【Objective】 The aim of this study was to explore genes associated with brucellosis resistance or susceptibility in sheep，in order to provide molecular markers for brucellosis resistance breeding in sheep. 【Method】 In this study，sheep naturally infected with brucellosis and not vaccinated were used as research subjects，and blood samples were collected for competitive enzyme-linked immunosorbent assay (cELISA). The whole-genome resequencing was conducted on the blood DNA samples. The results of cELISA were used as quantitative trait phenotypes，and GEMMA software was used for genome-wide association study. The clusterProfiler package was used to perform enrichment analysis on significant genes. 【Result】 A total of 56 genes were annotated from the Top 1 000 loci selected by genome-wide association study. GO functional enrichment results showed that annotated genes were mainly concentrated in β-selection and αβ T cell proliferation and differentiation. KEGG pathway enrichment analysis showed that annotated genes were mainly concentrated in Th1/Th2/Th17 cell differentiation and natural killer cell-mediated cytotoxicity. It mainly involved 6 genes，including interferon gamma receptor 1 (IFNGR1)，nuclear factor of activated T cells 2 (NFATC2)，NF-κB inhibitor beta (NFKBIB)，spleen associated tyrosine kinase (SYK)，transforming growth factor beta receptor 2 (TGFBR2)，and Zeta chain of T cell receptor associated protein kinase 70 (ZAP70). The screened items，pathways，and genes were closely related to Brucella infection or the host’s anti-infection response. 【Conclusion】 6 genes related to sheep brucellosis were screened，which laid a foundation for further functional verification at the cellular or individual level，and provided a reference for molecular markers for breeding sheep brucellosis resistance.

【Objective】 The purpose of this study was to construct expression vectors for chicken Caspase 3 (chCaspase 3) gene，identify the protease activity of the expressed protein，and conduct bioinformatics analysis，so as to lay a foundation for further research into the function of chCaspase 3. 【Method】 The cDNA from chicken bursa of Fabricius tissue was used as template for amplification of the chCaspase 3 gene via PCR. The recombinants of both prokaryotic and eukaryotic expression vectors for chCaspase 3 gene were then constructed. The recombinant prokaryotic expression vector was transformed into Escherichia coli BL21(DE3). Following IPTG induction，prokaryotic expression products of chCaspase 3 were subjected to Western blotting analysis. DF-1 cells were transfected with the eukaryotic expression vector in varying amounts(0，0.5，1.0，1.5 and 2.0 μg). The expression products were then analyzed using Western blotting and indirect immunofluorescence assay (IFA). Enzyme activity of the expressed proteins，both prokaryotic and eukaryotic，was assessed using Caspase 3 activity assay kits. Bioinformatic software was employed to assess the similarity of the chCaspase 3 and to forecast its protein’s structural domains and tertiary structure. 【Result】 The CDS region length of the chCaspase 3 gene was 852 bp，encoding 283 amino acids. Both prokaryotic pET28a(+)-chCaspase 3 and eukaryotic p3×Flag-CMV7.1-chCaspase 3 recombinant plasmids were constructed successfully. The prokaryotic expression product had a molecular weight of 36 ku，matching expectations. The expression of eukaryotic expression products were increased with the increase of transfected eukaryotic expression vectors. Caspase 3 enzyme activity was observed in both prokaryotic and eukaryotic expression products. Bioinformatics analysis showed the chCaspase 3 had low similarity with other species and distant phylogenetic relationships. The chCaspase 3 protein contained the conserved QACRG pentapeptide and CASc domain，characteristic of the Caspase family，mirroring the three-dimensional structure of human Caspase 3 (hCaspase 3). 【Conclusion】 The chCaspase 3 gene was cloned and expressed successfully，and its recombinant protein demonstrated Caspase 3 enzyme activity. Bioinformatics predictions revealed the three-dimensional structure of the chCaspase 3 protein closely resembled that of hCaspase 3. This similarity provided a basis for uncovering the function of chCaspase 3 and investigating its antiviral innate immunity.

【Objective】 This study was aimed to explore the regulatory mechanism of miR-141 in glucolipid metabolism and bone metabolism，and provide a basis for establishing animal model related to diabetes mellitus with osteoporosis and researching the regulation mechanism of miR-141 related gene expression. 【Method】 Mature miR-141 sequences of different species were obtained from miRbase database for sequence alignment and conservative analysis used BioEdit and WebLogo softwares. The PROMO and JASPAR online softwares were used to predict the transcription factor binding site of miR-141 in Capra hircus. The target genes of intersection were predicted by TargetScan and miRwalk online tools. GO function and KEGG pathway enrichment analysis of the predicted target genes were performed by DAVID and KOBAS online softwares. The functional protein interaction was analyzed with Networkanalyst database，meanwhile drawing the network regulation diagram. The YM500V2 online database was used to analyze the expression of miR-141 target genes in different tissues of Capra hircus. 【Result】 miR-141 mature sequences among different species were highly conservative. There were many transcription factor binding sites such as C/EBPα，MyoD，YY1 and Sp1 in the promoter region of miR-141 in Capra hircus. A total number of 89 target genes included Nfatc2，Egr2 and Fasl were predicted using different databases. GO function enrichment analysis results showed that the predicted target genes of miR-141 mainly enriched in the regulation of transcription from RNA polymerase Ⅱ promoter and regulation of transcription-DNA-templated for biological process，nucleus and cytoplasm for cellular component，protein binding and DNA binding for molecular function. KEGG pathway enrichment analysis results showed that the target genes were significantly enriched in regulation of actin cytoskeleton，tight junction，axon guidance，Hedgehog signaling pathway，cGMP-PKG signaling pathway，microRNA in cancer and other signaling pathways. miR-141 target genes interaction analysis results revealed that Nfatc2 and Egr2 genes were connected by Fasl gene，which had more targeted relationships with other proteins，and involved in several signaling pathways，such as TGF-β，Hedgehog，Wnt/β-catenin and other pathways. miR-141 was expressed in different tissues of Capra hircus，and its expression exhibited tissue differences. 【Conclusion】 miR-141 participated in regulating some signaling pathways，represented by Hedgehog and Wnt/β-catenin signaling pathways with target genes Nfatc2，Egr2 and Fasl，and further regulated the glucolipid metabolism as well as physiological and biochemical reactions of musculoskeletal system in Capra hircus.

【Objective】 This study was aimed to investigate the role of oxidative stress-responsive 1 (OXSR1) in the apoptosis of Madin-Darby canine kidney cells (MDCK)，so as to provide a reference for better understanding the apoptotic pathway in MDCK and study on interventional therapies targeting OXSR1 gene for MDCK apoptosis. 【Method】 OXSR1 gene knockout cell lines were produced using CRISPR/Cas9 technology on MDCK-Cas9 cells. OXSR1 gene competent and inactivae cell lines were generated by transfecting OXSR1 gene backtill and inactivation plasmids into OXSR1 gene knockout cells. MMC，JNJ-26854165 and Rotenone were used to stimulate wild-type，OXSR1 gene knockout，gene competent and inactive cells，and the apoptosis through p53-dependent and -independent pathways and ROS signaling pathway were detected using flow cytometry. The expression of relevant apoptotic genes were detected by Real-time quantitative PCR. 【Result】 There was apparent Indel features in the target sequences，it confirmed OXSR1 gene knockout cell lines were successfully constructed. Western blotting results indicated that OXSR1 gene competent and inactive cell lines were successfully constructed. Flow cytometry determined that MMC，JNJ-26854165 and Rotenone could induce varying degrees of cell apoptosis. When stimulated with MMC and Rotenone，the apoptosis rate of OXSR1 gene knockout cells was extremely significantly or significantly lower than that of wild-type and OXSR1 gene competent cells (P＜0.01 or P＜0.05)，and there was no significant difference with OXSR1 gene inactive cells (P＞0.05). When stimulated with JNJ-26854165，the apoptosis rate of OXSR1 gene knockout cells was extremely significantly or significantly lower than that of wild-type，OXSR1 gene competent and inactive cells (P＜0.01 or P＜0.05). Real-time quantitative PCR results showed that when cells were stimulated with MMC and Rotenone，the expression of BCL-XL gene in OXSR1 gene knockout cells was extremely significantly or significantly higher than that of wild-type and OXSR1 gene competent cells (P＜0.01 or P＜0.05)，and there was no significant difference with OXSR1 gene inactive cells (P＞0.05). When cells were stimulated with JNJ-26854165，the expression of BCL-XL gene in OXSR1 gene knockout cells was extremely significantly higher than that of wild-type，OXSR1 gene competent and inactive cells (P＜0.01). The expression of NOXA gene in OXSR1 gene knockout cells was extremely significantly lower than that of wild-type，OXSR1 gene competent and inactive cells (P＜0.01). 【Conclusion】 The kinase activity of OXSR1 gene regulated the transcription of BCL-XL gene in MDCK after adding MMC，JNJ-26854165 and Rotenone，and influenced the apoptosis through p53-dependent and-independent pathways and ROS pathway.

