainly include the traditional crude antigen screening method,cDNA library screening method,proteomics screening method,bioinformatics and multi-omics technology screening method.Many methods of antigen screening were developed along with the process of parasite vaccine research.The crude antigen screening method is based on the immunological principle of antigen-antibody interaction,and natural antigen screened by this method can cause a strong immune response.The advantage of cDNA library screening is that the screening is more targeted,so the composition of the candidate product is more single and clear.Proteomic screening technique is based on mass spectrometry,which can not only identify the unknown proteins,but also compare the differences of the identification results.It plays an important role in the discovery of unknown molecules and the screening of target molecules with special functions.With the advent of the post-genetic era,the combined screening technology of bioinformatics and multi-omics makes antigen screening turn into a multi-dimensional screening mode,and also makes the functional studies of candidate antigens and their epitopes more in-depth,which provides a technical means for the screening of candidate molecules of genetically engineered vaccines and polypeptide vaccines.

The purpose of this study was to evaluate the immunoprotective efficacy of all 9 members of Pasteurella glycolytic enzymes as an antigen.Firstly,the glycolytic enzyme genes of Pasteurella multocida C51-17 strain were amplified by PCR and linked to pET-28a(+) plasmid.The recombinant plasmid was transferred into Escherichia coli BL21(DE3) strain and induced to express overnight.The recombinant proteins of Pasteurella glycolytic enzymes were purified from the supernatant of the collected strain after ultrasonic crushing.After SDS-PAGE,the Pasteurella glycolytic enzymes were transferred to PVDF membranes,and the recovered sera from C51-17 strain infection was incubated.ECL staining was performed after incubating the second antibody.At last,C57BL/6 mice were immunized with glycolytic enzymes.After high level of specific antibody was produced in the immunized groups,C51-17 strain was used to challenge.Sequencing results showed that the genes of all 9 members of the Pasteurella glycolytic enzymes were successfully cloned into pET-28a(+) plasmid,and the recombinant protein was obtained after induced expression and purification.Western blotting results showed that phosophoglucose isomerase (PGI),triose phosphate isomerase (TPI),glyceraldehyde-3-phosphate dehydrogenase (GPD),phosphoglycerate kinase (PGK),and pyruvate kinase (PYK) could react with the recovered sera from Pasteurella multocida C51-17 strain.The results of immunoprotection test showed that PGI,aldolase (ALD),GPD,PGK,phosphoglycerate mutase (PGM) and enolase (ENO) had some protective effects.In conclusion,the results of this study showed that some members of the Pasteurella glycolytic enzymes had a certain degree of immune protection,which provided a new basis for the prevention and control of pasteurellosis.

This study was aimed to establish the artificial disease model of Equine herpesvirus type 1 (EHV-1),and confirm the half infective dose (ID₅₀) of equine infected with EHV-1 and the criteria for determining the incidence of infection,and lay the foundation for prevention of the disease and therapeutic drugs.Taking EHV-1 XJ2015 strain isolated from the aborted fetuses of a racetrack in Yili area of Xinjiang as the research object,4 groups of different virus dose challenge and control group were set up,and 5 mL of virus was sprayed into each horse’s nasal cavity.The clinical symptoms and pathogenesis of the horses were observed every day.14 days later,the pathological changes of tissues and organs were observed and the nasal detoxification and virus distribution were detected by Real-time PCR method.The results showed that the ID₅₀ of EHV-1 XJ2015 strain was 10^-6.67/5 mL for the horse,and the virus content was 10^4.33 TCID₅₀/mL.Compared with control group,the clinical scores of horses in 1×10⁶ and 1×10⁵ TCID₅₀/mL challenge groups increased significantly,high body temperature (up to 39.5 ℃,generally lasting for 2-6 days),loss of appetite,fluid nasal and mandibular lymphadenopathy.And the test horses in 1×10⁶ and 1×10⁵ TCID₅₀/mL challenge groups showed different levels of detoxification,a large number of viruses could be detected in the lung and brain tissues compared with control group (P<0.01;P<0.05).Histopathological examination revealed non-suppurative encephalitis and neuronal edema in the brain tissue of the horse,interstitial pneumonia,neutrophils,inflammatory cell infiltration,hemorrhage,and alveolar septal thickening occur in lung tissue.The above results showed that EHV-1 XJ2015 strain had strong pathogenicity to horses,the clinical symptoms of diseased horses were typical,the virus was mainly excreted with nasal fluid,and enriched in lung and brain tissue,and the criteria of EHV-1 infection in horses could be determined through the above indexes.The disease model of EHV-1 infected animals was successfully established in this experiment.

