【Objective】 The objective of this study was to investigate the effects of Toll-like receptor 7(TLR7)gene knockdown on the replication of Vesicular stomatitis virus (VSV).【Method】 The stable porcine renal epithelial cells (PK15) with TLR7 gene knockout were constructed using lentivirus-mediated CRISPR/Cas9 gene editing technique.The recombinant plasmid pTLR7-sgRNA was constructed and transfected into HEK293T/17 cells.Lentivirus was harvested and infected with PK15 cells.PK15-TLR7^-/- polyclonal cells were obtained after screening by purinomycin.PK15-TLR7^-/- monoclonal stable cell lines were obtained by limited dilution method after Western blotting.To verify the successful construction of TLR7 gene stabilized cell lines, optical microscopy and cytotoxicity test (CCK-8) were used to observe and detect the differences in morphology and viability of PK15 and PK15-TLR7^-/- cells.The proliferation of VSV-GFP infected PK15 and PK15-TLR7^-/- cells was observed by inverted fluorescence microscope and flow cytometry.Western blotting and Real-time quantitative PCR were used to detect the expression of GFP protein and VSV-N gene mRNA in PK15 and PK15-TLR7^-/- cells infected with VSV-GFP.Virus titers were used to detect the progenitor virus production of PK15 and PK15-TLR7^-/- cells infected with VSV-GFP.【Result】 Three sgRNAs constructed by CRISPR/Cas9 gene editing technology could effectively edit TLR7, and sgRNA2 had the highest editing efficiency.However, TLR7 knockout did not affect the morphology and viability of PK15 cells.When infected with VSV-GFP, the fluorescence intensity increased with time, and the GFP fluorescence intensity of PK15-TLR7^-/- cells was stronger than that of PK15 cells.Flow cytometry results showed that the proportion of PK15-TLR7^-/- cells infected with VSV-GFP was significantly or extremely significantly higher than that of PK15 cells at the same time (P<0.05;P<0.01).Real-time quantitative PCR results showed that the mRNA relative expression of VSV-N gene increased gradually with time from 4 to 36 h after infection, and the mRNA relative expression of VSV-N gene in PK15 cells was significantly or extremely significantly lower than that in PK15-TLR7^-/- cells (P<0.05;P<0.01).Western blotting results showed that VSV-GFP GFP was expressed in PK15 and PK15-TLR7^-/- cells at 6 h, and the expression level increased gradually with time, and the content of VSV-GFP GFP in PK15-TLR7^-/- cells was higher than that in PK15 cells.After 6 h of VSV-GFP infection, the progeny virus was released and the titer of VSV-GFP progeny virus in PK15 cells was lower than that in PK15-TLR7^-/- cells (P>0.05).The virus titer of VSV-GFP progeny in PK15 cells was significantly or extremely significantly lower than that in PK15-TLR7^-/- cells with increasing infection time (P<0.05;P<0.01).【Conclusion】 TLR7 gene knockout could promote VSV replication in PK15 cells, which preliminarily verified the role of TLR7 in natural immunity, providing new ideas and strategies for the prevention and control of VSV and other RNA viral diseases.

【Objective】 This study was aimed to identify immune-related circRNA of thymus in chickens, and investigate the role of circRNA in immune regulation of chickens.【Method】 The transcriptome of thymus in Yellow-feather broilers and mountain Black-bone chickens was sequenced by high-throughput sequencing platform Illumina PE150.Bioinformatics software find_circ and CIRI were used to identify circRNA molecules and analysis their basic structural characteristics.Difference significant analysis was performed to screen differenttially expressed circRNA, GO function and KEGG pathway enrichment analysis conducted to reveal the immune-related circRNAs, and the potential translation ability were predicted by IRESfinder and PFAM softwares, and it was verified by Real-time quantitative PCR.【Result】 There was abundant expression of circRNA in thymus of chickens.A total of 8 015 circRNAs were identified, and 115 circRNAs were significant differentially expressed in thymus of chickens (log₂|FoldChange|>1, P<0.05), 54 circRNAs upregulated and 61 circRNAs downregulated in Yellow-feather broilers(mountain Black-bone chickens was used as control).The GO function enrichment showed that there were 104 GO terms related to immune, involved 23 differential expression circRNAs, up to 20%(23/115).KEGG pathway analysis showed that there were 7 immune related pathways, including cytokine-cytokine receptor interaction, RIG-Ⅰ-like receptor signal pathway and Toll-like receptor signal pathway, which covered 7 differentially expressed circRNAs.5 circRNAs came out of GO function and KEGG pathway enrichment analysis were identical:circ_0002478(IL-1R1), circ_0003208(MAPK11), circ_0001674(STX8), circ_0005105(RIPK2) and circ_0003590(BRAF), which might play an important role in the regulation of immune system.Real-time quantitative PCR results showed that 5 circRNAs difference expression trend in thymus of two chicken breeds were consistent with the sequencing.circ_0002478(IL-1R1) not only had extremely significant difference between the two varieties (P<0.01), but also had the potential to encode peptides, it might participate in immune regulation in many ways.【Conclusion】 circRNA was rich in thymus of chickens, the immune-related circRNA circ_0002478(IL-1R1) might affect immune regulation in many ways, and could be a key target gene in immunity research of chickens.

【Objective】 The purpose of this study was to amplify LIM domain gene 1 (LMCD1) in Nubian goats and analyze using bioinformatics, construct eukaryotic expression vector, and detect the expression of LMCD1 gene, so as to provide a reference for studying the function of LMCD1 gene in Nubian goats and exploring the role of LMCD1 gene in skeletal muscle development of goats.【Method】 Total RNA was extracted from the longissimus dorsi muscle in Nubian goats.The CDS region of LMCD1 gene in Nubian goats was amplified by RT-PCR and analyzed by bioinformatics, and LMCD1 gene was connected to pEGFP-N1 vector by homologous recombination.After enzyme digestion and sequencing identification, the positive plasmid was named pEGFP-N1-LMCD1 recombinant plasmid.pEGFP-N1-LMCD1 recombinant plasmid was transfected into skeletal muscle satellite cells in goats, the expression of LMCD1 gene in Nubian goats was detected by Real-time quantitative PCR.【Result】 The total length of LMCD1 gene CDS in Nubian goats was 1 092 bp, encoding 363 amino acids.The molecular formula of LMCD1 protein was C₁₇₇₅H₂₈₁₈N₅₀₈O₅₃₃S₂₉, and the molecular weight was 40.73 ku.The sequence of LMCD1 gene CDS in Nubian goats had the highest similarity with Capra hircus (99.8%) and the lowest similarity with Danio rerio (55.4%), and the similarity with other species was 87.0% to 98.8%.LMCD1 protein had no signal peptide and transmembrane domain, and was a hydrophilic protein.pEGFP-N1-LMCD1 eukaryotic expression vector in Nubian goats was constructed and transfected into skeletal muscle satellite cells.LMCD1 gene was overexpressed to produce green fluorescence signal.【Conclusion】 In this study, LMCD1 gene CDS in Nubian goats was amplified successfully, the pEGFP-N1-LMCD1 eukaryotic expression vector was constructed, and its biological function was analyzed, which provided a theoretical basis for the follow-up study of the mechanism of LMCD1 gene in skeletal muscle development of goats.

【Objective】 Analyzing the X chromosome of Bos grunniens and other subfamilies of cattle from the perspective of genome alignment and codon bias was helpful to understand the variety differences and phylogenetic status of Bos grunniens, and provided reference for its adaptation to high altitude low-pressure and hypoxia environment and codon optimization.【Method】 Taking the coding region sequence of Bos grunniens X chromosome reference gene as the reference panel, genome alignment and gene collinearity analysis were carried out with the coding region sequence of X chromosome reference gene of Bos taurus and Bubalus bubalis.At the same time, gene annotation was carried out.The filtered coding region files were analyzed for preference, such as relative synonymous codon usage (RSCU), ENC-plot, PR2-plot and optimal codon determination.【Result】 In the coding region of X chromosome gene of Bos grunniens, genes involved in tracheal contraction, lung respiration and body metabolism were found to be different from Bos taurus and Bubalus bubalis, such as KLHL13, CENPI and PGK1 genes.The collinearity results indicated that the long segment showed an exchange linear region due to strong natural selection and mutation pressure.In codon analysis, the codon usage bias of three species of bovine subfamily was similar, their bias was weak, and their codons were biased towards the end of G/C.Their strong biased codons (RSCU ≥ 1.5) were all CUG, GUG, AGA, AGG and UGA.Among them, 16 optimal codons were selected for Bos grunniens, 13 for Bos taurus and 9 for Bubalus bubalis.These codons all end in A/U.【Conclusion】 By comparing Bos taurus and Bubalus bubalis, Bos grunniens might be under greater natural selection, so its cumulative degree of variation in the process of evolution was also greater.The codon bias of Bos grunniens, Bos taurus and Bubalus bubalis were more affected by natural selection than mutation.These results would provide reference and guidance for Bos grunniens genetic breeding, codon optimization and the development and utilization of genetic resources.

【Objective】 The experiment was aimed to study the application potential of recombinant Adenovirus with E0 and E2 tandem genes of Bovine viral diarrhea virus (BVDV) as genetic engineering vaccine.【Method】 The recombinant Adenovirus was amplified by PCR, E0-E2 gene fusion was performed by overlapping extended PCR and the recombinant shuttle plasmid pDC316-E0-E2 was constructed, which was co-transfected into HEK293T cells with AdMax skeleton plasmid for packaging, and verified by Western blotting.The titer of the virus was determined by Reed-Muench method.The immune effect was studied by ELISA and flow cytometry after the mice were injected by muscle and subcutaneous injection.【Result】 The target fragments of E0 and E2 genes were successfully amplified, which were 681 and 1 122 bp, and the complete Adenovirus AD5-E0-E2 with titer of 1.1×10¹⁰ PFU/mL was obtained.Western blotting was used to detect the expression of Ad5-E0-E2 exogenous gene in HEK293T cells, and the target band (65 ku) was obtained, which was consistent with expectations.ELISA results showed that high antibody levels could be generated by both muscle and subcutaneous injection.Flow cytometry results showed that the ratios of CD4⁺ and CD4⁺/CD8⁺ in groups of intramuscular and subcutaneous injection Ad5-E0-E2 were both extremely significantly higher than that in PBS group (P<0.01).【Conclusion】 Adenovirus Ad5-E0-E2 was successfully constructed in this experiment, which had good reactivity and immunogenicity, and could induce the body to produce specific antibodies against BVDV.