【Objective】 The aim of this study was to investigate the inhibitory effect of herbacetin on RSL-3-induced ferroptosis in RAW264.7 macrophages，and explore the effect of herbacetin on the ferroptosis of macrophages. 【Method】 After treatment with 0，5，10，20，40 and 80 μmol/L herbacetin for 48 h，the viability of RAW264.7 macrophage was detected using CCK-8 assay. The cells were divided into control and RSL-3 (15 μmol/L)，herbacetin (20，40 and 80 μmol/L)，ferroptosis inhibitor ferrostatin-1 (10 μmol/L Fer-1) groups. The herbacetin and Fer-1 groups were co-incubated with RSL-3 stimulation for 48 h，and the cell damage effect of each group was detected by lactate dehydrogenase (LDH) assay. The contents of iron ion，glutathione and malondialdehyde (MDA) in cells of each group were measured by colorimetric method. The intracellular Fe²⁺ fluorescence intensity，mitochondrial membrane potential (MMP) and lipid reactive oxygen species (ROS) levels in each group of cells were detected using fluorescence probe method. Western blotting was used to detect the expression of nuclear factor erythroid 2 related factor 2 (Nrf-2)，heme oxygenase-1 (HO-1) and glutathione peroxidase 4 (GPX-4) in cells of all groups. 【Result】 Compared with 0 μmol/L herbacetin，40 μmol/L herbacetin could significantly enhance macrophage viability (P＜0.05). Compared with 40 μmol/L herbacetin，80 μmol/L herbacetin could significantly decrease macrophage viability (P＜0.05). Therefore，5-40 μmol/L could be used as a safe drug concentration. Compared with control group，the LDH activity，the concentrations of total iron ion，Fe²⁺ and Fe³⁺，the average fluorescence intensity of Fe²⁺，GSSG content，the percentage of GSSG in total glutathione，and MDA level of cells in RSL-3 group were significantly increased (P＜0.05). The contents of GSH and total glutathione，the percentage of GSH to total glutathione，the relative percentage of MMP，and lipid ROS fluorescence ratio of cells in RSL-3 group were significantly decreased (P＜0.05)，suggesting the occurrence of ferroptosis. Compared with RSL-3 group，the activity of LDH，the concentrations of total iron ion，Fe²⁺ and Fe³⁺，the average fluorescence intensity of Fe²⁺，the content of GSSG，the percentage of GSSG to total glutathione，and MDA level of cells in 40 μmol/L herbacetin group were significantly decreased (P＜0.05). The contents of GSH and total glutathione，the percentage of GSH to total glutathione，the relative percentage of MMP，and lipid ROS fluorescence ratio of cells in 40 μmol/L herbacetin group were significantly increased (P＜0.05). Compared with Fer-1 group，40 μmol/L herbacetin group showed no significant difference in all the above indicators (P＞0.05). Western blotting results showed that compared with RSL-3 group，40 μmol/L herbacetin could significantly increase the expression of Nrf-2，HO-1 and GPX-4 (P＜0.05). Compared with Fer-1 group，40 μmol/L herbacetin group showed no significant difference in all the above indicators (P＞0.05). 【Conclusion】 Herbacetin regulated the molecular indicators related to ferroptosis induced by RSL-3 in RAW264.7 macrophages，increased the expression of GPX-4，and activated its Nrf-2/HO-1 pathway to exert an inhibitory effect on ferroptosis in RAW264.7 macrophages. Through in vitro experiments，this study preliminarily revealed that 40 μmol/L herbacetin could significantly improve the ferroptosis of macrophages and its mechanism.

【Objective】 The aim of this experiment was to study the protective effect of tannic acid (TA) on oxidative stress injury of mouse oocytes induced by sodium fluoride (NaF),and provide theoretical basis for developing antidotes for fluorosis.【Method】 Mice oocytes were added with 0 (control),0.25,0.50 and 0.75 mmol/L NaF in vitro maturation medium.The optimal concentration of NaF was determined by detecting the first polar body excretion (PBI) rate of oocytes.After treating mouse oocytes with the optimal concentration of NaF,the optimal concentration of TA was determined by adding different concentrations of TA (0,1,10 and 100 μg/L).On the basis of the above tests,control group,toxicity group (NaF) and antidote group (NaF＋TA) were set up.The levels of reactive oxygen species (ROS),glutathione (GSH) and mitochondrial membrane potential (MMP) in mouse oocytes were detected by immunofluorescence staining.The fertilization rate,cleavage rate and blastocyst rate of mouse oocytes in each group were measured.The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured.【Result】 Compared with control group,the excretion rate of the first polar body in 0.25-0.75 mmol/L NaF groups was extremely significantly decreased (P＜0.01).Then 0.25 mmol/L NaF was used as the effective toxic dose for testing.After NaF treatment,the excretion rate of first polar body in 10 μg/L TA group was extremely significantly higher than that in 0 μg/L TA group (P＜0.01).The results of immunofluorescence detection showed that,compared with control group,the ROS levels of oocytes in NaF group were significantly increased (P＜0.05),while the levels of GSH and MMP and the activities of SOD and GSH-Px were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).Compared with NaF group,the levels of GSH and MMP and the activities of SOD and GSH-Px of mouse oocytes in NaF＋TA group were significantly or extremely significantly increased (P＜0.05 or P＜0.01),ROS level and MDA content were significantly or extremely decreased (P＜0.05 or P＜0.01).The statistical results of early embryonic development showed that the fertilization rate,cleavage rate and blastocyst rate in NaF group were extremely significantly lower than those in control group (P＜0.01).The fertilization rate,cleavage rate and blastocyst rate of mice in NaF＋TA group were extremely significantly higher than those in NaF group (P＜0.01),but had no significant difference compared with control group (P＞0.05).【Conclusion】 NaF could induce oxidative stress in mouse oocytes by increasing ROS and MDA levels in oocytes,and reduce MMP levels,which affect early embryonic development.The addition of 10 μg/L TA could relieve the oxidative stress induced by NaF in mice and improve the maturation quality of mouse oocytes,thereby promoting in vitro fertilization and embryo development potential.

【Objective】 This experiment was aimed to investigate the effects of immunomodulatory and nutritional combined additive (INC) on the rumen microbiota and its metabolism of transition dairy cows，so as to improve the rumen health of dairy cows during the periparturient period and increase the milk yield. It also provided technical support for the use and promotion of new type additives. 【Method】 Fourteen transition dairy cows were randomly divided into two groups，with 7 heads in each group according to the milk yield and body condition of the last parity. The experiment lasted for 42 days. Dairy cows in control (CON) group were fed a basal diet，and dairy cows in treatment (TR) group were fed a basal diet supplemented with 60 g/d of INC. Milk samples were collected at 7，14 and 21 d postpartum for determination of milk composition，and milk yield was recorded daily. Rumen fluid was collected from all dairy cows using a rumen catheter after morning feeding at 21 d postpartum for 16S rRNA sequencing and metabolome analysis. 【Result】 ①No significant differences were found in the percentage of milk protein，milk lactose and milk fat between the two groups (P＞0.05)，the milk yield of dairy cows in TR group in the 1st week after delivery was extremely significantly higher than CON group (P＜0.01). ②At the phylum level，Firmicutes and Bacteroidetes were the dominant bacteria in the rumen of dairy cows in both groups. At the genus level，Prevotella and Vibrio succinicum were the dominant bacteria genera. ③A total of 19 different bacteria were detected in the two groups (LDA≥2，P＜0.05)，of which 4 genera，including Rikenellaceae-RC9-gut-group，were significantly enriched in CON group (P＜0.05)，and 15 genera，including Ruminococcaceae，were significantly enriched in TR group (P＜0.05). ④The metabolomics results showed that the relative concentrations of L-formylkynurenine，1H-indole-3-acetic acid and 3-methylindole in TR group were significantly increased. ⑤Lachnospiraceae_UCG_010 in TR group was significantly positively correlated with metabolites (P＜0.05), such as L-formylkynurenine，1H-indole-3-acetic acid and 3-methylindole. Lachnospiraceae_UCG_010 was positively correlated with the tryptophan metabolic pathway and linolenic acid metabolic pathway (P＜0.05)，and was significantly positively correlated with milk production in the 1st week after delivery (P＜0.05). 【Conclusion】 Addition of INC to the diet could increase the abundance of relevant nutrient-degrading bacteria in the rumen of transition dairy cows，improve cow health and rumen fermentation by regulating the flora structure of rumen microorganisms and their metabolism，and ultimately increase milk production of dairy cows.