In order to explore the morphological and biological characteristics of coliphages,55 strains of avian pathogenic Escherichia coli stored in the laboratory were used as the host bacteria in this study.A coliphage with a wide lysis spectrum was obtained from the sewage and sludge of a chicken farm by the double-layer agar plate method,named Bp16,after negative staining with 2% phosphotungstic acid,the morphology and structure were observed by transmission electron microscope.The temperature and pH stability,optimal multiplicity of infection (MOI),and one-step growth curve were measured by the double-layer agar plate method.Transmission electron microscopy showed that the isolated broad lysis spectrum phage Bp16 had an icosahedral structure in the head,with tails and filaments,and the body length was about 220 nm.According to the classification characteristics of Virus Classification—The 9th Report of the International Committee on Classification of Viruses,the bacteriophage was a member of the Caudovirales and the Mycobacteriophages family.Using 55 avian pathogenic Escherichia coli stored in the laboratory as indicator bacteria,its cracking efficiency was only 12.73%.The results of temperature stability showed that the titer of the phage remained basically unchanged at 37-60 ℃ for 30 min or 1 h,and the titer could still reach more than 1×10⁴ PFU/mL after treatment at 80 ℃ for 1 h,indicating that the phage was not sensitive to temperature changes and had heat resistance.The results of pH stability showed that the titer remained basically unchanged between pH 5.0 to 10.0,and it was relatively tolerant to acid and alkali.The results of the best MOI showed that the best MOI was 1.One-step growth curve according to the best MOI was drew,and it was measured that the incubation period was 20 min,the outbreak period was 50 min,and the lysis amount was 73 PFU/cell.In summary,the bacteriophage Bp16 had thermal stability,acid-base stability and strong lysis ability,which laid the foundation for the development of coliphage preparations for subsequent experiments.

In order to investigate the differential expression profile of circular RNA (circRNA) in the lung tissue of pigs infected with Mycoplasma hyopneumoniae (Mhp) as well as its role in Mhp infection.Jiangquhai pigs were used as subjects in this experiment,which were divided into Mhp-infected and control groups.On the 28th day post inoculation,the lung tissues were collected from all of the experimental pigs for analysis of circRNA expression by high-throughput sequencing and bioinformatics software.Data comparison showed that a total of 23 632 circRNAs were identified,with 213 differentially expressed circRNAs(DEcircRNAs).Among them,97 up-regulated and 116 down-regulated.Four from 213 DEcircRNAs were randomly selected and verified by Real-time quantitative PCR,and the results were consistent with those obtained by sequencing.Host genes of DEcircRNAs were annotated to immune-related signaling pathways,including antigen processing and presentation,lysosome and leukocyte transendothelial migration.The target relationship analysis of circRNA-miRNA-mRNA showed that three DEcircRNAs with the highest number of sponge miRNAs were circRNA-17284 (21),circRNA-04848 (19) and circRNA-17270 (19),of which 6 targeted miRNAs related to immune regulation were predicted,including ssc-miR-4331,ssc-miR-370,ssc-miR-328,ssc-miR-30c-3p,ssc-miR-122 and ssc-miR-125b.In this study,the expression profile of circRNA in the lung tissue of pigs infected with Mhp was determined,and the DEcircRNAs related to immune regulation was screened,which helped to clarify the mechanism of susceptibility of Jiangquhai pigs to Mhp infection,and provide scientific basis for disease-resistant breeding research.

This study was aimed to explore the effect of compatibility of Rehmannia glutinosa and Aristolochia on the liver and kidney of chickens,and provide modern theoretical basis for the compatibility and detoxification of traditional Chinese medicine.80 male chickens were randomly divided into 10 groups:Control group,Aristolochia high,medium and low dose groups,Rehmannia glutinosa high,medium and low dose groups,Aristolochia with Rehmannia glutinosa high,medium and low dose groups.After decocting,filtering and centrifuging,the impurities were removed and the decoction was made.The drug was given by gavage every day for 28 days.The liver and kidney indexes of AST,ALT,Cr and BUN, and T-AOC,SOD,GSH-Px and MDA were detected by kit.The liver and kidney were collected and sectioned to observe the histopathological changes.The results showed that compared with Aristolochia group,the contents of AST,ALT,Cr and BUN were significantly decreased (P<0.05),and the contents of T-AOC,SOD and GSH-Px were significantly increased after compatibility of Rehmannia glutinosa.The combination of Rehmannia glutinosa and Aristolochia could significantly reduce the content of MDA (P<0.05),and slow down the oxidative damage of Aristolochia.The pathological results showed that the compatibility of Rehmannia glutinosa and Aristolochia could significantly reduce the pathological damage of liver and kidney caused by Aristolochia decoction.In conclusion,the combination of Aristolochia and Rehmannia glutinosa could alleviate the liver and kidney injury in mice,and improve the drug safety of Aristolochia.