【Objective】 The purpose of this study was to clone and prokaryotic expression of heme oxygenase-1 (HO-1) gene, a protective factor of porcine mucous membrane, so as to provide technical support for studying the protective effect of highly expressed HO-1 in intestinal mucosal injury.【Method】 According to the HO-1 sequence published in GenBank (accession No:NM_001004027.1), a pair of specific primers were designed using Primer Premier 6.0, and the HO-1 gene fragment was amplified by RT-PCR, and then linked to the pMD19-T cloned vector, the recombinant vector was transformed into E.coli DH5α, and the positive clones were screened and identified by PCR.T4 DNA ligase was used to link the double digestion (Sal Ⅰ and Kpn Ⅰ) products of pNCMO2 vector and pMD19-T-HO-1 recombinant vector.The recombinant expression vector pNCMO2-HO-1 was transferred into the receptive Bacillus pumilus by electric shock transformation technology, and induced expression was performed by IPTG.The fusion expression of HO-1 in Bacillus pumilus was analyzed by SDS-PAGE and Western blotting.【Result】 The total length of porcine HO-1 gene was 897 bp, encoding 298 amino acids.The gene expression vector pNCMO2-HO-1 was double digested by restriction enzyme Sal Ⅰ and Kpn Ⅰ, the pNCMO2 vector fragment and HO-1 gene fragment were observed at 5 200 and 897 bp, respectively, which proved that the gene expression vector pNCMO2-HO-1 was successfully constructed.After electric transfer, double enzyme digestion results showed that pNCMO2 and HO-1 fragments were observed in similar locations as above, which proved that the recombinant expression vector pNCMO2-HO-1 was successfully introduced into Bacillus pumilus.SDS-PAGE and Western blotting results showed that obvious protein imprinting was appeared at 36.5 ku, indicating that HO-1 recombinant protein was successfully expressed and secreted.【Conclusion】 The recombinant prokaryotic expression vector of HO-1 was successfully constructed, and the recombinant vector of pNCMO2-HO-1 could be induced to express in Bacillus pumilus.

【Objective】 The aim of this experiment was to study the effects of prolactin (PRL) on the growth and morphological changes of primary and secondary hair follicles in Inner Mongolia cashmere goats in vitro.【Method】 The primary and secondary hair follicles of Inner Mongolia cashmere goats were isolated by mechanical method combined with cutting method.The primary hair follicle culture medium was supplemented with 0, 5, 10, 50 and 100 ng/mL prolactin, respectively, with 24 roots in each group for 5 days in vitro culture.The morphology of the hair follicles was observed and photographed under a microscope every day.The growth length, growth rate and survival rate were calculated to screen out the optimal concentration of prolactin.Then, the primary and secondary hair follicles were divided into primary hair follicle control group (PF-K), primary hair follicle experimental group (PF-PRL), secondary hair follicle control group (SF-K) and secondary hair follicle experimental group (SF-PRL), with 24 hairs in each group.The control group was cultured with the basal medium, and the experimental group medium was supplemented with the optimal concentration of prolactin.The hair follicles were cultured for 5 days, the morphology of hair follicles were observed and took photos every day, and the growth length of each group was measured at the same time.【Result】 The average daily growth length of hair follicle in 10 ng/mL prolactin group was extremely significantly higher than that in other concentration groups, the final growth length and survival rate of hair follicles in 10 ng/mL prolactin group were the highest (P<0.01).Therefore, 10 ng/mL prolactin was selected for subsequent experiments.The stem and root sheath of primary/secondary hair follicles in experimental and control groups were elongated simultaneously, and curved to different degrees with the increase of culture time.The total length of hair follicles PF-PRL and SF-PRL groups at 2-5 days was significantly higher than that in PF-K and SF-K groups, respectively (P<0.01).The total length of hair follicles in PF-K group increased significantly from day 1 to 5, except that there was no significant difference between day 1 and 0 (P<0.05), the total length of hair follicles in PF-PRL group was increased significantly from 0 to 5 days (P<0.05).The total length of hair follicles in SF-K group on the 5th day was significantly higher than that in 0-4 days (P<0.05).The total length of hair follicles in SF-PRL group was significantly higher than 0-3 days on day 4 and 5 (P<0.05), and the total length of hair follicles on day 3 was significantly higher than 0-2 days (P<0.05).The average daily growth length of hair follicles in PF-PRL and SF-PRL groups was significantly higher than that in PF-K and SF-K groups (P<0.01).【Conclusion】 10 ng/mL prolactin was the optimal concentration to promote hair follicle growth in vitro.10 ng/mL prolactin could promote the growth of primary and secondary hair follicles of Inner Mongolia cashmere goats in vitro.

As the junction of the internal and external environment in the body, the intestine is susceptible to various toxic and harmful substances from outside, which leads to damage to the epithelial structure, function and microecosystem, and then reduces growth performance of animals and economic benefit.Intestinal stem cell (ISC)-driven intestinal cell renewal at the base of the crypt is the driving force for the epithelial structure and is necessary for the repair after intestinal injury.ISCs activity are synergistically regulated by various molecular signals in the ISCs microenvironment.And the Janu kinase/signal transducer and activator of transcription (JAK/STAT) pathway is a common pathway for multiple cytokine signals, which bind to tyrosine kinase-related receptors to activate JAK, and then phosphorylate STAT and trigger the transcription of target genes such as cycle-related and anti-apoptotic proteins to control ISCs proliferation, apoptosis, and differentiation fate and ultimately maintains the ordered structure of the crypt villi axis.In addition, helper T cells (Th cells) around the ISCs secrete interleukins (ILs) and interferons (IFNs) in crosstalk with the JAK/STAT signaling pathway in ISCs to achieve a rapid response of intestinal cells to injury stimuli, which accelerates ISCs division and promote intestinal epithelial regeneration.This review focused on the mechanisms of JAK/STAT-mediated ISCs in maintaining the structural and functional integrity of the intestinal epithelium and how Th cells regulate the differentiation of ISCs to functional cells.Meanwhile, the authors described abnormal changes in this signaling pathway during the occurrence of certain livestock and poultry diseases, and identify emerging research models such as intestinal organoids into the study and practice of animal intestinal development and disease diagnosis.The accurate control of JAK/STAT is expected to reveal the updating regularity of the crypt-villus axis and provide the theoretical basis for establishing the scientific nutritional technology scheme and improving intestinal health.

【Objective】 This study was aimed to explore whether puerarin could promote the differentiation of Songliao Black pig precursor adipocytes through the AMPK pathway, in order to provide a reference for adding puerarin to diet to improve pork meat quality.【Method】 When the confluence degree of precursor adipocytes of Songliao Black pig reached 90%, the differentiation was induced for 4 days.When inducing differentiation, the cells were divided into control group (Con), puerarin group (Pue), puerarin+AMPK activator AICAR group (AICAR) and puerarin+AMPK inhibitor CompoundC group (CompoundC).Puerarin was not added to the induction solution in the control group, while 40 μmol/L puerarin was added in the Pue group, 40 μmol/L puerarin +500 μmol/L AICAR were added in AICAR group, 40 μmol/L puerarin +20 μmol/L CompoundC were added in CompoundC group.After induction and differentiation, the cells of each group were collected and the triglyceride (TG)concentration was detected by triglyceride enzyme method, the expression levels of AMPK subunits and adipogenic differentiation related genes were detected by Real-time quantitative PCR, peroxisome proliferator activated receptor γ (PPARγ) AMP-dependent protein kinase (AMPK), p-AMPK (Thr-172), acetyl-CoA carboxylase (ACC) were detected by Western blotting.Autodock Vina 1.20 was used to predict the molecular docking of puerarin and AMPKα subunits.【Result】 The results of triglyceride determination showed that compared with Con group, the triglyceride concentration of Pue group was significantly up-regulated (P<0.05);Compared with Pue group, the triglyceride concentration of AICAR group was significantly decreased (P<0.05).The results of Real-time quantitative PCR showed that compared with Con group, the expressions of PPKAG2, PPKAB1, PPKAB2, PPKAA1 and PGC-1α in Pue group were significantly decreased (P<0.05), the expressions of C/EBPα, PPARγ and ACC in Pue group were significantly increased (P<0.05);Compared with Pue group, the expressions of PPKAG1, PPKAG2, PPKAB1, PPKAB2 and PPKAA1 in AICAR group were significantly increased (P<0.05), the expressions of C/EBPα, PPARγ and ACC in AICAR group were significantly decreased (P<0.05), the expressions of PPKAG1, PPKAG2 and PPKAB2 in CompoundC group were significantly decreased (P<0.05), the expressions of C/EBPα and ACC in CompoundC group were significantly increased (P<0.05).Western blotting results showed that compared with Con group, the expressions of PPARγ and ACC protein in Pue group were significantly up-regulated(P<0.05), the expressions of AMPK, p-AMPK protein were significantly down-regulated (P<0.05), and p-AMPK/AMPK was significantly decreased (P<0.05);Compared with Pue group, the expressions of PPARγ and ACC protein in AICAR group were significantly decreased (P<0.05), while AMPK and p-AMPK protein were significantly up-regulated (P<0.05), the expressions of PPARγ and ACC protein in CompoundC group were significantly increased (P<0.05), while the expressions of AMPK, p-AMPK protein and p-AMPK/AMPK were significantly decreased (P<0.05).【Conclusion】 40 μmol/L puerarin could significantly increase the content of triglycerides in adipocytes and significantly up-regulate the expressions of the adipogenic differentiation genes PPARγ, C/EBPα and ACC, significantly down-regulate the expressions of AMPK subunits.AMPK activator AIRCR could significantly inhibit the effect of puerarin, indicating that puerarin could regulate the adipogenic differentiation of Songliao Black pig precursor adipocytes by inhibiting AMPK pathway.

【Objective】 The objective of this study was to analyze the effect of different training stages on the performance and heart rate variability(HRV) of Yili horses and to provide data for reference in the conditioning training of 1 600 m distance Yili horses.【Method】 Eight 3-year-old Yili horses (stallions) were selected as test subjects.A 3-month speed specific performance training program was conducted, and a speed test race was organized in the last week of each month of training, and HRV was collected before, immediately after, 0.5 h after, and 1 h after 1 600 m test race.Among them, the time-domain indexes included the mean of all R-R intervals (Mean RR), the standard deviation of all R-R intervals (SDNN), the mean heart rate (Mean HR), the root mean square of the difference between adjacent R-R intervals (RMSSD), the number of adjacent R-R intervals with a difference greater than 50 ms (NN50), and the percentage of adjacent R-R intervals with a difference greater than 50 ms to the total number of heartbeats (pNN50).Frequency domain metrics included very low frequency (VLF), low frequency power (LF), high frequency power (HF) and nonlinear metrics:standard deviation (Y) of all R-R-spacing (SD1), standard deviation (X) of all R-R-spacing (SD2).Consequently, the variability of HRV indexes in horses at different training stages was analyzed.【Result】 The race time in the post training period in 1 600 m speed test race of Yili horses was significantly lower than that in the early training period (P<0.05), and Mean RR, NN50 and pNN50 in the middle and post training periods were significantly lower than that in the early training period (P<0.05).Mean HR in the beginning of training was significantly lower than that in post training (P<0.05).VLF and LF in the end of training were significantly lower than that in the beginning of training (P<0.05).【Conclusion】 Under the conditions of this test, the HRV indexes of 1 600 m test race of Yili horses in different conditioning training stages were analyzed.The outcomes demonstrated that the types of neural activity in Yili horses at the beginning, middle and end of training presented some differences, showing changes of increased sympathetic excitability and decreased parasympathetic excitability, and the horses' athletic performance improved.Therefore, HRV could be considered as an effective tool to evaluate the training load and intensity of Yili horses during training.