【Objective】 This study was conducted to investigate the effects of grape seed proanthocyanidins (GSPs) on colon epithelial barrier of sheep fed a high-concentrate diet and the mechanism of alleviating colon epithelial injury through Nrf2/ARE pathway.【Method】 A total of forty-eight 4-month-old 1/2 Dorper × 1/2 thin-tailed Han crossed ram lambs with similar body weight (22.75 kg±1.20 kg) were randomly assigned into four experimental treatment groups，with 12 lambs in each group. Lambs in each group were supplemented with the basal diets of 0 (CON group)，10(10GSPs group)，20(20GSPs group) and 40 mg/kg BW GSPs (40GSPs group)，respectively.The feeding experiment lasted for 60 days，including 15 days adaptation period and 45 days experimental period.On the morning after the trial period ended，6 sheep in each group were slaughtered and colon tissues were collected for morphological and inflammatory cell infiltration analysis.The levels of interleukin-2 (IL-2),IL-4,prostaglandin E₂ (PGE₂) and other inflammatory factors，and the expression levels of nuclear factor-like-2 factor (Nrf2)，heme oxygenase-1 (HO-1)，glutathione peroxidase 4 (GPx4) and other related genes and proteins were detected in colon tissues.【Result】 ①In CON group，colon mucosal epithelial cells were seriously missing，lamina propria was exposed，intestinal gland arrangement was loose，spaces were widened，tight junction protein was missing and damaged. Whereas，the colonic morphological structure and tight junction protein distribution was normal in 20GSPs and 40GSPs groups.②The enzyme activities of myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS)，and the concentrations of nitric oxide (NO)，IL-2 in colon tissues were linearly decreased with the increase of GSPs supplementation (P＜0.05).③Compared with CON group，the gene and protein expression of Nrf2 and HO-1 in colon tissues in 20GSPs and 40GSPs groups were significantly increased (P＜0.05)，and the expression of GPx4 gene in colon tissues in 40GSPs group was significantly increased (P＜0.05).④Compared with CON group，the gene and protein expression of Toll-like receptor 4 (TLR4) in colon tissues of lambs in 20GSPs and 40GSPs groups were significantly decreased，and the gene and nuclear protein expression of nuclear factor kappa B (NF-κB) p65 in colon tissues of lambs in 40GSPs group were significantly decreased (P＜0.05). The gene and protein expression of tumor necrosis factor α (TNF-α) and protein expression levels of IL-1β in colon tissues of lambs in 20GSPs and 40GSPs groups were also significantly lower than those in CON group (P＜0.05).【Conclusion】 GSPs could reduce local inflammation of colon tissue and alleviate colon epithelial tissue damage by activating Nrf2 pathway and inhibiting the activation of NF-κB inflammatory signaling pathway in sheep fed high-concentrate diet.

【Objective】 This study was aimed to investigate the effects of different levels of capsaicin (CAP) supplementation on production performance，egg quality，follicle development and reproductive hormones of Changshun Green-shell laying hens at the late laying period，and estimate the appropriate dose of CAP in diet of laying hens. 【Method】 Five hundred Changshun Green-shell laying hens (56 weeks of age) were chose and randomly allocated into 5 treatments，containing 10 replicates per treatment and 10 hens per replicate. Hens in group Ⅰ were supplied with the basal diet，and those in groups Ⅱ，Ⅲ，Ⅳ and Ⅴ were separately fed with diets containing 100，150，200 and 400 mg/kg CAP based on the basal diet. The experimental period was 8 weeks. At the end of the experiment，the production performance and egg quality of hens were determined，the blood and ovarian tissue were collected to determine the levels of reproductive hormones and the indicators of reproductive organ development. 【Result】 Compared with group Ⅰ，①The laying rate and egg mass of hens in groups Ⅱ-Ⅴ were significantly increased at 1-4，5-8 and 1-8 weeks of the experimental period (P＜0.05). The average daily feed intake of hens at 1-4 and 5-8 weeks of the experimental period were significantly increased (P＜0.05). ② The yolk color of eggs in groups Ⅱ-Ⅴ was significantly enhanced at the 4th week of the experimental period，while at the 8th week of the experimental period，the yolk color of eggs was significantly improved only in groups Ⅱ and Ⅲ (P＜0.05). Besides，the Haugh unit of eggs in group Ⅱ was significantly improved at the 4th and 8th week of the experimental period (P＜0.05). In addition，the Haugh unit of eggs in group Ⅲ was significantly improved at the 4th week of the experimental period (P＜0.05). ③The relative weights of ovarium，large yellow follicles and small yellow follicles of hens in groups Ⅱ-Ⅴ were significantly increased (P＜0.05). Moreover，the number of small yellow follicles，and the contents of follicle-stimulating hormone，luteinizing hormone，progesterone and estradiol-17 β in serum of hens in groups Ⅱ-Ⅳ were significantly increased (P＜0.05). 【Conclusion】 Taken together，dietary CAP supplementation contributed to improving the production performance and egg quality，promoting follicle development and increasing the levels of productive hormone in Changshun Green-shell laying hens at the late laying period，and the better addition dose was 242-281 mg/kg.

【Objective】 The purpose of this experiment was to study the stability and metabolism of red clover isoflavones in rumen. 【Methods】 In in vivo experiment，3 fistulated Holstein cows were selected and fed 2 g of red clover extract (isoflavone content 22.91%) into the rumen before morning feeding. Rumen fluid was collected at 0，0.5，1，2，4，8，12 and 14 h，respectively，and HPLC was used to analyze the isoflavone contents. In in vitro rumen fermentation，3 treatment groups were set up，0.1 g/120 mL (high-dose group)，0.01 g/120 mL (low-dose group)，and no red clover extract group (control group)，with 5 replicates in each group，and fermented at 39 ℃. Rumen fluid was collected at 0，0.5，1，2，4，8，12 and 14 h，respectively. HPLC was used to analyze the contents of isoflavones. 【Results】 In in vivo experiments，biochanin A and formononetin were metabolized at 14 and 8 h,respectively. After 2 h，the metabolite S-equol was present in the rumen. The half-lives of biochanin A and formononetin were 1.02 and 2.25 h，respectively，while those of daidzein and genistein were 3.38 and 0.77 h，respectively.In in vitro experiment，the half-lives of the four isoflavones in the high-dose group and the low-dose group were different. In the high-dose group，the half-lives of biochanin A，formononetin，daidzein and genistein were 2.45，0.70，12.62 and 5.74 h，respectively. In the low-dose group，the half-lives of biochanin A，formononetin，daidzein and genistein were 2.82，0.87，13.11 and 3.45 h，respectively. 【Conclusion】 Red clover isoflavones were unstable in the rumen of dairy cows and could be catabolized by microorganisms. The half-lives of the main isoflavones，biochanin A and formononetin were all less than 4 h. It was speculated that the metabolic end products of the two isoflavones were S-equol and ethylphenol，respectively.

As an unconventional protein feed，mulberry leaves have high nutritional and economic value. Mulberry leaves are palatable and rich in protein，amino acids，flavonoids，alkaloids，polyphenols，polysaccharides and other bioactive substances. In recent years，with the development and utilization of mulberry resources，the research on the application of mulberry leaves and its extracts in the field of animal husbandry has become more and more in-depth. As a high-quality feed source in livestock and poultry production，mulberry leaves are mainly processed by drying，silage，fermentation，extraction of active substances，etc. Different processing methods have different effects on the composition and content of nutrients in mulberry leaves. Adding mulberry leaf or its extract to the diet of livestock and poultry can improve the immunity，survival rate，production performance and antioxidant capacity of young livestock and poultry，improve intestinal microecology，regulate metabolism，and improve the quality of livestock and poultry products. The application of mulberry leaves varies among different animals，and different developmental stages of the same animal species. The authors summarized the feeding value，processing methods and application of mulberry leaves in livestock and poultry production，in order to provided the basis for the rational development and utilization of mulberry leaves and was conducive to promoting the high-quality development of animal husbandry.

【Objective】 This study was aimed to explore the organism response of dairy cows with different parities to 25 hydroxyvitamin D₃，analyze the influence of parity on the production performance of dairy cows，postpartum disease incidence and the growth performance of progeny calves，and then reveal the specific role of periparturient dairy cows parity on the nutritional effect of 25 hydroxyvitamin D₃. 【Method】 Twenty-six healthy Holstein dairy cows with a consistent body condition score (3.25) and 21 days before the expected date of calving were selected and assigned to primiparous group (parity=1，n=11) and mulitiparous group (parity≥2，n=15) according to parity size. All dairy cows were fed a uniform ration，which was supplemented with 3 mg of 25-hydroxyvitamin D₃ per head per day for 82 days (the 22nd days was the expected calving day). Daily milk production was recorded，and milk samples were collected at 0，7，and 21 days post-calving for milk composition testing. Feed and blood samples were collected at 21 and 7 days prior to calving (―21 and ―7 days)，on the day of delivery (0 days)，and 7 and 21 days after calving. Blood samples were collected from dairy cows and progeny calves to test the routine nutrients in feed，blood immunity and antioxidant indexes，and blood bone and collagen metabolism indexes，respectively. The body weight and body size of progeny calves were measured at both 0 and 60 d. 【Result】 ①Compared with primiparous dairy cows，the milk yield of mulitiparous dairy cows at 7 and 21 d postpartum was extremely significantly or significantly increased (P＜0.01 or P＜0.05)，the non-fat milk solids of mulitiparous cows at 7 d postpartum were significantly decreased (P＜0.05). ②Compared with mulitiparous dairy cows，the immunogbulin G (IgG) content of blood in primiparous dairy cows was significantly decreased (P＜0.05). The calcitonin (CT) contents of blood in mulitiparous dairy cows at ―21 and ―7 d of calving were significantly increased (P＜0.05). The tartrate-resistant acid phosphatase (TRAP) levels of blood in mulitiparous dairy cows at 7 d of calving were extremely significantly increased (P＜0.01)，the contents of CT and IgM were significantly decreased (P＜0.01)，while the tumor necrosis factor-α (TNF-α) content was extremely significantly increased (P＜0.01). The contents of IgM and TNF-α of blood in mulitiparous dairy cows at 21 d of calving were significantly or extremely significantly increased (P＜0.05 or P＜0.01). ③At 60 days of age，the body height of progeny calves of mulitiparous dairy cows was extremely significantly higher than that of progeny calves of primiparous dairy cows (P＜0.01). 【Conclusion】 A significant difference was observed in the nutritional effects of 25-hydroxyvitamin D₃ in primiparous and mulitiparous periparturient dairy cows. The administration of 25-hydroxyvitamin D₃ during the perinatal period resulted in an enhancement of haematological immunocompetence in primiparous dairy cows，as well as an acceleration of bone and collagen metabolism in mulitiparous dairy cows. Additionally，the progeny calves of these dairy cows exhibited an increase in body height at 60 days of age.