To find out the cause of mink death in Hebei,pathogen identification was conducted by histopathological examination,bacterial isolation and culture,morphological observation,biochemical identification,drug susceptibility test,drug resistance gene and virulence gene detection and animal regression experiment.The results showed that 3 strains of Pseudomonas aeruginosa were isolated,3 strains had 100% similarity with strains of Pseudomonas aeruginosa in GenBank,and all 3 strains could cause the death of the rats.Drug susceptibility test results showed that the isolates were highly susceptible to levofloxacin,gentamycin,amikacin,polymyxin B and ceftriaxone,and were resistant to extended-spectrum β-lactamases (ESBLs),aminoglycosides,macrolides,quinolones and tetracyclines.The isolates carried ESBLs genes (bla_CTX-M1,bla_OXA-2,bla_OXA-10),carbapenemases (bla_VIM-1,bla_SPM-1,bla_KPC-1),and 17 virulence-related markers including adhesion,T1SS-T3SS,oxidativestress,quorum sensing and regulation,and phospholipids.The results would provide references for disease control,therapeutical guidance and drug administration in mink.

The study was aimed to investigate the effect of Passiflora edulis polysaccharide extract (PEPE) on oxidative stress-related factors of RAW264.7 cells infected with Porcine circovirus type 2 (PCV2) and explore the role of PEPE on oxidative stress.First,100 μL RAW264.7 cells of 1×10⁶ cells/mL were added into 96-well cell culture plate in each well.Cell control group and PEPE groups (25,50,100,200,400,800 and 1 600 μg/mL) were respectively set up and cultured in the incubator for 24 and 48 h,respectively.MTT assay was used to detect cell activity and screen the safe concentration range of PEPE.The experiment was then divided into the following six groups:Cell control group,virus group,PEPE high (400 μg/mL),medium (200 μg/mL) and low (100 μg/mL) dose groups and vitamin C group,among them,the cell control group was added with 10% FBS-containing DMEM culture medium,and the other groups were added with PCV2 virus liquid.After incubating for 2 h,the corresponding concentration of PEPE solution were added to the PEPE groups,the prepared VC solution was added to the VC group,and DMEM culture medium containing 10% FBS was added to cell control group and virus group,and cultured for 48 h.MTT assay was also used to detect cell activity to study the effect of PEPE on the activity of RAW264.7 cells infected with PCV2.Finally,in accordance with the above 6 test component groups,the treatment was the same as above,the cells were cultured for 12 h,and samples were collected to detect the levels of oxidative stress related factors.Griess method and DCFH-DA fluorescent probe label method were used to analyze the levels of nitric oxide (NO) and reactive oxygen species (ROS) in RAW264.7 cells supernatant.The levels of glutathione (GSH) and oxidized glutathione (GSSG) were analyzed by OPT fluorescence assay.The activities of xanthine oxidase (XOD),myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) were analyzed by chemiluminescence technique.The results of this study showed that the safe concentration range of PEPE on RAW264.7 cells was 25-400 μg/mL.After PCV2 infected RAW264.7 cells,the cell viability was significantly reduced (P<0.05),and the PEPE group with different concentrations could improve the cell viability.The secreting levels of NO and ROS,GSSG content and activities of XOD,MPO and iNOS were significantly increased (P<0.05),while GSH content was significantly decreased (P<0.05) after PCV2 infected RAW264.7 cells.When PCV2-infected cells were treated with PEPE,the production of NO,ROS,GSSG,as well as the activities of XOD,MPO and iNOS were significantly decreased (P<0.05),while the level of GSH in 100 μg/mL PEPE treatment group was significantly increased (P<0.05).The results showed that PEPE enhanced antioxidant capacity of PCV2-infected RAW264.7 cells,which was conducive to easing the oxidative stress caused by virus infection.

The puopose of the study was to analyze the effect of aroA gene on the biological characteristics and virulence of Salmonella Enteritidis.The aroA gene of Salmonella Enteritis isolated from poultry in the field was knocked out using λ-Red homologous recombination.The genetic stability of the gene deletion strain was tested by continuous passages.The differences between the wild strain and the gene deletion strain were compared by bacterial growth curve,the utilization of different biochemical reaction tubes,the ability of biofilm formation,the resistance to environmental stress,virulence to 1-day-old chicks.The results showed that the gene deletion strain was successfully constructed,and no revert mutation was found in the target gene after 30 successive generations.The deletion strains entered the logarithmic growth phase and stable phase at the same time compared with the wild strains under the same culture conditions,but the growth rate of the deletion strains was lower than that of the parent strains.The biochemical characteristics of the deletion strains were not changed compared with the wild strains.The ability of biofilm formation and resistance to acid,alkali,high osmolar and heat stress of the deletion strain were significantly lower than those of the wild strain (P<0.05).1-day-old chicks were inoculated through the mouth with the deletion strain and the wild strain respectively.All the chickens in the wild strain group were died after 14 days of observation,while none in the deletion strain group.In conclusion,the aroA gene deletion strain of Salmonella Enteritis with good genetic stability was successfully constructed and obtained in this experiment.The biochemical characteristics and in vitro growth trend of the deletion strain were not significantly changed,but the biofilm formation ability,resistance to environmental stress and virulence were significantly decreased.This study laid a foundation for the further study of the characteristics and immunogenicity of aroA gene in Salmonella Enteritis.