【Objective】 This study was conducted to investigate the effects of slow-release urea (SRU) and rumen-protected glucose (RPG) supplementation on performance, serum biochemical indices and rumen fermentation of heat-stressed sheep.【Method】 A total of forty 3-month-old Hu male lambs with similar body weight were randomly selected and were divided into 4 groups equally (n=10):Control group (CON group, fed a basal diet), slow release urea group (SRU group, fed a basal diet supplemented with 15 g/d slow release urea), rumen-protected glucose group (RPG group, fed a basal diet supplemented with 10 g/d rumen-protected glucose), urea and glucose group (UG group, fed a basal diet supplemented with 15 g/d slow-release urea and 10 g/d rumen-protected glucose).The feeding experiment was lasted for 55 days with 15 d of adaptation and followed by 50 d for trial period.Before morning feeding on the last day of normal feeding period, blood and rumen fluid were collected, and the contents of immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G (IgG), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) in blood samples were determined.pH, ammonia nitrogen, microbial protein and volatile fatty acid in rumen fluid were measured.【Result】 Compared with CON group, ①The respiratory rate of heat stress sheep was significantly decreased in both SRU and RPG groups, the rectal temperature of heat stress sheep was significantly decreased in UG group (P<0.05).②The final body weight and average daily gain of heat stressed sheep in SRU, RPG and UG groups were significantly increased (P<0.05), and the feed to gain ratio was significantly decreased (P<0.05).③The contents of IgG and IgM in serum of heat stressed sheep in RPG and UG groups were significantly increased (P<0.05), and the contents of TNF-α and IL-2 in serum were significantly decreased (P< 0.05).④The ration of acetic acid to propionic acid in the rumen was significantly increased in the three treatment groups (P<0.05), the content of total volatile fatty acid and proportions of acetic acid, isobutyric acid and isovalerate acid in the rumen were significantly increased in SRU group (P<0.05).The pH of rumen fluid and proportion of isovalerate acid were significantly increased in RPG group (P<0.05), while the content of ammonia nitrogen and proportions of propionic acid and butyric acid in the rumen were significantly decreased in RPG group (P<0.05).The contents of the proportion of acetic acid and total volatile fatty acid and microbial protein in the rumen were significantly increased in UG group (P<0.05).【Conclusion】 The growth performance and rumen fermentation could be improved, the stability of rumen environment was maintained, and heat stress was effectively relieved in lambs fed diets supplemented with slow-release urea and rumen-protected glucose.

Quercetin is a natural flavonoid organic compound, which is rich in natural resources, safe and non-toxic.Quercetin has many physiological functions:It can directly remove free radicals in cells, activate antioxidant enzyme system, inhibit signal transduction of oxidative stress pathway, and subsequently exert antioxidant function.It can limit the production of inflammatory factors by inhibiting the activation of inflammatory pathways and reducing the activation of white blood cells, and thus achieve the anti-inflammatory effect.It can inhibit the proliferation, migration and invasion of tumor cells, promote their apoptosis and finally play an important role in anti-tumor.It can exert antibacterial function by blocking the ways related to biofilm formation of potential pathogenic bacteria, such as the destruction of the cell wall and cell membrane structure, the synthesis of protein and nucleic acid and the binding of ATP.In the application of chickens production, quercetin can improve the growth of broilers by increasing the transport and absorption of nutrients in ileum and reducing the damage caused by intestinal oxidative stress.It can improve the laying performance by promoting the development of reproductive organs of laying hens and enhancing the secretion of reproductive and growth-related hormones.It can improve the product quality of eggs and chickens, regulate the metabolism of calcium, protein and lipid, improve the immune function of organism, and regulate the intestinal microflora.The authors reviewed the important physiological functions of quercetin, such as antioxidation, anti-inflammation and antibacterial actions and underlying mechanisms, as well as the application progress of quercetin in chickens production in recent years, in order to provide theoretical basis for promoting the application of quercetin in chickens production and the development in chicken feed.

【Objective】 This study was aimed to investigate the effects of mogrosides on improving the lipid metabolism abnormality in obese mice caused by high-fat diet, and explore its mechanism of action.【Method】 60 healthy male Kunming mice were randomly divided into 6 groups after being fed with high-fat diet for 4 weeks, including normal group, model group, simvastatin group, and high (200 mg/(kg·d)), medium (100 mg/(kg·d)) and low (50 mg/(kg·d)) dose mogrosides groups.The mogrosides dose groups were administered by gavage for 4 weeks while maintaining high-fat feeding.After the administration was completed, the body weight of mice in each group and the weight of adipose tissue were obained, and the fat coefficient was calculated.The contents of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in serum and liver, the contents of apolipoprotein A1 (ApoA1) and apolipoprotein B (ApoB) in serum, and the activities of lipoprotein lipase (LPL) and hepatic esterase (HL) in liver were measured.【Result】 Compared with blank group, the body weight, fat weight and fat coefficient of mice in model group were extremely significantly increased (P<0.01).Compared with model group, the fat weight and fat coefficient of mice in high and medium dose mogrosides groups were significantly reduced (P<0.05);The contents of TC and LDL-C in serum and liver of mice in high dose mogrosides group were significantly or extremely significantly reduced (P<0.05 or P<0.01), and the content of HDL-C in serum was extremely significantly increased (P<0.01);The content of ApoB in serum of mice in high and medium doses mogrosides groups was extremely significantly reduced (P<0.01), and the activities of LPL and HL in liver of mice were extremely significantly or significantly increased (P<0.01 or P<0.05).【Conclusion】 The mogrosides could prevent the increase of blood lipid, liver fat and body fat in obese mice induced by high-fat diet, its possible mechanism was by affecting the synthesis of ApoB and the activities of HL and LPL.The results provided a reference for the development of the mogrosides as a potential weight lowering and lipid lowering agent.

【Objective】 The aim of this study was to study the effects of glucomannan-modified montmorillonite and chitosan-modified montmorillonite on the growth performance and immune function of chicks.【Method】 252 8-day-old laying chicks were randomly divided into 7 groups with 6 replicates in each group and 6 chicks in each replicate.The chicks in group 1 were fed with basal feed, the chicks in group 2 were fed with mildewed feed, and the chicks in groups 3 to 7 were fed with mildewed feed supplemented with 0.15% glucomannan-modified montmorillonite, 0.15% chitosan-modified montmorillonite, 0.15% high purity nano montmorillonite, 0.15% glucomannan and 0.15% chitosan, respectively.The test period was 42 days.At the end of the experiment, the growth performance, the immune organ index, the plasma immunoglobulins and immune factors, the spleen immune factors of test chicks in each group were measured.【Result】 Compared with chicks fed with basal diet, the average daily gain, contents of IL-2, IL-4, IL-6 and IFN-γ in serum and spleen and contents of IgA, IgG and IgM of chicks fed with mildewed feed only were significantly decreased (P<0.05).Compared with chicks fed with mildewed feed only, the average daily gain of chicks fed with mildewed feed supplemented with 0.15% glucomannan-modified montmorillonite and 0.15% chitosan-modified montmorillonite were significantly increased, and the F/G and immune organ index were significantly decreased (P<0.05).The contents of IL-2, IL-4, IL-6 and IFN-γ in serum and spleen and the contents of IgA, IgG and IgM in serum of chicks were significantly increased (P<0.05).【Conclusion】 The immune function of chicks were decreased due to mildewed feed, and the two organic modified montmorillonite could alleviate the toxic effect of mycotoxins on chicks and improve the growth performance and immune function of chicks.

【Objective】 The purpose of this experiment was to study the effects of fermented traditional Chinese medicinal on the growth performance, antibody level and intestinal flora of layer hens.【Method】 84 1 day old Cobb laying hens were selected and pre fed to 13 days old.The formal experiment was conducted at 14 days old.The chickens were randomly divided into 7 groups, each group was raised in a single cage, with 12 per cage.They were blank control group (group A), compound traditional Chinese medicine group (group B), low-dose fermented traditional Chinese medicine group (group C), middle-dose fermented traditional Chinese medicine group (group D), high-dose fermented traditional Chinese medicine group (group E), fermentation broth group (group F) and control drug Astragalus polysaccharide group (group G).The daily feed and leftover was recorded at each stage during the test.The chicks were weighed on an empty stomach in the early morning on the 1st (14 days of age), 14th (28 days of age) and 28th (42 days of age) days, and the average daily gain (ADG), average daily feed intake (ADFI) and feed-to-gain ratio (F/G) were calculated at each stage.On the 7th, 14th, 21st and 28th days of the experiment, blood was collected from the veins of chicken wings in each group for the detection of antibody titer.At the end of the experiment, the chickens of groups A, B, D, F and G were subjected to necropsy, the cecal contents of 5 chickens in each group were subjected to high-throughput sequencing analysis.【Result】 During the 1st to 14th day, the ADFI of groups D and E were extremely significantly higher than that of groups A, F and G (P<0.01), the ADG of group E and D were extremely significantly or significantly higher than that of groups A and F (P<0.01 or P<0.05).The F/G of groups F and G were extremely significantly lower than that of other groups (P<0.01).During the 15th to 28th day, the ADFI of groups C and E were significantly higher than that of groups B and F (P<0.05), the ADG of group B was extremely significantly higher than that of other groups (P<0.01), and the ADG of group E was significantly higher than that of group A (P<0.05).The F/G of group B was extremely significantly lower than that of other groups, and the F/G of groups D, E, F and G were extremely significantly lower than that of group A (P<0.01).On the 7th day, there was no significant difference of serum antibody titer in chickens in each group (P>0.05).On the 14th day, the antibody level of group E was significantly higher than that of groups A, F and G (P<0.05).On the 21st and 28th days, the antibody level of group E was significantly higher than taht of group A (P<0.05).Group D had the highest Chao1 and Observed species indexes, but had no significant relationship with the other groups (P>0.05).At the phylum level, the relative abundance of Bacteroidetes in cecum of groups D and G were significantly higher than that of group A (P<0.05).At the genus level, the relative abundance of Bacteroides in cecum of group D was significantly higher than that of group A (P<0.05).【Conclusion】 Fermented traditional Chinese medicine could improve the growth performance and antibody level of chickens, and it also had a significant regulatory effect on the intestinal flora.

【Objective】 The composition of colostrum of Dulong cattle (Bos frontalis) and Yunling cattle were determined to obtain the difference of colostrum composition between these two breeds.【Method】 45 colostrum samples of Dulong cattle and Yunling cattle (under the same condition of age, parity and feeding management) were collected to determinate milk protein, milk fat, lactose, total solids (TS), non-fat solids (SNF), milk urea nitrogen (MUN), somatic cell counts (SCC) and freezing point (FP) on the first day after delivery.The independent t-test was subsequently utilized to compare the colostrum composition between Dulong cattle and Yunling cattle on the first day after postpartum.【Result】 The contents of protein, fat, TS and SNF in colostrun of Dulong cattle were 16.77%, 4.70%, 27.82% and 22.39%, which were 28.11% (P<0.01), 5.38% (P>0.05), 22.45% (P<0.01) and 26.93% (P<0.01) higher than those of Yunling cattle, respectively.The contents of lactose and MUN and SCC were 2.98%, 354.2 mg/L and 35.73×10⁴/mL, respectively, which were 10.38% (P<0.05), 39.63% (P<0.01) and 78.06% (P<0.01), respectively, lower than those of Yunling cattle.FP of Dulong cattle was -0.5337 ℃, which was 0.1383 ℃ (P<0.01) higher than that of Yunling cattle.Milk fat-protein ratio (FPR) was 0.266, which was 0.131 lower than that of Yunling cattle (P>0.05).【Conclusion】 The colostrum compositions of the two breeds were significantly different on the first day after postpartum, and the colostrum of Dulong cattle had notably higher nutritional value.