【Objective】 The effects of different levels of Allium mongolicum Regel powder on carcass characteristics and meat quality of Angus calves were studied in order to provide reference for the application of Allium mongolicum Regel powder in calves production.【Method】 24 calves aged (14±2) months，with good health and similar body weight (271.17 kg±17.60 kg)，were randomly divided into 4 groups，with 3 replicates in each group and 2 calves in each replicate. The calves in control group were fed with basic diet (CON group)，and the calves in experimental groups were fed with Allium mongolicum Regel powder of 10 (low level group，LAMR)，15 (middle level group，MAMR) and 20 g (high level group，HAMR) on the basis of CON group. The experiment lasted for 135 days，pre-test period lasted for 15 days，and experimental period lasted for 120 days. After the experiment，the live body weight before slaughter was measured，and the carcass characteristics，high-grade meat and high-quality meat yields were measured after slaughter. The longissimus dorsi muscle of the left thoracic segment of the carcass was collected to determine the meat quality related indicators. 【Result】 Compared with CON group，① The live body weight of calves before slaughter in LAMR group was significantly increased (P＜0.05)，and the renal index in HAMR group was significantly decreased (P＜0.05). ② The crude ash contents of longissimus dorsi muscle of calves in LAMR and MAMR groups were extremely significantly increased (P＜0.01)，the crude fat content in HAMR group and the crude protein content in LAMR group were significantly and extremely significantly increased (P＜0.05，P＜0.01). ③ With the increase of Allium mongolicum Regel powder，the Topside in high-quality meat showed a linear increase (P＜0.05)，and the weight of Outside flat in high-quality meat and the ratio of high-quality meat to net meat both decreased first and then increased (0.05＜P＜0.10). ④ In LAMR，MAMR and HAMR groups，the muscle fiber area and muscle fiber diameter of the longissimus dorsi muscle were extremely significantly and significantly decreased (P＜0.01，P＜0.05). ⑤ The shearing force of longissimus dorsi muscle in LAMR，MAMR and HAMR groups were decreased extremely significantly (P＜0.01)，the cooking loss in LAMR group decreased significantly (P＜0.05)，and the cooked meat rate of longissimus dorsi muscle in LAMR and HAMR groups increased significantly (P＜0.05).【Conclusion】 Adding 10 g/day Allium mongolicum Regel powder to the diet was beneficial to muscle protein deposition，muscle growth and final weight improvement of Angus calves. Adding 20 g/day Allium mongolicum Regel powder to the diet was beneficial to intramuscular fat deposition and beef tenderness.

【Objective】 To investigate the effects of vitamin D₃(VD₃) supplementation on growth performance,apparent digestibility of nutrients and serum biochemical indexes of 10-12 months old sika deer (Cervus nippon).【Method】 Twenty-four healthy male sika deers aged 10 months with an average body weight of 45.32 kg±5.23 kg were randomly divided into 4 groups with 6 deers in each group and 1 deer per replicate,which were fed basal diet supplemented with 0 (control group)，2 000，4 000 and 8 000 IU/kg VD₃,respectively. The experiment lasted for 90 days. At the ending of the experiment，the body weight and feed intake of sika deer were counted to determine the growth performance.Blood samples were collected to determine the serum levels of glutathione peroxidase,total superoxide dismutase(T-SOD) and alkaline phosphatase, and the levels of malondialdehyde,immunoglobulin A,immunog-lobulin G,immunoglobulin M,parathyroid，calcitonin，1,25 hydroxyvitamin D₃,calcium and phosphorus. Since the 75th day，fecal samples were collected for 3 days，and the contents of dry matter,crude protein,crude fat,acid detergent fiber,neutral detergent fiber,calcium and phosphorus were determined.【Result】 ①Compared with control group,diet supplemented with 4 000 IU/kg VD₃ significantly increased the average daily gain of sika deer (P＜0.05),but there was no significant difference among other groups (P＞0.05). ②The apparent digestibility of Ca in 4 000 IU/kg group was significantly higher than that in control group (P＜0.05)，but there was no significant difference among other groups (P＞0.05).③The serum T-SOD levels of sika deer in 2 000，4 000 and 8 000 IU/kg groups were significantly higher than that in control group (P＜0.05),but there was no significant difference among 2 000，4 000 and 8 000 IU/kg groups (P＞0.05). Diet supplemented with 8 000 IU/kg VD₃ significantly increased serum 1,25 hydroxyvitamin D₃ level (P＜0.05)，but there was no significant difference among other groups (P＞0.05).【Conclusion】 Dietary VD₃ supplementation was beneficial to improve growth performance，antioxidant capacity and serum 1,25 hydroxyvitamin D₃ content of sika deer. According to the indexes determined in this experiment,4 000 IU/kg VD₃ had a good application effect on 10-12 months old sika deer.

【Objective】 The aim of this experiment was to study the effects of dietary nutrient levels on the growth performance，body measurement traits，slaughter performance，meat quality，serum biochemical indices and rumen fermentation parameters of Duolang sheep，so as to provide an important reference for the scientific feeding of sheep. 【Method】 Eighteen healthy，3-4 month old Duolang sheep with similar body weight were randomly divided into 3 groups with 6 replicates per group and 1 sheep per replicate. The concentrate-to-roughage ratio of the diets Ⅰ，Ⅱ and Ⅲ were 35∶65，50∶50 and 65∶35，respectively. The experiment period lasted for 74 days，including 14 days of adaption period and 60 days of formal period. Sheep were weighed，measured body size and collected blood from the jugular vein at the 60th days before morning feeding. After slaughter，rumen contents were collected，the slaughter performance and meat quality were determined. 【Result】 ① Compared with diet Ⅰ，diet Ⅲ could significantly increase the average daily gain and chest circumference of sheep (P＜0.05). ② Compared with diet Ⅰ，diet Ⅱ could significantly increase the dressing percentage of sheep (P＜0.05)，and diet Ⅲ could extremely significantly increase the carcass weight，dressing percentage and liver weight of sheep (P＜0.01). ③ Compared with diet Ⅱ，diet Ⅰ could extremely significantly or significantly increase a^*_{24 h}，pH_{24 h} and dripping loss rate of longissimus dorsi muscle in sheep (P＜0.01 or P＜0.05)，and extremely significantly or significantly increase pH_{45 min} and pH_{24 h} of biceps femoris muscle in sheep (P＜0.01 or P＜0.05). Diet Ⅲ could extremely significantly or significantly increase a^*_{24 h} and b^*_{24 h} of longissimus dorsi muscle in sheep (P＜0.01 or P＜0.05). Compared with diet Ⅱ，diet Ⅰ could extremely significantly or significantly increase L^*_{24 h} and b^*_{45 min} of biceps femoris muscle in sheep (P＜0.01 or P＜0.05). ④ Compared with diet Ⅱ，diet Ⅲ could significantly increase the urea nitrogen of serum in sheep (P＜0.05). ⑤ Compared with diet Ⅰ，diet Ⅲ could extremely significantly or significantly increase the contents of ruminal valerate，lactic acid，isobutyrate and isovalerate of sheep (P＜0.01 or P＜0.05)，significantly decrease A/P (P＜0.05)，and diets Ⅱ and Ⅲ could significantly increase the ammonia nitrogen level (P＜0.05). 【Conclusion】 In conclusion，fed total mixed ration with a concentrate-to-roughage ratio of 65∶35 diet could improve the growth performance，slaughter performance and meat quality，promote liver development，change rumen fermentation type，and increase the content of total volatile fatty acid in Duolang sheep.