This experiment was aimed to study the effects of oxidative stress on the offspring of mice.The male and female mice were injected 12.5 μg/g 3-nitropropionic acid (3-NPA) intraperitoneally,which were divided into normal male mice and normal female mice of control group (C),normal male and the 3-NPA treated female (FM),normal female and the 3-NPA treated male mice (MM) and 3-NPA treated male mice and the 3-NPA treated female mice mating group (DM) for 3 times.The number of progeny fetal mice was counted by the method of secting fetal mice.The heart,liver and lung of the progeny fetal mice were collected and the activities of superoxide dismutase (SOD),catalase (CAT),glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) of the progeny fetal mice were determined.The results showed that compared with the control group,the number of fetal mice in FM group was not significantly decreased (P>0.05),while the number of fetal mice in MM and DM groups was significantly decreased (P<0.05).The CAT activity in liver and lung of fetal mice in FM and DM groups was significantly increased (P<0.05).The CAT activity in liver and lung of fetal mice in MM group was increased,but the difference was not significant (P>0.05).CAT activity in heart increased in all groups,but there was no significant difference (P>0.05).T-AOC activity in liver,lung and heart was significantly increased (P<0.05),except for in lung in MM group (P>0.05).The activity of GSH-Px in fetal liver of DM group was significantly increased (P<0.05),and increased in FM and MM groups,but the difference was not significant (P>0.05).The activity of GSH-Px in lung of fetal mice in FM and DM groups was significantly increased (P<0.05).There was no significant difference in GSH-Px activity among all groups in heart (P>0.05).The activity of SOD in liver,lung and heart of fetal mice in DM group was significantly increased (P<0.05),and increased in liver,lung and heart in FM and MM groups,but the differences were not significant (P>0.05).Therefore,the oxidative stress mouse model of 3-NPA was successfully established in this study,and the oxidative stress of parental male mice had a greater effect on fetal mice.

To explore the feasibility and safety of Vitex negundo L.var.cannabifolia (Sieb.et Zucc.)Hand. -Mazz.(V.negundo) as a traditional Chinese veterinary medicine,the acute toxicity test and 30-day feeding test were carried out on V.negundo water extract.In the acute toxicity experiment,20 SPF Kunming mice were randomly divided into drug group and blank control group.The drug group was intragastrically administered at a crude drug concentration of 40 mg/g body weight,and the blank control group was intragastrically administered with an equal volume of normal saline for continuous observation for 7 days.Repeat the test once more.The 30-day feeding test used 80 SPF SD rats,which were randomly divided into four groups,high (40 mg/g body weight),medium (20 mg/g body weight) and low (10 mg/g body weight) dose groups,and blank control group,half male and female in each group.Drug feeding were continuously administed for 30 days.Weighed once a week,blood was collected after weighing on the 30th day to test hematological and blood biochemical indicators,organs were weighed to calculate the organ coefficient,the main organs of high-dose group and the blank control group were fixed and then were made slices to observe the tissue lesions.The results showed that LD₅₀ of V.negundo water extract was >40 000 mg/kg in the acute toxicity test.In 30-day feeding test,the overall condition of the rats was good throughout the test period.The body weight of high-dose group was extremely significantly lower than those in blank control group (P<0.01),there was no significant difference in other groups.The organ coefficients of spleen of male rats in the high-dose group and testis of male rats in middle-dose group were significantly higher than that in blank control group (P<0.05),the organ coefficient of testis of male rats in high-dose group was extremely significantly higher than that in blank control group (P<0.01),there was no significant difference in the organ coefficient between the groups of female rats.The number of monocytes in middle-dose and low-dose groups were extremely significantly and significantly higherthan that of blank control group (P<0.01;P<0.05),respectively,there was no significant difference between other hematological indexes and blood biochemical indexes (P>0.05),and they were all within the normal range.Histopathological examination showed no siginificant changes.It showed that V.negundo water extract had no obvious acute and subacute toxicity under the experimental conditions,and had high safety.V.negundo water extract could be used as a safe Chinese veterinary medicine for further development and utilization.