【Objective】 The purpose of this study was to explore the difference in nutritional composition of Lueyang Black-bone chicken muscles at different ages, providing theoretical support for determining the best listing date and scientific breeding.【Method】 1 200 Lueyang Black-bone chickens, which were born on the same day, were randomly divided into four replicates, with 300 chickens in each replicate.All chickens were caged on the net after being desuperheated at 42 days of age, and raised off the ground on the net after 90 days of age.The basic diet of each stage was special feed for poultry, and the whole process was free to eat and drink.Weekly body weight was recorded and Gompertz model was used to fit the growth curve.At 90, 120, and 150 days of age, 8 males and 8 females with similar body conditions were randomly selected and slaughtered by neck bloodletting, the breast muscles were collected to detect pH, shear force, moisture, dry matter, amino acid, fat, protein, inosinic acid and fatty acid content at different growth periods.【Result】 The body weight of Lueyang Black-bone chickens increased slowly from 1 to 4 weeks, entered the rapid growth period after 4 weeks, and gradually slowed down after 17 weeks.The Gompertz model could fit the body weight growth curve of Lueyang Black-bone chickens well.The fitting degrees of hens and roosters were both>0.99, the inflection point weight were 760.75 and 940.52 g, and the inflection point weeks old were 8.19 and 8.49 weeks, respectively.The shear force of hens and roosters was increased gradually with the increase of age, and the shear force at 150 days of age was extremely significantly higher than that at 90 and 120 days of age (P<0.01).Breast muscle dry matter of hens at 150 days of age was extremely significantly higher than that at 90 and 120 days of age (P<0.01).The contents of Glu, Ala, and Asp in the flavoring amino acids in breast muscle of hens at 150 days of age were extremely significantly higher than that at 90 and 120 days of age (P<0.01), while the content of Tyr at 150 days of age was significantly lower than that at 90 and 120 days of age.The contents of Thr, Ser, Met, Leu, Lys and the total amount of amino acids at 150 days of age were significantly higher than those at 90 days of age (P<0.05).The contents of Glu, Ala, Phe, Cys, Pro in the flavor amino acids of rooster breast muscle at 120 days of age were extremely significantly higher than those at 90 days of age (P<0.01), while the contents of Tyr, Cys, Pro and Phe at 120 days of age were extremely significantly higher than those at 150 days of age (P<0.01), the content of His was the lowest at 120 days of age.The fat content of breast muscle of 120-day-old hens was extremely significantly lower than that of 150-day-old hens (P<0.01), and the protein content of breast muscle of 120-day-old hens was significantly lower than that of 150-day-old hens (P<0.05).The contents of C18:2n6c and C14:0 in breast muscle of hens at 150 days of age were extremely significantly higher than those at 120 days of age (P<0.01), and the content of C16:1 at 150 days of age was extremely significantly lower than that at 120 days of age (P<0.01).The contents of inosinic acid and C16:0 in rooster breast muscle at 120 days of age were significantly higher than that at 150 days of age (P<0.05), while the content of C18:2n6c at 150 days of age was significantly higher than that at 120 days of age (P<0.05).【Conclusion】 The nutrient contents of hens were gradually increased with the increase of age.The nutrient content of breast muscle of 120-day-old cock was the highest, and the nutrient contents were decreased gradually with the increase of day age after 120-day-old.Therefore, it was recommended that hens should be listed between 120 and 150 days of age, and roosters should be listed at 120 days of age.

【Objective】 This study was aimed to screen long non-coding RNA (lncRNA) related to inbreeding depression of reproduction in chickens, so as to provide reference for further exploring the regulation mechanism of lncRNA on inbreeding depression of reproduction in chickens.【Method】 The strongly and weakly inbreeding populations of Langshan chickens were used as materials, lncRNA expression in hypothalamus and ovary of Langshan chickens was analyzed by transcriptome sequencing.Differentially expressed lncRNA between strongly and weakly inbreeding populations were identified, and cis regulation target gene prediction and function analysis were performed.【Result】 A total of 4 222 lncRNAs were identified in hypothalamus and ovary of Langshan chickens.Comparing the strongly and weakly inbreeding populations, 35 and 215 differentially expressed lncRNAs were found in hypothalamus and ovary, respectively.In hypothalamus, 98 cis regulation target genes were predicted in differentially expressed lncRNAs, which were significantly enriched in reproduction-related biological processes (P<0.05), such as embryo development ending in birth or egg hatching, embryonic heart tube development and response to retinoic acid, involving MSTRG.9196.4, MSTRG.9195.2, MSTRG.6254.2 lncRNAs and their corresponding target genes DNAAF2 and FKBP1B.In ovary, 414 target genes were predicted, which were enriched in oocyte meiosis, MAPK and folic acid synthesis signaling pathways, including MSTRG.7683.1, MSTRG.13604.4, MSTRG.16570.1, MSTRG.8330.5, MSTRG.8330.4 and other lncRNAs related to neuroendocrine regulation and gametogenesis.【Conclusion】 In this study, a series of differential lncRNAs related to embryo development and gametogenesis in chickens were screened from hypothalamus and ovary.These lncRNAs could be used as candidate lncRNAs for inbreeding depression of reproduction in chickens, and provided clues for revealing the regulation mechanism of inbreeding depression in reproduction of chickens.

【Objective】 The aim of the expriment was to study on the relationship between PDK4 gene polymorphism and meat quality traits in Chinese Steppe Red cattle.【Method】 120 Chinese Steppe Red cattle were selected as the research object.The single nucleotide polymorphism (SNP) of exons 1-11 of PDK4 gene was detected by Sanger sequencing, and the genotype frequency, gene frequency and population genetic parameters of SNP were analyzed.SPSS 21.0 software was used to analyze the association between SNP polymorphism of PDK4 gene and meat quality traits (cooked meat rate, meat tenderness, water loss rate, pH, drip loss, intramuscular fat content, initial water content and protein content) in Chinese Steppe Red cattle.【Result】 Three SNPs were detected in exons 8 and 11 of PDK4 gene in Chinese Steppe Red cattle.There was a SNP site at 57 bp (G57C) in exon 8 of PDK4 gene, which caused the change of coding amino acids.There were GG, GC and CC genotypes at G57C site.There were two SNPs at 330 bp(G330T) and 389 bp(C389T) in exon 11 of PDK4 gene.There were three genotypes of GG, GT and TT at G330T and three genotypes CC, CT and TT at C389T.Chi-square test results showed that the G57C site of exon 8 of Chinese Steppe Red cattle deviated from Hardy-Weinberg equilibrium (P<0.05), and the G330T and C398T of exon 11 conformed to Hardy-Weinberg equilibrium (P>0.05).The analysis of population genetic parameters showed that the G57C site in exon 8 belonged to low degree polymorphism (PIC<0.25), and the number of alleles was 1.1429, indicating that it had little variation in Chinese Steppe Red cattle.Exon 11 G330T and C398T belong to moderate polymorphism sites (0.25<PIC<0.5), and the number of alleles were 1.6431 and 1.6447 respectively, indicating that the genetic marker could provide genetic information.The association analysis results showed that the intramuscular fat content of GG and CC genotypes at G57C site in exon 8 of PDK4 gene was significantly higher than that of GC genotype (P<0.05).The drip loss and initial water component of GG genotype at G330T in exon 11 were significantly higher than those of GT genotype (P<0.05).The meat tenderness of GG genotype was significantly higher than that of TT genotype (P<0.05).The intramuscular fat content of GT genotype was significantly higher than that of TT genotype (P<0.05), and the dehydration rate of CT genotype at C389T was significantly lower than that of TT genotype (P<0.05).【Conclusion】 PDK4 gene polymorphism was associated with meat quality traits in Chinese Steppe Red cattle, and could be used as a candidate gene for meat quality traits.

【Objective】 The aim of this study was to investigate the role and mechanism of miR-181a and miR-181d-5p in the differentiation of primary precursor adipocytes in Rongchang pigs.【Method】 The sequence conservation of miR-181a and miR-181d-5p among different species of pig, human, rat and mouse was analyzed by comparing seed sequence differences.miR-181a and miR-181d-5p expression in pig muscle and subcutaneous adipose tissue was detected by Real-time quantitative PCR.miRandn and TargetScan online softwares were used to predict miR-181a and miR-181d-5p target genes with GO and KEGG enrichment analysis.The free energy of miR-181a and miR-181d-5p binding to their target genes was predicted using RNAhybrid online software.Primary precursor adipocytes were isolated from subcutaneous adipose tissue on the back of 7-day-old male Rongchang pigs.Wild-type and mutant vectors of miR-181a and miR-181d-5p target genes were designed and cotransfected with the corresponding miRNAs in the precursor adipocytes.A dual luciferase reporter gene assay was performed to verify the targeting relationship between miRNA and target genes, and the binding of miR-181a and miR-181d-5p to their target genes was measured.miR-181a and miR-181d-5p mimics (miR-181a mimics, miR-181d-5p mimics), inhibitor (miR-181a inhibitor, miR-181d-5p inhibitor), negative control (NC) and interfering siRNAs of their target genes (Gene-siRNA) were constructed, transfected the cells.The transfected cells were induced to differentiate 6 h later.After 6 d, gene expression and triglyceride production were determined by Real-time quantitative PCR, oil red O staining was used to detect triglyceride in the treated cells, respectively.【Result】 The results of conservativeness analysis showed that miR-181a and miR-181d-5p were highly sequence conserved among pig, human, rat and mouse.miR-181a and miR-181d-5p were quantified in different tissues of pigs, which showed that miR-181a and miR-181d-5p were specifically highly expressed in porcine adipose tissues.The results of target gene prediction showed that the target genes of miR-181a and miR-181d-5p were mitochondrial glycerol 3-phosphate dehydrogenase (GPD2) and cyclic adenylate response element (CREB1), respectively.GO and KEGG functional enrichment analysis revealed that the target genes of miR-181a and miR-181d-5p could inhibit triglyceride production and regulate adipocyte differentiation, and miR-181a and miR-181d-5p.The free binding energy of both miR-181a and miR-181d-5p with their target genes was less than -87.8 kJ/mol.The results of dual luciferase reporter gene assay showed that mimics transfected with miR-181a and miR-181d-5p could extremely significantly inhibit luciferase activity in the target gene 3'-UTR wild-type dual luciferase reporter vector (P<0.01), but did not affect the target gene mutant type.However, it did not affect the luciferase activity in the target gene mutant dual luciferase reporter vector.The induction of differentiation showed that miR-181a was significantly higher at day 4 (P<0.05) and reached a extremely significant difference at day 6 (P<0.01) compared with undifferentiated adipocytes (0 d).miR-181a and miR-181d-5p, as well as GPD2 and CREB1 genes, were extremely significantly higher at days 4 and 6 of differentiation (P<0.01).miR-181d-5p cell transfection assays both yielded similar results.miR-181a and miR-181d-5p expressions were both extremely significantly increased in mimics group (P<0.01) and miR-181a and miR-181d-5p expressions were both extremely significantly decreased in inhibitor group (P<0.01) compared with the NC group.The expressions of GPD2 and CREB1 genes were extremely significantly decreased in both mimics and siRNA groups (P<0.01), and the expressions of GPD2 and CREB1 genes were extremely significantly increased in inhibitor group (P<0.01).The relative expressions of PPARγ and C/EBPα genes were significantly or extremely significantly increased in both mimics and siRNA groups (P<0.05 or P<0.01), and the relative amounts of PPARγ and C/EBPα genes were significantly decreased in inhibitor group (P<0.05).The results of oil red O staining showed that the number of cellular lipid droplets increased in mimics and siRNA groups and decreased in inhibitor group compared with NC group.【Conclusion】 miR-181a and miR-181d-5p could target and bind GPD2 and CREB1 genes 3'-UTR region sequences, respectively, reduce the expression of GPD2 and CREB1 genes, and promote lipogenic differentiation of porcine precursor adipocytes.