【Objective】 The experiment was conducted to investigate the effects of feed supplementation with avian autochthonous Bacillus coagulans BC-362 on the growth performance，antioxidant capacity and immune function of laying chicks infected with Salmonella Pullorum (SP).【Method】 150 one-day-old healthy Jinghong-1 SPF laying chicks with similar body weights were randomly divided into control group (group A)，probiotic group (group B)，SP + probiotic group (group C)，SP + antibiotic group (group D)，and SP model group (group E). Each group was comprised of 6 replicates，with five chicks per replicate. The basal diet was given to groups A，D，and E，while groups B and C were supplemented with 1.0×10⁸ CFU/g of Bacillus coagulans in the basal diet. At 7 days，groups C，D，and E were gavaged with 1.0×10⁹ CFU of SP (0.5 mL)，and groups A and B were gavaged with the same dose of sterile water. Additionally，group D was supplemented with 0.375 g/kg of neomycin sulphate in the basal diet starting from the second day of gavage. The experimental period lasted for 14 days. At 14 days，the growth performance of the chicks was measured; Blood，immune organs，and jejunum tissues were collected to determine levels of serum antioxidant and immunity，immune organ index，jejunum morphology，CD3 immunohistochemistry expression，and transcript levels of inflammatory and antioxidant-related genes.【Result】 ①Compared with group A，the average daily gain (ADG) of laying chicks in group B was significantly increased，and the feed to gain ratio (F/G) was significantly decreased (P＜0.05). In group E，both the average daily feed intake (ADFI) and ADG of laying chicks were significantly decreased (P＜0.05). Compared with group E，the ADFI and ADG of laying chicks in groups C and D were significantly increased (P＜0.05). There were no significant differences in ADFI，ADG，and F/G of laying chicks between groups C and D (P＞0.05).②Compared with group A，the activities of serum superoxide dismutase (SOD) and catalase (CAT) of laying chicks were significantly higher (P＜0.05) in group B, and the activities of serum SOD，CAT and glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) of laying chicks were significantly decreased (P＜0.05)，while the malondialdehyde (MDA) content were significantly increased (P＜0.05) in group E.Compared with group E，serum SOD activity and T-AOC of laying chicks were increased significantly (P＜0.05)，and the MDA content of laying chicks were decreased significantly (P＜0.05) in both groups C and D. The activities of serum CAT and GSH-Px of laying chicks in group C were significantly higher than those in group D (P＜0.05). ③Compared with group A，the serum IgA and IgM contents of laying chicks were significantly higher in group B (P＜0.05)，while the serum IgA，IgM and IgG contents of laying chicks were significantly lower in group E (P＜0.05). Compared with group E，the serum IgA，IgM，and IgG contents of laying chicks were significantly increased in groups C and D (P＜0.05)，with the IgM content of laying chicks in group C was significantly higher than that in group D (P＜0.05).④Compared with group A，the thymus index of laying chicks were significantly higher in group B (P＜0.05)，and the immune organ index of laying chicks were significantly lower in group E (P＜0.05). The thymus and bursa indices of laying chicks were significantly higher in groups C and D compared to group E (P＜0.05). The spleen index of laying chicks were significantly higher in group C than that in group D (P＜0.05). ⑤Compared with group A，there was no significant difference in the number of goblet cells and CD3 expression in jejunal tissues of laying chicks in group B (P＞0.05)，while the numbers of goblet cells and CD3 expression were significantly reduced in group E (P＜0.05). The expression of CD3 in jejunal tissues of laying chicks were significantly higher in groups C and D compared to group E (P＜0.05)，and the number of goblet cells in jejunal tissues of laying chicks in group C was significantly higher than that in group D (P＜0.05).⑥Compared with group A，the relative expression of inflammation-related genes Toll-like receptor 4 (TLR4)，myeloid differentiation factor 88 (MYD88)，nuclear factor kappa-B (NF-κB) and interleukin-6 (IL-6) in jejunal tissues of laying chicks were significantly downregulate in group B (P＜0.05)，while the relative expression of antioxidant-related genes Kelch-like ECH associated protein 1 (Keap1)，glutathione peroxidase 1 (GPX1)，and glutamate-cysteine ligase catalytic subunit (GCLC) were significantly upregulate (P＜0.05). The relative expression of inflammation-related genes TLR4，MYD88，IL-1β，NF-κB and IL-6 in jejunal tissues of laying chicks in group E was significantly upregulated (P＜0.05)，while the relative expression of antioxidant-related genes NADH quinone dehydrogenase 1 (NQO1)，heme oxygenase 1 (HO-1)，GCLC，and nuclear factor E2-associated factor (Nrf2) were significantly downregulated (P＜0.05). In groups C and D，the relative expression of inflammation-related genes TLR4，MYD88，IL-1β，NF-κB and IL-6 of laying chicks were significantly lower than those in group E (P＜0.05)，whereas the relative expression of antioxidant-related gene NQO1 and GCLC was significantly higher than in group E (P＜0.05). Compared with group D，the relative expression of TLR4，MYD88，and IL-1β was significantly downregulate (P＜0.05)，and the relative expression of Keap1 and GCLC was significantly upregulate in group C (P＜0.05).【Conclusion】 The supplementation of avian autochthonous Bacillus coagulans BC-362 to diets improved the growth performance of SP-infected laying chicks and enhanced the body’s antioxidant and immune capabilities，thereby reducing the intestinal inflammatory response infected with SP in laying chicks.

【Objective】 The aim of this study was to compare and analyze the nutrient content of the whole plant，stem，leaves and flowers of different growing stages of Sophora alopecuroide，determine the most suitable harvesting period，and use different strains to ferment the cut of Sophora alopecuroide. 【Method】 The whole plant，stem，leaves and flower samples of Sophora alopecuroide at different growth stages were collected and the contents of crude protein (CP)，crude fat (EE)，neutral detergent fiber (NDF)，acid detergent fiber (ADF) and crude ash (Ash) were determined. A single-factor completely randomized experiment design was adopted，and the fermentation experimental groups were control group (CK group without adding bacterioides)，EM bacterial solution group (E group，added 2 mg/kg EM bacterial solution)，Bacillus licheniformis group (D group，added 31.25 mg/kg Bacillus licheniformis powder)，Bacillus subtilis group (K group，added 31.25 mg/kg Bacillus subtilis powder) and Enterococcus faecalis group (F group，added 25 mg/kg Enterococcus faecalis powder)，with 4 replicates in each group. The prepared fermentation material was fermented at 37 ℃ for 96 h，the sensory quality of the fermentation material was evaluated，and the nutrient contents of the fermented feed were determined. 【Result】 CP content in whole plant and stem of Sophora alopecuroide at non-flowering stage and initial flowering stage was significantly higher than that at full flowering stage，ADF content in whole plant and NDF content in leaves was significantly lower than that at full flowering stage (P＜0.05)，but there was no significant difference between non-flowering stage and initial flowering stage (P＞0.05). The NDF content in whole plant of Sophora alopecuroide in non-flowering stage was significantly lower than that in initial flowering stage and full flowering stage (P＜0.05)，but there was no significant difference between initial flowering stage and full flowering stage (P＞0.05). The content of NDF in the stems of Sophora alopecuroide at the non-flowering stage was significantly lower than that at the full flowering stage (P＜0.05)，but there was no significant difference between the initial flowering stage and the non-flowering stage and the full flowering stage (P＞0.05). The significant changes of ADF content in the stems of Sophora alopecuroide were full flowering stage ＞ non-flowering stage ＞ initial flowering stage (P＜0.05). The content of CP in Sophora alopecuroide flowers at initial flowering stage was significantly higher than that at full flowering stage，while the contents of NDF and ADF were significantly lower than that at full flowering stage (P＜0.05). After fermentation by Bacillus licheniformis and Bacillus subtilis，the Sophora alopecuroide had no butyric acid odor and aromatic fruit or bread flavor，and the leaf structure was good，showing light brown，and the score was excellent. The content of CP in fermented feed of Sophora alopecuroide showed significant changes： F＜CK＜E,D and K groups (P＜0.05)，but there was no significant difference among groups E，D and K groups(P＞0.05). The content of EE in fermented feed of Sophora alopecuroide showed significant changes： F＜D＜CK groups (P＜0.05).The NDF in fermented feed of Sophora alopecuroide showed significant changes： D＜CK,E and K＜F groups (P＜0.05)，but there was no significant difference among CK，E and K groups(P＞0.05). The ADF in fermented feed of Sophora alopecuroide showed significant changes： D，K and F＜CK and E groups (P＜0.05)，while there was no significant difference between E and CK groups，and no significant difference among groups D，F and K (P＞0.05). There was no significant difference in Ash content among the groups (P＞0.05).【Conclusion】 There were some differences in the nutrient content of Sophora alopecuroide in different growing stages，and the nutritional value of Sophora alopecuroide was higher in the non-flowering stage and the initial flowering stage，so the selection of cutting at this stage was suitable. The fermentation effect of different strains on Sophora alopecuroide at the initial flowering stage was different，among which Bacillus subtilis and Bacillus licheniformis had the best fermentation effect.

With strict restrictions on the use of antibiotics in animal husbandry worldwide，seeking safe and effective alternatives has become an important issue in the field of animal production. Plant derived antibacterial substances have attracted much attention due to their natural，safe，and environmentally friendly characteristics. Among them，cinnamaldehyde，as an active ingredient from cinnamon essential oil，has shown great potential for application in pig production due to its significant biological activity. The article analyzes the antibacterial，anti-inflammatory，antioxidant and immunomodulatory activities of cinnamaldehyde，reviews the research progress of its application in pig production，and explores its practical effects and mechanisms in promoting growth performance，improving intestinal health，optimizing growth environment，and preventing and treating diseases. In terms of growth performance，adding cinnamaldehyde to feed can improve the average daily weight gain and feed conversion rate of pigs，thereby bringing economic benefits to producers. In terms of improving intestinal health，cinnamaldehyde can reduce the occurrence of intestinal diseases such as diarrhea by maintaining the balance of intestinal microbiota and enhancing intestinal barrier function. In terms of optimizing the growth environment，the use of cinnamaldehyde reduces the emission of harmful gases and improves the quality of the aquaculture environment. In terms of disease prevention and treatment，the application of cinnamaldehyde has also shown positive effects. In the future，further research is needed on the mechanism of action of cinnamaldehyde to better understand and utilize its biological functions in pig production，and more clinical trials should be actively conducted to verify its effectiveness and safety in different breeding environments and pig breeds. The article aims to provide useful references for researchers and producers in related fields，and lay a solid foundation for further research and application of cinnamaldehyde in pig production.

【Objective】 The aim of this study was to investigate the effects of ellagic acid (EA) on the quality and antioxidant properties of boar semen during room temperature preservation，in order to provide a theoretical basis for the promotion and application of EA in semen preservation.【Method】 Semen was collected from four healthy adult Duroc boars aged 2 to 3 years. The qualified semen was divided into five portions and diluted with BTS (Beltsville thawing solution) diluted at room temperature，which contained different concentrations of EA (0 (control),5，10，15 and 20 μg/mL). Sperm motility，average path velocity，swing amplitude，whipping frequency，plasma membrane integrity rate，acrosome integrity rate，and antioxidant capacity were assessed on the 1st，3rd，5th，and 7th days of preservation. 【Result】 On the 1st day of preservation，plasma membrane integrity was significantly higher in 15 μg/mL EA group compared to control group (P＜0.05)，and there was no significant difference in other indicators among experimental groups and control group (P＞0.05). On the 3rd day of semen preservation，the average path velocity，catalase (CAT) activity，and total antioxidant capacity (T-AOC) of sperm in 10 and 15 μg/mL EA groups were significantly higher than those in control group (P＜0.05). On the 5th day of semen preservation，the swing amplitude of sperm in 15 and 20 μg/mL EA groups was significantly lower than that in control group and the 5 and 10 μg/mL EA groups (P＜0.05)，and the sperm motility in 5，10 and 15 μg/mL EA groups was significantly higher than that in control group (P＜0.05). The CAT，glutathione peroxidase (GSH-Px) activitives，and T-AOC in the semen of 10，15 and 20 μg/mL EA groups were significantly higher than those in control group (P＜0.05). On the 7th day of semen preservation，the sperm motility，average path velocity，plasma membrane integrity，acrosome integrity，CAT activity，and T-AOC in each experimental group were significantly higher than those in control group (P＜0.05). 【Conclusion】 The incorporation of EA into BTS dilution solution was found to offer a protective effect on semen during room-temperature storage，with the optimal outcome observed at an EA concentration of 10 μg/mL.

【Objective】 The purpose of this experiment was to screen the key genes and related pathways that affected the litter size of Yangyuan donkeys，and improve the reproductive performance of Yangyuan donkeys. 【Method】 The venous blood samples of healthy Yangyuan donkeys with twins and singleton were used for transcriptome high-throughput sequencing. After comparison with the reference genome of the African wild donkey (Equus asinus (ass))，the differentially expressed genes were screened,GO function and KEGG pathway enrichment analysis were performed on the differentially expressed genes to screen the genes related to the litter size of Yangyuan donkeys. Five differentially expressed genes were randomly selected,and Real-time quantitative PCR was used for expression quantitative verification. 【Result】 A total of 1 336 differentially expressed genes were screened out between twins and singleton groups，among which 723 genes were upregulated，and 613 genes were downregulated. GO function enrichment analysis showed that the differentially expressed genes were mainly enriched in receptor activity，signal transduction and other terms. KEGG pathway enrichment analysis showed that ovarian steroidogenesis and estrogen signaling pathways were in top 20 enriched pathways，and a total of 4 candidate genes related to the litter size of Yangyuan donkeys were screened out，including IGF1，IGF1R，INSR and ADCY3. Real-time quantitative PCR results were consistent with the transcriptome sequencing results，and the transcriptome sequencing results were reliable. 【Conclusion】 The enrichment of differentially expressed genes in Yangyuan donkeys with different number of foals was found in ovarian steroidogenesis and estrogen signaling pathways，IGF1，IGF1R，INSR and ADCY3 genes were involved in ovarian hormone secretion and up-regulated in twins group. The results provided a reference for further clarifying the molecular mechanism of different litter sizes in Yangyuan donkeys.

Spermatogenesis is a highly heterogeneous process in time and space that occurs in the mammalian testis through a series of cellular differentiation and development，culminating in the formation of spermatozoa. With the continuous updating and development of bioinformatics，most previous studies have relied on the analysis of RNA from various cell types. However，isolating each cell type from the testis is very challenging，and therefore relatively few studies have been conducted on different types of testicular germ cells at the transcriptome level. In recent years，due to the progress and development of science and technology，single-cell RNA sequencing (scRNA-Seq) has introduced a variety of sequencing platforms，and its scope of use and scenarios continue to expand，with a wide range of applications in the fields of neurology，oncology，immunity，growth and development and disease，and the research objects are mainly related to human，model organisms and domestic animals. This technology can efficiently capture individual cells in the testis for high-throughput sequencing analysis to obtain their transcriptome-level information，finely characterize their cell types，reveal their cellular heterogeneity，and infer their developmental trajectories. Currently，scRNA-Seq has begun to explore the mechanisms related to spermatogenesis in livestock from the perspective of livestock germ cells，so as to further study the processes related to spermatogenesis in livestock. Currently，scRNA-Seq has begun to explore the mechanisms of spermatogenesis in livestock from the perspective of livestock germ cells，so as to further study the process of spermatogenesis in livestock. Therefore，based on the description of scRNA-Seq technology，the author summarizes and discusses its application and research progress in the field of spermatogenesis in domestic animals such as cattle，sheep and swine，so as to provide a theoretical basis for the study of spermatogenesis in domestic animals.

The cryopreservation is an effective method commonly used in production to preserve animal semen，while cryopreservation process of animal sperm can lead to sperm structure，metabolic pathways，physiological functions and vitality alterations caused by oxidative stress，subsequently affecting sperm quality and fertilization rate. Melatonin，which is widely used in the cryopreservation of animal semen in animal production，exhibits potent antioxidant properties，scavenges free radicals generated by oxidative stress，and improves the viability and fertilization rate of sperm after cryopreservation. The mechanism of melatonin protects sperm mainly through the following pathways. Firstly，melatonin can increase the activities of antioxidant enzymes glutathione peroxidase (GSH-Px)，catalase (CAT) and superoxide dismutase (SOD) in semen，and reduces lipid oxidation and oxidative products in semen. Secondly，melatonin can improve oxidative phosphorylation，electronic transport chain and tricarboxylic acid cycle，and ATP production in mitochondria. Thirdly，melatonin can improve the integrity of sperm plasma membrane，acrosomal membrane，mitochondrial membrane and DNA structure. Finally，melatonin also can enhance the anti-apoptotic effect and inhibit the expression of apoptotic proteins and genes. The authors review an overview of the antioxidant properties of melatonin，antioxidant enzyme activities，antioxidant enzyme gene expression，signaling pathways，and the effects on the structure and function of sperm plasma membrane, DNA，mitochondria，and related molecular mechanisms，in order to provide a theoretical reference for the use of melatonin as an antioxidant in the preservation of semen in the future.

【Objective】 This study was aimed to develop an ELISA detection method for serum antibodies against Ornithobacterium rhinotracheale (ORT).【Method】 The outer membrane protein OR02 of ORT was cloned and expressed using prokaryotic expression methods.The immunogenicity of the purified expression product was confirmed by Western blotting.The purified product was used as an antigen coated on a solid-phase carrier,and the antigen coating concentration and serum dilution ratio were determined using checkerboard titration method.In addition,the reaction conditions for ELISA detection were optimized,and the cut-off value for the detection method was determined using ORT negative serum.The specificity of the method was evaluated by testing positive sera for other pathogens,and the sensitivity was assessed by testing ORT positive sera at different dilution ratios.Furthermore,a comparison was made with a commercial assay kit to evaluate the concordance of the ELISA method.【Result】 The recombinant protein OR02 was expressed in inclusion bodies with a molecular weight of 58 ku,consistent with the expected size.Western blotting revealed specific binding of the OR02 recombinant protein with positive serum samples for,indicating good immunoreactivity.Using the purified OR02 protein as the coating antigen,the optimal antigen coating concentration was determined to be 2 ng/μL,the dilution of serum sample was 1∶50,the incubation time was 30 min,the dilution of enzyme-linked immunosorbent assay secondary antibody was 1∶5 000 with the incubation time of 30 min,and the optimal substrate color development conditions were incubated for 15 min under dark conditions.The criteria for determining the critical value of positivity and negativity were:D_{450 nm} value of serum≥0.266 was considered positive,while D_{450 nm} value of serum＜0.266 was considered negative.No cross-reactivity was observed when testing positive sera for other pathogens such as Avibacterium paragallinarum (Apg),Newcastle disease virus (NDV),and Infectious bursal disease virus (IBDV).Positive results was still detected when the positive serum with a titer of 1∶64 was diluted at 1∶800.The coincidence rate with commercial kits for the same clinical samples was 91.25%.【Conclusion】 In this study,an indirect ELISA method was developed using recombinant OR02 protein for the detection of serum antibodies against ORT.The method demonstrated good specificity and sensitivity,with a high coincidence rate compared to a commercial kit.It could be considered a reliable tool for the detection of ORT antibodies in chicken serum.