【Objective】 The CDS region of porcine beta-defensin-124 (PBD-124) gene was cloned and its polymorphism was explored.The expression of PBD-124 gene in different breeds of pigs and different tissues of the same kind of pigs was analyzed.【Method】 The CDS region of PBD-124 gene was amplified by RT-PCR and cloned.PCR-RFLP was used to detect the polymorphism of PBD-124 gene (Bln Ⅰ) in Large White, Min and wild hybrid pigs.The expression difference of PBD-124 gene in liver, spleen and blood of different breeds of pigs was detected by Real-time quantitative PCR.【Result】 The CDS region of PBD-124 gene was successfully cloned, with a length of 423 bp and a total of 140 amino acids.Sequencing results showed that there were two mutation sites of c.257 G>A and c.263 T>G.Because there was linkage between the two mutation sites, only the first mutation site was digested.Three genotypes of GG, GA and AA were detected in Large White pigs, while only two genotypes of GA and AA were detected in Min and wild hybrid pigs.AA genotype was the dominant genotype in three pig populations.The genotype frequency of Large White, Min and wild hybrid pigs were 0.5261, 0.9412 and 0.6452, respectively, and three pig populations were in Hardy-Weinberg equilibrium (P>0.05).The polymorphism of three pig populations was not high.Large White pigs were in moderate polypeptide (0.25<PIC<0.5), while Min and wild hybrid pigs were in low polypeptide (PIC<0.25).Real-time quantitative PCR results showed that PBD-124 gene was expressed in spleen, liver and blood of Large White, Min and wild hybrid pigs, and there were expression differences.【Conclusion】 The length of PBD-124 gene CDS was 423 bp, and encoded 140 amino acids.Compared with the reference sequence, two SNP mutation sites were found, which did not cause amino acid mutation.The polymorphism of three pig populations was not high.The expression of PBD-124 gene was different in different breeds of pigs and different tissues of the same kind of pigs.

【Objective】 The purpose of this experiment was to detect the single nucleotide polymorphism (SNP) of connective tissue growth factor (CTGF) and G protein-regulated inducer of neurite outgrowth family subtype 3 (Gprin3) genes in yaks, and analyze the association between SNP and body size traits in yaks.【Method】 The SNP of CTGF and Gprin3 genes were detected by gene pool screening technique in 260 individuals of Sibu, Pali, Leiwuqi, Shenzha yaks in Tibet and Maiwa yaks in Sichuan.The polymorphic information content (PIC), number of effective alleles, homozygosity and heterozygosity were analyzed, and the association between SNP and body size traits in yaks was analyzed by least square method (LMS).【Result】 Three SNPs were detected in intron region of CTGF gene in yaks, which were T1537A, C2195T and C2421T.T1537A was in Hardy-Weinberg equilibrium in Sibu and Maiwa yaks, and deviated from Hardy-Weinberg equilibrium in Pali, Leiwuqi and Shenzha yaks, which was significantly associated with body height (P<0.05).C2195T was in Hardy-Weinberg equilibrium and moderately polymorphic in 5 yak populations (0.25<PIC<0.5).C2421T was in Hardy-Weinberg equilibriumin in 5 yak populations, which was significantly associated with the body weight, body length, heart girth, circumference and forelimb length (P<0.05).Four SNPs were identified in Gprin3 gene, which were G310C, C414T, C1100T and G1213A.G310C, C414T and C1100T were in Hardy-Weinberg equilibrium in 5 yak populations, G1213A deviated from Hardy-Weinberg equilibrium in Leiwuqi yaks, and accorded with Hardy-Weinberg equilibrium in other 4 yak populations.G310C was low polymorphic in 5 yak populations (PIC<0.25).C414T and C1100T were moderately polymorphic in Leiwuqi and Sibu yaks, G1213A was moderately polymorphic in Leiwuqi yaks, but was low polymorphic in other populations.C414T was significantly associated with the body weight, body height, body length and heart girth in 5 yak populations, C1100T was significantly associated with the circumference and forelimb length in Sibu and Shenzha yaks (P<0.05).【Conclusion】 CTGF and Gprin3 genes had abundant polymorphism, T1537A and C2421T of CTGF gene, C414T and C1100T of Gprin3 gene, were associated with body size traits in yaks, which could be used as candidate genes and loci affecting the growth and development traits for directional breeding of yaks.

【Objective】 The aim of this study was to investigate the conservative status and phylogenetic relationship of 5 Tibet yak populations (Ali, Sibu, Niangya, Leiwuqi and Pali yaks) by estimating the genetic diversity of main yak populations and combing the genetic structure among yak populations from different areas.【Method】 A total of 195 individuals from 5 yak populations were genotyped using 13 microsatellite markers (SSR), and a series of genetic parameters were evaluated, such as the number of alleles, gene heterozygosity, polymorphism information content and genetic distance between populations.【Result】 Ali yak population had the largest number of alleles (6.43), while the Leiwuqi yak had the least (5.00).The observed heterozygosity distribution ranges from 0.5311 (Niangya yak) to 0.5995 (Leiwuqi yak).The number of markers deviating from Hardy-Weinberg equilibrium in each population ranges from 5 (Leiwuqi yak) to 9 (Ali yak), the highest inbreeding coefficient within population was 0.172 (Ali yak), and 4 populations (Ali, Niangya, Sibu and Pali yaks) with a significant risk of inbreeding (P<0.05).According to the pairwise difference of populations, there were significant genetic divergence (P<0.05) between each population pair.The results of STRUCTURE software analysis revealed that 5 yak populations were divided into 3 clusters.Among them, Ali yak had richer diversity within populations than that of others.In addition, the phylogenetic tree indicated that the independent phylogenetic relationship between populations as well as inconsistent with their habitat geographical distribution.The principal coordinate analysis (PCoA) results revealed that there were some individuals from different population carry closely kinship, indicating that there was genetic material exchange between populations.【Conclusion】 5 Tibet yak populations had rich genetic diversity and relatively independent population phylogenetic relationship.However, most populations were under risk of population events.Therefore, this study would not only help to clarify the genetic diversity of Tibetan yak, but also provided a theoretical reference for improvement of future conservation strategies.

【Objective】 The purpose of this study was to explore the effect of gonadotropin inhibitory hormone (GnIH) on gonadal reproductive function and glucose metabolism of SD rats, and the correlation between gonadal reproductive function and glucose metabolism of SD rats.【Method】 36 SD rats were randomly divided into control group (0.9% normal saline), 1 μg/100 μL GnIH (group Ⅰ) and 10 μg/100 μL GnIH (group Ⅱ), 12 rats in each group with half male and half female.Saline or GnIH (200 μL each time) was injected at 07:00 and 19:00 every day, after 14 days of continuous injection, the weight of rats was measured to calculate the degree of obesity.After death under anesthesia, ovaries and testis were collected, weighed, and the egg body ratio and testis body ratio were calculated.The changes of estrus cycle of female rats were observed by vaginal smear.The sperm motility of male rats was calculated under microscope.HE staining was used to observe the changes of ovarian and testicular tissues.The expression levels of glucose metabolism genes IR and GLUT4 as well as inflammation-related factor genes TNF-α and IL-1β were detected by Real-time quantitative PCR in ovary and testis of SD rats.The correlation between reproductive function and glucose metabolism was analyzed by SPSS 22.0 software.【Result】 Compared with control group, the degree of obesity of female SD rats in group Ⅰ and male SD rats in group Ⅱ was increased significantly, the ovarian size/weight and ovary-to-body ratio of group Ⅱ were increased significantly, the testicular weight and testis-to-body ratio was significantly decreased (P<0.05).HE staining results showed that compared with control group, the follicles of female rats in group Ⅱ showed cystic expansion, the granulosa cell layer decreased and the follicular cavity became larger.The spermatogenic tubules of male rats in groups Ⅰ and Ⅱ were vacuolated, and spermatogenic cells were disordered and their levels decreased.Compared with control group, the proestrus period of rats in group Ⅱ was significantly prolonged, the sperm motility was significantly decreased (P<0.05).Real-time quantitative PCR results showed that compared with control group, the expression level of GLUT4 gene in female rats of group Ⅰ and group Ⅱ were extremely significantly decreased (P<0.01), the expression of IR gene of group Ⅱ was significantly decreased (P<0.05), the expression of GLUT4 gene in male rats of groups Ⅰ and Ⅱ was significantly decreased (P<0.01), the expressions TNF-α and IL-1β genes of female and male rats in group Ⅰ were increased significantly (P<0.05), the expression of TNF-α gene in female and male rats of group Ⅱ was significantly increased (P<0.05).The results of correlation analysis showed that the ovary-to-body ratio of female rats was extremely significantly positively correlated with the expression levels of GLUT4 gene (P<0.01), and significantly positively correlated with the expression levels of IR gene (P<0.05), the estrous cycle of female rats was significantly negatively correlated with the expression levels of GLUT4 gene (P<0.01), and the testis-to-body ratio of male rats was significantly positively correlated with the expression levels of GLUT4 gene (P<0.05), while the sperm motility was extremely significantly positively correlated with the expression levels of GLUT4 gene (P<0.01).【Conclusion】 Intraperitoneal injection of GnIH could inhibit reproductive function and lead to the disorder of glucose metabolism in rats, and GnIH might participate in the cross-talk between gonadal energy metabolism and reproductive function, was a new connecting factor between energy metabolism and reproductive function.

【Objective】 The aim of this expremient was to study the effect of superovulation on the changes of the morphology and hormone levels of the reproductive organs of rabbits.【Method】 Twelve rabbits were divided into experimental group and control group, 6 rabbits in each group.The rabbits in the experimental group was injected 70 IU pregnancy mare serum gonadotropin(PMSG) into the inner thigh, 72 h after the injection, 100 IU human chorionic gonadotropin (hCG) was injected at the same injection point.The control group was injected with the same volume normal saline at the same time.Rabbits in both the experimental group and the control group were caged with male rabbits to stimulate ovulation.After 24 hours, the rabbits were killed and the ovaries, fallopian tubes and uterus were collected, the length, width and thickness of ovary, the length, width and circumference of uterus, the thickness of uterine wall, the length of fallopian tube, the width of funnel, isthmus and ampulla were measured.The ovaries were made into tissue sections, and the changes of ovarian follicles were studied by HE staining.The ear vein blood of rabbits in the control group and the experimental group were collected before PMSG injection, hCG injection and death, inhibin (INH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E₂) and progesterone (P₄) in serum were detected by ELISA.【Result】 Compared with control group, the weight, length, width, thickness, the number of hyperemic follicles, the number of ovulation points and the number of atretic follicles of ovarian tissues in the experimental group were extremely significantly increased (P<0.01);The diameter of secondary follicles in the ovary was significantly decreased (P<0.05);The length of the fallopian tubes, the width of the ampulla of the fallopian tubes and the length of the uterus were significantly increased (P<0.05);The width of the infundibulum of the fallopian tubes and the length of uterus were significantly increased (P<0.01).At three different blood collection times, the serum INH level of the rabbits in control group showed an increasing trend, Before hCG injection and before death, the INH content in the experimental group was significantly lower than that in control group (P<0.05).Before hCG injection, the serum FSH content of the experimental group was significantly higher than that of control group (P<0.05), while the content of FSH in serum of the experimental group was significantly lower than that of the control group before death(P<0.05).Before hCG injection and before death, the content of LH in serum of the experimental group was significantly higher than that of control group (P<0.05).There was no significant difference in the contents of P₄ and E₂ between the experimental group and the control group at each time point (P>0.05).【Conclusion】 The superovulation treatment of 70 IU PMSG+100 IU hCG increased the ovarian volume and the number of ovulation points on the surface of rabbits, and cooperated with endogenous hormones to act on the ovary to obtain a better superovulation effect in rabbits.