【Objective】 This study was aimed to establish an immunocolloid gold assay for Bovine ephemeral fever virus (BEFV),and provide a research basis for the diagnosis of bovine epidemic fever (BEF).【Method】 The prokaryotic expression vector pET32a-BEFV-G was constructed and transformed into Escherichia coli BL21 (DE3) receptor cells for expression,and the expression product after successful induction was purified and identified by SDS-PAGE and Western blotting.The purified protein was used as the specific antigen-coated detection line,streptococcal G protein was used as the gold-labeled antigen,which was labeled on a gold-labeled pad after binding with gold nanoparticles,and goat anti-mouse IgG was used as the quality-control line.By optimizing the different reaction conditions,the immuno-colloidal gold assay method was established for the detection of BEFV,and the test strips were examined for their sensitivity,specificity and reproducibility.【Results】 SDS-PAGE results showed that the recombinant BEFV G protein was expressed in the form of inclusion bodies with a molecular mass of about 46 ku at a final concentration of IPTG of 0.75 mmol/L and induced at 37 ℃ for 8 h.Western blotting result showed that the concentrated and purified recombinant BEFV G protein had good reactivity and could be used as an encapsulated antigen for the detection of BEFV.It could be used as an encapsulated antigen for the establishment of immunocolloid gold test strips.Under the conditions that the concentration of the antibody sheep anti-mouse IgG encapsulated in the quality control line was 2 mg/mL,the concentration of the BEFV G protein encapsulated was 0.6 mg/mL,and the amount of gold sprayed was 2.5 μL/cm,the detection could be completed in 3-5 min.The prepared test strip had high sensitivity and specificity,and the coloration was close to the elimination line when the serum was diluted 40 times,and the test strip only reacted with the BEFV positive serum,and did not cross-react with the positive sera of other viruses,such as Bovine coronavirus (BCoV),Bovine rotavirus (BRV),and Bovine infectious rhinotracheitis virus (IBRV),etc.The test strips had been proved to be more reproducible after repeated tests of several positive serum samples.The reproducibility of the test strips was proved to be high,and the immunocolloid gold detection method was successfully established.【Conclusion】 In this study,a new type of immunocolloid gold test strip was successfully prepared to detect BEFV using prokaryotic G protein as antigen,which had high specificity and good stability,and was suitable for the rapid detection of BEF in the clinic.

【Objective】 This study was aimed to establish an indirect ELISA method for detecting antibodies against African swine fever virus (ASFV) with good specificity and sensitivity.【Method】 According to the gene sequence of ASFV P54 protein, E.coli codon optimisation was carried out,and the optimised P54 sequence was cloned into the pET-28a(+) plasmid to construct the recombinant plasmid pET28a-P54,which was used to express the target proteins using the E.coli expression system.Protein purification was carried out with a His nickel column,and after identification by SDS-PAGE and Western blotting,the purified P54 protein was encapsulated as a detection antigen to establish an indirect ELISA method,which was validated for its critical value,specificity,sensitivity and reproducibility after optimisation of the reaction conditions such as encapsulation conditions,closure solution,closure time and serum incubation.【Result】 The results of SDS-PAGE showed that the molecular weight of P54 protein was 42 ku,and the purity was above 95%.The results of Western blotting showed that P54 protein could react specifically with ASFV positive serum.The optimized ELISA conditions were as follows:0.3125 μg/mL antigen was coated at 37 ℃ for 1 h,5% skimmed milk powder was blocked at 37 ℃ for 1 h,serum diluted at 1∶800 was incubated at 37 ℃ for 2 h,and enzyme-labeled secondary antibody diluted at 1∶15 000 was incubated at 37 ℃ for 1 h.The ELISA method only reacted specifically with ASFV positive sera,and did not cross-react with positive sera of Swine fever virus,Porcine parvovirus,Porcine reproductive and respiratory syndrome virus,Porcine circovirus type 2,Bovine viral diarrhoea virus,Porcine pseudorabies virus and Encephalitis B virus.The sensitivity of the method was high,and the ASFV-positive serum was still significantly responsive when the dilution was 1∶3 200.The intra-batch and inter-batch duplicates were both＜10%.Compared with the commercial kits,the positive compliance rate was 95.45%,the negative compliance rate was 96.05%,and the total compliance rate was 95.83%.【Conclusion】 In this study,an indirect ELISA method using ASFV P54 protein as antigen was established.The method had good specificity and sensitivity,and could be used for serological detection of ASFV,providing more options for rapid diagnosis of African swine fever.

【Objective】 This study was aimed to analyze the effects of Transmissible gastroenteritis virus (TGEV) infection on circular RNA (circRNA) expression in porcine kidney epithelial cells (PK-15) and the potential regulatory functions of differentially expressed circRNA. 【Method】 Illumina HiSeq 4000 sequencing platform was used to sequence circRNA transcriptome of PK-15 cells (control) and TGEV-infected PK-15 cells，then screened differentially expressed circRNA. GO function and KEGG pathway enrichment analysis of differentially expressed circRNAs-derived genes were performed. TGEV infection-associated circRNA-miRNA-mRNA regulatory network was predicted. Moreover，12 differentially expressed circRNAs were randomly selected to verify their expression by Real-time quantitative PCR. 【Result】 Sequencing results revealed 1 029 novel circRNAs. Compared with PK-15 cells，a total of 128 significantly differentially expressed circRNAs were identified in TGEV-infected PK-15 cells，of which 70 circRNAs were upregulated and 58 circRNAs were downregulated. GO functional analysis showed that the genes originating from differentially expressed circRNAs in TGEV-infected PK-15 cells were mainly enriched in biological processes such as cellular macromolecular metabolic processes and cellular metabolism，cellular components such as intracellular fractions and intracellular membrane-bound organelles，and molecular functions such as organic ring compound binding and nucleic acid binding. KEGG pathway enrichment results showed that the differentially expressed circRNAs source genes in TGEV-infected PK-15 cells were mainly enriched in actin cytoskeleton regulation，influenza A，hepatitis C，cAMP signaling pathway，mTOR signaling pathway，etc. The circRNA-miRNA-mRNA regulatory network revealed that there was a large and complex circRNA regulatory network in TGEV-infected PK-15 cells，and a variety of circRNAs regulated innate immunity and transmembrane ion transport by targeting genes IFNAR1，IFNAR2 and NHE3 via miRNAs，thereby affecting TGEV infection and symptoms. Real-time quantitative PCR showed that the expression of 12 differentially expressed circRNAs were consistent with the basal trend of RNA-Seq results. 【Conclusion】 TGEV altered circRNA expression in PK-15 cells. Differentially expressed circRNA-derived genes were widely involved in cellular macromolecular metabolism，viral infection and cAMP signaling pathways. Multiple target genes IFNAR1，IFNAR2 and NHE3 associated with innate immunity and TGEV pathogenesis were regulated by circRNA. The results revealed the potential function of circRNA in TGEV-host interactions.

【Objective】 This study was aimed to identify the species of ticks that parasitized on the body surface of a stray dog from Jiaxing,Zhejiang,and its pathogen carrying status,and to understand the life cycle of Rhipicephalus sanguineus and the harm of the pathogens to humans and animals,so as to provide follow-up control measures for related diseases.【Method】 The morphology and molecular species of ticks were identified by optical microscopy and PCR.At the same time,18S rRNA gene of Pyriformida and Babesia,ompA gene of spotted fever group Rickettsia,dsb gene of Ehrlichia and 5S-23S rRNA gene of Borrelia burgdorferi were amplified by PCR to detect the pathogens carried by ticks.【Result】 The 82 ticks collected from the stray dog were all Rhipicephalus sanguineus,of which 29.27%(24/82) were positive for Theileria,4.88% (4/82) were positive for Colpodella sp.,2.44% (2/82) were positive for Babesia,25.61% (21/82) were positive for spotted fever group Rickettsia,and 18.29% (15/82) were positive for Ehrlichia,and no ticks were found to carry Borrelia burgdorferi.The number and variety of pathogens detected in a single tick were varied,among which 24 ticks carried only one pathogen,12 ticks carried two pathogens simultaneously,6 ticks carried 3 pathogens simultaneously,and 40 ticks did not carry the pathogens tested. 【Conclusion】 The ticks parasitizing on the body surface of this stray dog were all Rhipicephalus sanguineus,which carried a variety of pathogens such as Tyleria,Babesia,spotted fever group Rickettsia and Ehrlichia,which could pose a great threat to humans and animals.The prevention and control of ticks in this area should be strengthened,especially the management of stray dogs.