Somatic cell nuclear transfer (SCNT) is a reproductive biotechnology that can reprogram differentiated somatic cells into totipotent embryos.It has a wide range of application prospects in the propagation of excellent breeding livestock, protection of endangered species and therapeutic cloning, However, the extremely low cloning efficiency, abnormal placenta of cloned animals and fetal malformation after birth seriously limit the practical application of this technology.The low cloning efficiency and abnormal embryonic development are mainly due to errors or incomplete reprogramming of donor nuclear epigenetics.In 1958, the first SCNT animal individual was obtained by transferring the intestinal nucleus of Xenopus laevis larvae into enucleated oocytes;In 1986, three surviving lambs were successfully obtained by electrofusion of a blastomere and enucleated oocytes;In 1997, the mammary epithelial cells of adult ewes were fused with enucleated egg cells to obtain the first SCNT mammal "Dolly", which opened the era of cloning.At present, cattle, mice, goats, pigs, European argali, rabbits, domestic cats, horses, rats, mules, dogs, ferrets, wolves, buffalo, red deer, humped camels, cynomolgus monkeys have been successfully cloned one after another.One of the most remarkable is the successful cloning of cynomolgus monkeys in 2018.By comparing the development of SCNT embryos with that of fertilized embryos, the author expounds the reprogramming process and defects of DNA methylation, histone modification, genomic imprinting and chromosome status in the process of SCNT.The effects of eliminating epigenetic reprogramming barriers, alone or in combination, on cloning efficiency are discussed in terms of epigenetic regulators, histone lysine demethylases, inhibition of Xist expression, supplementation of protamine and sperm RNA.With the development and improvement of low-sample-size sequencing technology, more detailed genome-wide epigenetic modification maps can be detected in SCNT embryos, further revealing defects in epigenetic reprogramming of SCNT embryos, and providing opportunities for improving cloning efficiency.Through the elaboration of the above content, hoping to provide strategies and ideas for the subsequent development of joint methods to eliminate multiple epigenetic barriers and improve cloning efficiency.

Reproductive traits are important economic traits of Bos taurus, which are closely related to production costs and economic benefits.The main reproductive traits include semen quality, follicular development, ovulation, embryo development, calving difficulty, calving interval, etc.The performance of reproductive traits is usually strictly controlled by genes.In-depth excavation and screening of gene functions of important reproductive traits is very important for cattle improvement and breeding, improving production performance and saving production cost.In this paper, the research progress of cattle reproductive traits genes at home and abroad were introduced, such as luteinizing hormone receptor (LHR) and sperm flagella 2 (SPEF2) are related to semen quality;Gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and anti-Müllerian hormone (AMH), bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9) are related to follicular formation and ovulation;JY-1, caudal type homeobox 2 (CDX2) and pappalisin 2 (PAPP-A2) are related to the early embryo development and the difficulty of calving;Sialic acid binding Ig like lectin 5 (SIGLEC5), CXC motif chemokine receptor 1 (CXCR1) and forkhead box O1 (FoxO1) are related to calving interval.By summarizing the research on these important reproductive traits related genes, the author introduced the influence of these important reproductive traits genes on cattle production performance, in order to provide theoretical reference for the research on related regulatory factors affecting cattle reproductive traits.

【Objective】 In order to investigate the genotypes and genetic variation of Porcine epidemic diarrhea virus (PEDV) strains spreading in parts of Southwest China.【Method】 200 anal swabs of diarrhea porcine which were collected in different regions of Sichuan and Yunnan from 2020 to 2021 were detected by RT-PCR methods.Then, the S and ORF3 genes of PEDV in positive samples were cloned and sequenced.MegAlign software was used for variability analysis, Mega 7.0 software was used for system evolution analysis, and RDP4 software was used for gene recombination analysis.【Result】 The positive rates of PEDV in all samples were 15.50%(31/200) and the positive rate of population was 100%(8/8).The PEDV S and ORF3 genes sequences of 8 isolates were 90.5%-99.4% and 92.9%-100%, respectively.Phylogenetic analysis based on the S gene showed that 8 PEDV strains were distributed in three subgroups, G2a, G2b, and G2c.Among them, the Sichuan SCWJSWUN02 strain was closed to Fujian CH/FJXM/1/2012 strain, Yunnan YNKMSWUN01 strain and Vietnamese strain were clustered in a separate group.It indicated that the current PEDV epidemic in Southwest China was characterized by inter-regional transmission.Compared with the previously circulating strains and vaccine strains, the amino acid of the S protein of 8 PEDV isolates were changed on different levels.However, ORF3 protein was relatively conserved.The continuous amino acid insertions and deletions were found in the S protein of parts of PEDV strains.Such as, 4 continuous amino acid deletions were found in 59-62 aimo acid of SCMYSWUN03 strain S protein.5 amino acid insertions and deletions were found in 78-82 and 84-88 aimo acid of YNKMSWUN01 strain, respectively.Some amino acid mutations were present in the identified receptor binding domains of the S protein.In addition, the recombination analysis showed that SCMYSWUN03 strain might have gene recombination events, with a probability of 98.2% (P<0.01).【Conclusion】 This study found that the current popular PEDV strains in Southwest China included three subgroups of G2a, G2b and G2c.The genotypes of the strains were diverse, and the variability among different strains was large, which enriched the molecular epidemiological investigation data of PEDV in Sichuan and Yunnan, and revealed the molecular genetic variation law and evolutionary characteristics of 8 PEDV strains prevalent in Southwest China, It provided a theoretical basis for the formulation of prevention and control measures of porcine diarrhea in this region.

【Objective】 To investigate the co-infection situation of Porcine parvovirus (PPV7) and Porcine circovirus type 2 (PCV2)in Fujian and Guangdong, and to understand the molecular genetic characteristics of PPV7 Cap gene.【Method】 The blood sample of 432 infected pigs from 69 pig farms in Fujian and Guangdong were collected to detect PPV7 and PCV2 by PCR.The PPV7 Cap gene of positive samples was cloned and sequenced.The DNAStar software was used to analyze the nucleotide and amino acid sequences of PPV7 Cap gene, and Mega 7.0 software was used to draw the genetic evolution tree.【Result】 The results showed that the positive rate of PPV7 was 21.99% (96/432), the positive rate of farms was 53.62%(37/69), and the positive rate of PCV2 was 54.17% (234/432), and the co-infection rate of PCV2 and PPV7 was 13.43% (58/432).The 17 PPV7 Cap gene sequences were amplified using PCR.Nucleotide homology analysis revealed that the homology of the 17 PPV7 Cap gene sequences was 85.6%-100%, and the homology with reference strains was 85.8%-99.0%.Amino acid sequence comparison analysis revealed that the amino acid homology of the 17 PPV7 Cap protein sequences was 87.6%-100%, and the amino acid homology with reference strains was 82.6%-98.7%.Phylogenetic analysis of Cap gene showed that PPV7 could be divided into five main evolutionary branches of PPV7a-PPV7e, among which 9 isolates belonged to PPV7a subtype, 3 isolates belonged to PPV7b subtype, 4 isolates belong to PPV7c subtype, and only 1 isolates isolates belonged to PPV7e subtype.【Conclusion】 This study indicated that PPV7 was widely prevalent in Fujian and Guangdong regions, and had a high co-infection rate with PCV2, which might be the pathogenic factor of Porcine circovirus associated disease(PCVAD).The genetic diversity of PPV7 isolates was abudant in both regions, and PPV7a was the dominant strain at present.The findings of this study provided theoretical basis and data reference for PPV7 prevention and control and vaccine research.

【Objective】 This study was aimed to construct the glycosyl-flippase gtcA gene deletion strain, and explore the effects of gtcA gene in the pathogenicity of Listeria monocytogenes.【Method】 The deletion strain ΔgtcA was constructed by homologous recombination, the growth ability of EGDe-prfA*, ΔgtcA and CΔgtcA was compared.The effects of gtcA gene deletion on the anchoring of InlA and InlB on bacterial surface was detected by Western blotting.The roles of gtcA gene in pathogenicity was validated by the adhesion and invasion assay on DF1 cells, the phagocytosis and multiplication assay in macrophages RAW264.7, and virulence assay conducted on chicken embryo.【Result】 The growth curve showed that gtcA gene deletion did not affect the growth ability of Listeria monocytogenes in BHI medium.Western blotting results showed that the anchor quantity of InlB in ΔgtcA surface was extremely significantly lower than that of EGDe-prfA* and CΔgtcA (P<0.01), but the anchor quantity of InlA in ΔgtcA surface was not significantly lower than that of EGDe-prfA* (P>0.05).The results of cell adhesion and invasion test showed that the average adhesion rate and invasion rate of ΔgtcA were significantly lower than that of EGDe-prfA* and CΔgtcA (P<0.05).The phagocytosis test results showed that the average phagocytosis rate of ΔgtcA by macrophage RAW264.7 was significantly lower than that of EGDe-prfA* and CΔgtcA (P<0.05), but there was no significant difference of proliferation multiple within 3 h in macrophage RAW264.7 between ΔgtcA and EGDe-prfA* or CΔgtcA (P>0.05).Virulence assay results showed that the average bacteria load of liver and spleen in ΔgtcA infected chicken embryo were 6.86×10³ and 3.69×10² CFU, respectively, which were extremely significantly lower than that of EGDe-prfA* and CΔgtcA infected chicken embryos (P<0.01).Moreover, the survival rate and survival time of ΔgtcA infected chicken embryo were higher than that of EGDe-prfA* and CΔgtcA infected chicken embryos.【Conclusion】 gtcA gene deletion did not affect the growth ability of EGDe-prfA*, while decreased the surface anchoring of InlB, infection ability and pathogenicity of EGDe-prfA*.The results provided a reference for further exploring the mechanisms of gtcA mediates pathogenicity of Listeria monocytogenes.

【Objective】 The study was aimed to screen vaccine candidate strains of Streptococcus suis serotype 3, prepare a bivalent inactivated vaccine of Streptococcus suis serotype 3+9 and evaluate its immunoprotective effect on BALB/c mice.【Method】 In this study, serotype 3 strain of Streptococcus suis isolated from diseased pigs were screened by the larvae of Galleria mellonella and BALB/c mice model and was studied as vaccine candidate strains.Then the biological characteristics of the candidate strains were studied.The serotype 3 vaccine candidate strains and the previously screened serotype 9 candidate vaccine strains were inactivated and concentrated, and the antigen concentration was 2×10¹⁰ CFU/mL.After the sterility test, the concentrated antigen liquid was mixed in a ratio of 1:1, and then mixed with the Summit Poly Solution adjuvant in a ratio of 4:1 to prepare bivalent inactivated vaccine of Streptococcus suis serotype 3+9, the bacterial content of Streptococcus suis serotype 3 and 9 were both 2×10⁹ CFU/mL.The prepared vaccine was inoculated into 6-week-old BABL/c mice.The second immunization was carried out on the 14th day after the first immunization, and the challenge was carried out on the 14th day after the second immunization.At the same time, a commercial vaccine immunization group and a negative control group were established to evaluate the safety and immune protection effect of the vaccine.【Result】 PCR identification result showed that all 23 clinical isolates were Streptococcus suis serotype 3, named KQ3-1 to KQ3-23 in succession.The virulent strain KQ3-1 was screened by primary screening and rescreening of Galleria mellonella and BALB/c mice.Virulence gene testing showed that the genotype of KQ3-1 was gapdh⁺/sly⁺/fbps^-/orf2^-/mrp^-/89K^-/gdh⁺/epf^-.The growth curve showed that the strain reached its peak when cultured at 37 ℃ for 8 to 10 h.The pathogenicity of BALB/c mice showed that intraperitoneal inoculation could cause clinical symptoms such as depression, pile up, hair erection, motor retardation and death within 12 h.The LD₅₀ of the strain was 5.2×10⁷ CFU/piece.After immunizing the mice with the prepared vaccine, the mental status and food intake of the mice were normal, there were no adverse reactions such as swelling and lumps at the injection site of the vaccine, and there was no death, indicating that the vaccine had good safety.After immunization, Streptococcus suis serotype 2 strain ZYS, Streptococcus suis serotype 3 strain KQ3-1 and Streptococcus suis serotype 9 strain YT were challenged.The mortality rate of control group was 90%, 100% and 100%, respectively.The protection rates of Streptococcus suis serotype 3+9 bivalent inactivated vaccine immunization group were 30%, 80% and 70%, respectively.The protection effects of commercial vaccine group were 70%, 0 and 10%, respectively.【Conclusion】 The vaccine developed in this study could provide good immune protection against Streptococcus suis serotype 3 and 9 strains and had potential value of vaccine market development.

【Objective】 The study was aimed to provide biological materials for developing phage "cocktail" preparations for the prevention and treatment of dairy mastitis.【Method】 Staphylococcus aureus 82 isolated from cow mastitis was used as the host bacteria, the phages were isolated and purified from the sewage, abandoned milk and feces of dairy farms by drip method and double-layer plate method, the morphological characteristics of the phage were observed by plaque and transmission electron microscope.The nucleic acid type of the phage was analyzed by nuclease treatment.The pyrolysis spectrum, MOI, one-step growth curve, thermal stability and pH stability were studied and analyzed.【Result】 The lytic phage P82 was isolated and screened.Under electron microscope, the head of the lytic phage P82 was icosahedral with a size of about 120 nm×83 nm, and the length of the lytic phage P82 was about 126 nm, it belonged to the order of Endurophage, and the family of Myoendurophage.The nucleic acid type was identified as dsDNA by agarose gel electrophoresis.The biological characteristics of the phage were determined:The best MOI of P82 was 0.001, and the cleavage rate of Staphylococcus aureus was 36.51%(23/63).The host spectrum was wide.The incubation period of growth was about 25 min, the outbreak period was about 45 min, and the cleavage volume was about 74 PFU/cell.P82 was stable at pH 4.0-12.0, and had high thermal stability.It completely lost activity at pH 2.0 and 13.0.It still had cracking ability at 70 ℃ for 60 min, but lost cracking ability at 80 ℃ for 40 min.【Conclusion】 The phage P82 isolated in this study had high titer, strong cleavage ability, fast proliferation speed, wide phage spectrum and good tolerance to different temperatures and pH.It could be used as a phage candidate strain for specific cleavage of Staphylococcus aureus from cow mastitis.It could be mixed with other phages to make a phage "cocktail" for the prevention and treatment of cow mastitis and provide materials for phage therapy of cow mastitis.

【Objective】 This study was aimed to establish a duplex TaqMan Real-time PCR method for rapid detection of Bovine viral diarrhea virus types 1(BVDV1) and 2(BVDV2).【Method】 Specific primers were designed based on the 5'-non-coding region of 95 strains of BVDV1 and BVDV2 in GenBank.The positive plasmids containing the target fragments of BVDV1 and BVDV2 were constructed.The reaction conditions were optimized, the standard curve was constructed, the specificity, sensitivity and reproducibility of the duplex TaqMan Real-time PCR method were tested, and the established duplex TaqMan Real-time PCR assay was used to detect the clinical samples collected from Jilin province.【Result】 The results showed that the optimal annealing temperature of the duplex TaqMan Real-time PCR was 57.0 ℃, the optimum primer concentration was 0.5 μmol/L, and the optimum probe concentration was 0.3 μmol/L.The standard curves of BVDV1 and BVDV2 were Y=-3.54X+37.36 (R²=0.990) and Y=-3.18X+35.95 (R²=0.997), respectively.There was no specific amplification of Infectious bovine rhinotracheitis virus (IBRV), Bovine respiratory syncytial virus (BRSV) and Bovine parainfluenza virus type 3 (BPIV3), with intra- and inter-batch CV less than 3%, and the lower limit of detection was 10 copies/μL.The results of clinical samples showed that the overall positive rate was 23.1% (36/156), of which 17.9% (28/156) were positive for BVDV1 and 5.1% (8/156) were positive for BVDV2.【Conclusion】 In this study, a duplex TaqMan Real-time PCR method was established, which could identify BVDV types 1 and 2 simultaneously, quickly and accurately, and provided technical support for the prevention, control and purification of BVDV.

Senecavirus A (SVA), also known as Seneca Valley virus (SVV), is a member of the genus Senecavirus in the family Picornaviridae.SVA mainly causes vesicular disease in pigs, which is similar to clinical symptoms such as foot-and-mouth disease, vesicular stomatitis and porcine vesicular disease, it can lead to acute death of newborn piglets and seriously affect the development of pig industry.Since the occurrence of SVA infection in Guangdong province in 2015, the disease has been reported in many provinces in China.At present, due to the lack of immune barrier against SVA in pigs in China and the strong infectivity of the disease, there is a potential risk of large-scale outbreak.How to effectively prevent and control SVA infection is an urgent problem to be solved.A variety of SVA diagnostic methods have been developed for early differential diagnosis under laboratory and field conditions.The isolation and identification of SVA, in situ hybridization and immunohistochemistry can be used to detect the existence of pathogens and their relationship with morphological changes in tissues.Serological diagnostic methods include indirect ELISA based on different structural proteins, competitive ELISA, homogeneous photoluminescence immunoassay and virus neutralization test.The detection of antibodies by immunological methods is helpful to understand the process of SVA infection and is the main means of clinical diagnosis.Virus nucleic acid detection methods mainly include PCR technology, isothermal amplification technology, genome sequencing and other molecular biological technologies, which play an important role in the early rapid detection of virus infection and the detection of new viruses.At present, there is still no commercial vaccine to prevent SVA infection, but researchers have developed a variety of potential candidate vaccines such as inactivated vaccine, attenuated vaccine, nucleic acid vaccine and subunit vaccine.The author systematically summarized the latest progress of SVA detection methods and vaccine research and development, in order to provide reference basis for the prevention and control of SVA infection.

【Objective】 This study was aimed to explore the mechanism of Forsythia suspense leaves extract (FSLE) against enrofloxacin (ENR)-induced liver injury (EILI) by network pharmacology and in vivo experiments.【Method】 The Forsythia suspense leaves were extracted by ethanol-enzymatic method, whose main chemical components were identified by liquid chromatography mass spectrometry (LC-MS).The effective active ingredients and targets of FSLE were analyzed and selected by PubChem, Swiss ADME and Swiss Target Prediction database, while the EILI targets were screened by GeneCards disease database.STRING online analysis and Cytoscape 3.8.0 software were applied to analyze and construct the core targets of FSLE treats EILI.Metascape website was used to perform GO function and KEGG pathway enrichment analysis of the targets, and Cytoscape 3.8.0 software was hired to construct the "component-target-signal pathway" visual network.The protective effects of FSLE were verified in EILI mice in vivo.The expression of AMP-activated protein kinase (AMPK) was tested by Real-time PCR and Western blotting, and the contents of glutathione (GSH), hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and the activity of total superoxide dismutase (SOD) were detected by commercial kits.【Result】 About 20 oral ingredients in FSLE and their related 338 drug targets were analyzed and screened by LC-MS and Network pharmacology, while 913 protein targeting EILI, 124 protein targets which contained 12 core targets, were screened from drug-disease targets intersection.20 signing pathways were obtained by KEGG pathway enrichment analysis.KEGG pathway showed that AMPK signal pathway had a good affinity to EILI.The animal model experiments showed that FSLE could significantly reduce the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum of mice (P<0.05), and could significantly increase the expression of gene and protein of AMPK (P<0.05).Compared with the normal control group, FSLE could significantly reduce the contents of MDA and H₂O₂ in liver of EILI mice (P<0.05), and significantly increase the GSH content and SOD activity (P<0.05), which reduced the EILI-induced oxidative stress significantly.【Conclusion】 FSLE could protect the liver against ENR induced injury by inducing the expression of AMPK to reduce the oxidative stress, which was helpful for the development of new drugs targeting liver injury.

【Objective】 The purpose of this study was to provide a reference for advancing the construction of Chinese food safety monitoring system and improving related regulations and standards, to ensure food safety while enhancing the competitiveness of Chinese poultry meat and egg products in the international market, breaking international technical trade barriers, and promoting the development of international trade in poultry products.【Method】 Firstly, this study comparatively analyzed the types of veterinary drug residues and the standards of veterinary drug maximum residue limits (MRLs) specified in poultry meat and eggs between the new national standard GB 31650-2019 National Food Safety Standards Maximum Residue Limits of Veterinary Drugs in Food and the abolished Ministry of Agriculture Announcement No.235 Maximum Residue Limits of Veterinary Drugs in Animal Food.Then compared the veterinary drug residue types and veterinary drug MRLs standards in poultry meat and eggs specified by the European Union and the United States.【Result】 The results showed that China's new national standard supplemented and modified the old standard to a certain extent, and most of the MRLs standards of poultry meat and eggs were the same or even stricter than those of the European Union and the United States, which indicated that China's veterinary drug residue standard system had entered a new stage.However, the MRLs standards of some veterinary drugs were still different from those of the European Union and the United States.There were 33 types of veterinary drugs including dicloxacillin whose MRLs standards were missing or looser than those of the European Union and the United States.【Conclusion】 In the future, China still needed to further improve the relevant standards for the limits of veterinary drug residues in poultry meat and egg products based on the production situation, the actual use of veterinary drugs, and the needed of people's consumption upgrades, so as to bring them closer to international standards and improve China's competitiveness of poultry meat and eggs in the international market, and strict monitoring of the residues of veterinary drugs in import and export products, to prevent them from affecting the health and safety of consumers and the development of international trade of poultry meat and eggs.

【Objective】 The aim of this study was to explore the effect of hydrogen sulfide (H₂S) on the acute kidney injury (AKI) of dog induced by cisplatin.【Method】 18 healthy adult Beagle dogs were randomly divided into three groups:Control group (C), cis group and H+cis group.The dogs in control group were injected with normal saline intravenously, the dogs in cis group were injected with 5 mg/kg cisplatin intravenously, the dogs in H+cis group were injected with 1 mg/kg NaHS intravenously, and after 30 min cisplatin was injected, NaHS solution was injected once every 24 h, a total of 3 times.The dogs were anesthetized 72 h after cisplatin injection, and blood was collected from the vein to test the serum creatinine (Scr) and urea nitrogen (BUN).The kidney pathological changes were observed by HE staining.The contents of glutathione (GSH), hydrogen peroxide (H₂O₂) and nitric oxide (NO) in kidney tissues of dog were detected by biochemical kits.The mRNA and protein expressions of IL-1β, IL-6, TNF-α, NF-κB, COX2, iNOS, IL-4 and IL-10 in kidney were detected by Real-time quantitative PCR and Western blotting.【Result】 The levels of Scr and BUN in cis group were extremely significantly higher than those in control group (P<0.01), and the levels of Scr and BUN in H+cis group were extremely significantly lower than those in cis group (P<0.01).The results of pathological examination showed that there were significant pathological changes in kidney of dogs in cis group, while the pathological changes in kidney of dogs of H+cis group were alleviated.The biochemical test results showed that compared with C group, the content of GSH in kidney of cis group was decreased extremely significantly (P<0.01), and the contents of H₂O₂ and NO were increased extremely significantly (P<0.01);Compared with cis group, the content of GSH in kidney of H+cis group was increased extremely significantly (P<0.01), while the contents of H₂O₂ and NO were decreased extremely significantly (P<0.01).The results of Real-time quantitative PCR and Western blotting showed that compared with C group, the mRNA and protein relative expressions of the proinflammatory factor IL-1β, IL-6, TNF-α, NF-κB and COX2 in cis group were increased significantly or extremely significantly (P<0.05;P<0.01), while the mRNA and protein relative expressions of anti-inflammatory factors IL-4 and IL-10 were decreased extremely significantly (P<0.01);Compared with cis group, the mRNA and protein expressions of anti-inflammatory factors IL-4 and IL-10 in kidney of dogs in H+cis group were extremely significantly higher (P<0.01), while the mRNA and protein relative expressions of pro-inflammatory factor IL-1β, IL-6, TNF-α, NF-κB and COX2 were decreased extremely significantly (P<0.01).【Conclusion】 H₂S could improve cisplatin induced AKI in dogs by enhancing the antioxidant capacity of kindey and inhibiting the expression of inflammatory factors.

【Objective】 The study was aimed to isolate and detect the serotype, virulence gene, drug resistance and drug resistance gene of Salmonella from sheep in Zhangjiakou, and analyze the correlation and difference between phenotype and gene.【Method】 The collected samples were inoculated and purified with selective medium.Suspected single colonies were selected for Gram staining, microscopic examination and biochemical identification.PCR identification and serotype identification were carried out according to the nucleotide sequence of Salmonella specific gene invA.SPF Kunming mice were used to test the pathogenicity of the isolates and determine the median lethal dose.Then the virulence island genes hilA, avrA, sseL, ssaQ, mgtC, siiD and sopB, enterotoxin gene stn, plasmid virulence gene spvR were amplified.Kirby-Bauer disk diffusion method was used for drug sensitivity test to amplify drug resistance β-lactamase genes including bla_TEM, bla_CMY, bla_OXA, fluoroquinolone genes qnrS, oqxA, oqxB, sulfonamide resistance genes sul1, sul2, sul3, tetracycline resistance gene tetB and aminoglycoside resistance gene aadA1.According to the PCR results, the target virulence gene and drug resistance gene were sent to sequence, and BLAST comparison analysis was carried out with the corresponding reference gene in NCBI.【Result】 4 strains of Salmonella from sheep were isolated and identified as Salmonella Typhimurium.The isolates were pathogenic to mice, and the median lethal doses of 4 isolates were between 5.67×10⁷ and 6.45×10⁷ CFU.The isolated strains could detect virulence island genes hilA, avrA, sseL, ssaQ, mgtC, siiD, sopB, enterotoxin gene stn and plasmid virulence gene spvR, and the positive detection rates were all >50%.The isolates had strong resistance to compound sulfamethoxazole, rifampicin, lincomycin, penicillin and ampicillin, and were sensitive to gentamicin and ciprofloxacin.Drug resistance was detected in the isolates, β-lactam gene bla_TEM, fluoroquinolone gene qnrS and sulfonamide resistance genes sul1 and sul2 were positive.【Conclusion】 The virulence phenotype of Salmonella isolated from sheep was related to the virulence genes carried by chromosomes and plasmids.The isolated strains carried β-lactam and sulfonamide resistance genes, which were consistent with the drug resistance phenotype.Gentamicin and ciprofloxacin could be used as the first choice in clinic.

【Objective】 The aim of this study was to develop a process for producing Porcine circovirus type 2(PCV2) vaccine based on serum-free suspension culture of PK-15 cells, improve the production efficiency, and reduce the production cost.【Method】 Adherent PK-15 cells were domesticated as suspension cells in serum-free medium by direct domestication method.Then, in serum-free suspension culture system, the domesticated PK-15 cells were continuously passaged to investigate the passage and growth stability.Further, the process parameter such as multiplicity of infection (MOI) (0.10, 0.05, 0.01 and 0.001) and cell density at infection (CDI) (1×10⁶, 3×10⁶ and 5×10⁶/mL) was explored in a 125 mL shake flask.At the same time, the difference of glucose, amino acid, and metabolic byproducts such as lacitc acid, ammonium metabolism in the culture medium before and after cell infection with virus was studied.【Result】 After 30 days direct domestication, PK-15 cells could completely adapt to serum-free suspension culture.The average specific cell growth rate was increased from 0.1 d^-1 to 0.6 d^-1 during domestication.Suspension PK-15 cells could be continuously and stably passaged 15 times, and the average specific growth rate was about 0.6 d^-1, and the cell viability was always>90%.At inoculation density 1.0×10⁶/mL, the peak viable cell density reached 6.2×10⁶/mL on the 4th day and maintained for 1 day.The viability before the 4th day was more than 90%, and then rapidly declined.In the process of virus proliferation, the optimal process parameters for virus production were determined:MOI=0.05, CDI=1.0×10⁶/mL.The virus titer at the final harvest reached 10^6.2 TCID₅₀/mL.Metabolic analysis of cells before and after infection showed that the metabolic rates of glucose, most amino acid were significantly accelerated after virus infection.In the infected group, glucose and glutamine depletion occurred around 72 h, accompanied by rapid accumulation of metabolic by-products lacitc acid and ammonia, then cells would change the metabolic pathway to use lacitc acid.【Conclusion】 Suspended PK-15 cells could be obtained after 30 days direct domestication, and suspended PK-15 cells could be used for proliferation of PCV2.The results provided a theoretical and practical basis for large-scale serum-free suspension culture of PK-15 cells to produce PCV2 vaccine.

【Objective】 This study was aimed to explore the extraction technology of total flavonoids from Vitex negundo L. var. cannabifolia (Sieb. et Zucc.) Hand.-Mazz. (V. negundo) and its antibacterial activity against Clostridium perfringens.【Method】 Rutin was used as reference substance and the content of total flavonoids in V. negundo was determined by spectrophotometry.Taking the content of total flavonoids as the investigation index, the effects of ethanol concentration, extraction time, extraction temperature and solid-liquid ratio on the extraction amount of total flavonoids from V. negundo were studied through single factor test, and the level range of the best process under the condition of single factor was determined.On the basis of single factor test, through the design of L9 (3⁴) orthogonal test, the content of total flavonoids in V. negundo was determined by NaNO₂-AL(NO₃)₃-NaOH color development method, the standard curve was drawn, the regression equation was calculated, and the standard curve was verified through the methodological investigation of precision, stability, repeatability and sample recovery.By substituting the data obtained by the experiment into the standard curve, the best preparation process of total flavonoids of V. negundo was obtained, and on this basis, the antibacterial activity of its extract against Clostridium perfringens was investigated by micro double dilution method.【Result】 The optimum extraction conditions of total flavonoids from V. negundo were ethanol concentration of 60%, extraction temperature of 50 ℃, extraction time of 2.5 h and solid-liquid ratio of 1:35.Under the optimum extraction conditions, the content of total flavonoids in V. negundo was the highest (43.17 mg/g).The order of influencing factors was solid-liquid ratio > extraction time > ethanol concentration > extraction temperature.The solid-liquid ratio had the greatest influence on the content of total flavonoids in V. negundo.The minimum inhibitory concentration (MIC) of V. negundo extract against Clostridium perfringens was 7.81 μg/mL.【Conclusion】 Under the conditions of ethanol concentration of 60%, extraction temperature of 50 ℃, extraction time of 2.5 h and solid-liquid ratio of 1:35, the content of total flavonoids extracted from V. negundo was the highest.The prepared extract had strong antibacterial effect on Clostridium perfringens, which provided a theoretical basis for the production of high-quality V. negundo extract.

【Objective】 The study was aimed to determine the extraction process of Jiawei Pingwei oral liquid.【Method】 The L₉(3⁴) orthogonal test method was used to conduct in-depth research on the extraction process.The volatile oil of medicinal materials containing volatile oil was extracted, and the drug residue was mixed with other medicinal materials for decoction and extraction.In the extraction process of volatile oil, firstly, the particle size of decoction pieces was investigated and the appropriate size of decoction pieces was selected.On this basis, three factors were selected:Solvent volume (A), soaking time (B) and decocting time (C).Three levels were taken for each factor for orthogonal test.Taking the extraction amount of volatile oil as the evaluation index, the extraction process of volatile oil from Atractylodes macrocephala, Magnolia officinalis, tangerine peel, Aucklandia and Fructus aurantii in the prescription was explored and studied, and the process was verified for three times.In the process of water decocting and extracting, three factors were selected:Solvent times (A), decocting time (B) and extraction times (C), and three levels were taken for each factor for orthogonal test.Taking the content of naringin and the total alkali content of matrine (total content of matrine and oxymatrine) as evaluation indexes, the water decoction extraction process was explored and studied, and the process was verified for 3 times.【Result】 Particle size screening results of pieces showed that 1-2 cm pieces were more reasonable.The verification analysis showed that the factors affecting the extraction of volatile oil were decocting time (C) > soaking time (B) > solvent times (A).The extraction process of volatile oil was 8 times water and decocting for 10 h.The factors affecting the water decoction extraction were extraction times (C) > decocting time (B) > solvent times (A).The water decocting extraction process was 8 times water, decocted for 1.5 h, and extracted 3 times.【Conclusion】 This study obtained the specific extraction method and process of Jiawei Pingwei oral liquid through experimental verification.The process had high extraction efficiency, stable and reliable preparation process, could meet the process standard of mass production, and had important practical value.

【Objective】 A clinical case of Bufo gargarizans infection with parasite in multiple organs was clinically diagnosed, and its species classification was studied through molecular biological identification.【Method】 An emaciated female Bufo gargarizans raised in the laboratory died after food refusal was dissected and observed, and the histopathology was studied.The morphology of the isolated parasite was observed and studied by helminth autopsy and nodule pressing method, and the molecular biology was identified.【Result】 Autopsy study showed that the toad' heart, liver, other organs and multi-mesangium were infected by Acanthocephala, and no other common worms such as nematodes and trematodes were found in the toad.HE staining results showed that the infection of Acanthocephala caused mechanical damage and inflammatory reactions to toad' lung, liver and other organs, in which the mucosa of the intestinal wall was seriously damaged and detached.Cytochrome c oxidase subunit Ⅰ (COⅠ) gene was amplified by PCR, five COⅠ gene segments of the species of interest were obtained from different parts, and the Acanthocephala was identified by comparison with the same species in GenBank, the GC contents ranged from 40.14% to 40.83%, and the similarity of JS-01 strain was the closest to Sphaerirostris lanceoides (accession No.:MG931943.1), which was 99.5%.By means of molecular biological analysis and morphological observation, it was determined to be Sphaerirostris lanceoides.【Conclusion】 A large number of infection and parasitism of Sphaerirostris lanceoides caused the death of toads.The results of this experiment could provide reference for the prevention and control of echinocephalosis in medicinal toads in terms of clinical symptoms, pathology and molecular biology, and had reference value for the diagnosis and research of Sphaerirostris lanceoides in amphibians.