【Objective】 The experiment was to study the interaction between chicken interferon γ (chIFN-γ) and chicken complement receptor 2 (chCR2) proteins，and lay a foundation for the research of new functions of chIFN-γ and chCR2.【Method】 chIFN-γ related expression plasmid was constructed by homologous recombination method. The interaction between chIFN-γ and chCR2 proteins was identified by co-immunoprecipitation，laser confocal and surface plasmon resonance techniques，and the interaction mode of chIFN-γ and chCR2 proteins was simulated by molecular docking.【Result】 The pCMV-Myc-chIFN-γ expression plasmid was constructed successfully. The plasmid of pCMV-Myc-chIFN-γ and pCMV-HA-chCR2-ΔTM were co-transfected into HEK-293FT cells and the results showed that chIFN-γ could interact with chCR2. The plasmid of pCMV-Myc-chIFN-γ and pCMV-HA-chCR2-ΔTM were co-transfected into DF-1 cells and the results showed that chIFN-γ and chCR2 proteins could co-locate in DF-1 cells. The results of surface plasmon resonance test showed that the equilibrium dissociation constant (KD) between chIFN-γ and chCR2 proteins was 0.362 μmol/L，and the affinity between the two proteins was high. The results of molecular docking experiments showed that the interaction between chIFN-γ and chCR2 proteins was 1 salt bridge between Asp23-His150 and 3 sets of hydrogen bonds between Asn37-Ser143，Glu57-Gly179 and Ile93-Tyr184.【Conclusion】 The results of this study proved that chIFN-γ and chCR2 proteins could interact，and the interaction mode between them was 1 salt bridge and 3 sets of hydrogen bonds.

【Objective】 This study was aimed to understand and master the biological characteristics and genetic variation of Bovine viral diarrhea virus(BVDV) epidemic strains in Shandong province，and provide theoretical basis and technical support for the prevention and control of BVDV.【Method】 The tissue supernatant was prepared from suspected BVDV-infected samples of a cattle farm.RT-PCR method was used for pathogen identification，MDBK cells were inoculated for virus isolation and culture，and then subjected to indirect immunofluorescence assay(IFA) and electron microscopy for identification，and the whole genome of the isolated strain was segmented and amplified，cloned and sequenced.The alignment and analysis of genetic evolution of the whole genome，N^pro and E2 genes were done by Lasergene and other softwares.【Result】 The RT-PCR results of BVDV-specific primers showed BVDV-specific bands of approximately 504 bp，which was consistent with the expected band size.The IFA results showed specific green fluorescence,and virus particles about 50 nm in diameter were visible by electron microscopy，indicating that a non-cytopathogenic BVDV strain named SDNF9 was isolated.The total sequence of the strain was 12 232 nt.Comparing to BVDV reference strains，the sequence similarity was 79.1% to 93.8%，with the highest homology of 93.8% observed with the 22AH-1 strain from China and the Bega-like strain from Australia.Multiple amino acid variations were found in the N^pro and E2 proteins.Phylogenetic analysis showed that SDNF9 was in a different branch of genetic evolution from the BVDV2 and BVDV3 strains，while it was in the same clade as BVDV1 and belonged to the BVDV 1c genotype.【Conclusion】 In this study，an epidemic strain of BVDV 1c was successfully isolated and genomic and genetic evolution analysis was carried out，it was closely related to Chinese isolated 22AH-1，which provided data and material support for the prevention，control and vaccine development in the region.

【Objective】 This study was to explore the biological function of chicken promyelocytic leukemia protein nuclear bodies (PML NBs) in the process of Marek’s disease virus (MDV) infection,and establish a method to visualization and detection of chicken promyelocytic leukemia protein (Ch-PML) based on a monoclonal anti-Ch-PML.【Method】 Recombinant Ch-PML protein was prepared by the prokaryotic expression system and was used as immunogen to immunize BALB/c mice.Myeloma cells and splenocytes of the immunized mice were fused by cell fusion technology,and a hybridoma cell line secreting specific antibodies anti-Ch-PML was screened by ELISA.The in vivo method for the production of ascites and the corresponding antibody specific anti-Ch-PML was obtained via purification with Protein A/G.Western blotting and ELISA were used to determine the specificity,affinity and subclass of the monoclonal antibody,and finally the monoclonal antibody was used to establish an indirect immunofluorescence assay (IFA) for the detection of chicken PML NBs.【Result】 Ch-PML was expressed soluble in E.coli with a molecular weight of 19 ku.An anti-Ch-PML monoclonal antibody was successfully produced which had high specificity and affinity,and was detected by subclass and type as an IgG2b subclass with a Kappa-type light chain.An IFA was developed using the prepared anti-Ch-PML monoclonal antibody.The results showed that endogenous PML NBs were distributed in the nucleus of host cells in a punctate pattern,and overexpressed Ch-PML formed clumped aggregates in the nucleus of host cells.The number of PML NBs in the nucleus of MDV-infected cells was significantly reduced compared with that of uninfected cells (P＜0.05),indicating that MDV infection led to the inhibition of PML NBs assembly in the nucleus.【Conclusion】 In this study,a fluorescence detection method for chicken PML NBs was established by the prepared anti-Ch-PML monoclonal antibody,and discovered the phenomenon that MDV inhibited the assembly of PML NBs in host cells,which provided a new research idea and tool for elucidating the pathogenic mechanism of MDV.

【Objective】 The aim of this study was to explore the biological and genomic characteristics of a lytic phage of multi-drug resistant Escherichia coli，so as to provide reference for the scientific prevention and control of multi-drug resistant strains. 【Method】 The phage was isolated and purified from farm sewage by double-plate method with Escherichia coli M719-6WT as host bacteria from Brown bear. The morphology of phage was observed by transmission electron microscopy. The host spectrum of phage was determined by plaque method and its biological characteristics were investigated. Phage bioinformatics analysis was conducted based on phage whole genome sequencing data，and bacteriostatic effects of phage were evaluated by in vitro and in vivo bacteriostatic tests.【Result】 Through double-plate method，a phage specific to Escherichia coli was isolated and purified，named pEC-M719-6WT.2，with clear and transparent plaques. Transmission electron microscopy and whole-genome analysis revealed that phage pEC-M719-6WT.2 belonged to the subfamily Tevenvirinae of the Myoviridae family. Host range determination showed pEC-M719-6WT.2 could lyse 11 strains of Escherichia coli. The optimum multiplicity of infection (MOI) was 0.1，the optimum temperature was 25 ℃，the optimum pH was 7.0，the incubation period was less than 10 min，the lysis period was 70 min，and the cleavage volume was about 190 PFU/cell. Genomic analysis showed that phage pEC-M719-6WT.2 had a genome size of 170.441 kb and no known genes related to drug resistance，lysogen or virulence were found. The results of receptor binding tests showed that phage pEC-M719-6WT.2 could bind to lipopolysaccharide receptors on the surface of host bacteria. In vitro bacteriostatic results showed，when MOI was 100，phage pEC-M719-6WT.2 combined with 8 μg/mL tetracycline could prolong the bacteriostasis time to 24 h. The results of in vivo protection test showed that after 2.8×10⁵ CFU/mL M719-6WT was infected with Galleria mellonella larvae，the survival rate of the larvae was increased to 100% and 70% within 24 and 48 h after injection of 2×10⁷ PFU/mL phage pEC-M719-6WT.2.【Conclusion】 A phage against multi-drug resistant Escherichia coli was isolated. The phage had good environmental stability and antibacterial activity，clear bioinformatics characteristics，and good antibacterial effect in vivo and in vitro. It could produce synergistic effect when combined with antibiotics，and had potential application value in the treatment of clinical drug-resistant Escherichia coli infection.

【Objective】 The purpose of this study was to express the N protein of Porcine epidemic diarrhea virus (PEDV) using a prokaryotic expression system，and prepare polyclonal antibody with high efficiency and specificity.【Method】 Bioinformatics tools were used to analyze the amino acid sequence of PEDV N protein and predict its antigenicity. Recombinant N protein was induced by prokaryotic expression vector pColdⅠ，and the recombinant protein was identified by SDS-PAGE and Western blotting. New Zealand White rabbits were immunized with the preparation adjuvant of rabbit polyclonal antibody mixed with purified recombinant N protein，and serum was collected to prepare polyclonal antibody. The titer of recombinant N protein polyclonal antibody was determined by indirect ELISA，and the polyclonal antibody was verified by Western blotting and indirect immunofluorescence assay (IFA).【Result】 The results of bioinformatics prediction show that N protein didn’t contain signal peptide and transmembrane structure，and had good antigenicity and solubility. SDS-PAGE results showed that the recombinant N protein approximately 58 ku in size，existed primarily in a soluble form. Western blotting revealed that the protein specifically react with anti-His labeled mouse monoclonal antibodies . Indirect ELISA results demonstrated that the titer of polyclonal antibody could reach 1∶ 204 800. Western blotting results showed that recombinant N protein and N protein in PEDV infected Vero cell samples could be specifically identified when the antibody dilution was 1∶5 000. IFA results showed that when the dilution was 1∶1 000，the antibody could effectively identify N protein in PEDV-infected cell samples.【Conclusion】 The rabbit anti-N protein polyclonal antibody with high efficiency and good specificity was successfully prepared，providing experimental materials for further study of PEDV N protein function，understanding PEDV replication mechanism and virus-host cell interaction，and laying a foundation for the development of PEDV diagnosis and detection methods.

【Objective】 The aim of this study was to investigate and analyse the prevalence and molecular genetic evolution of Porcine circovirus type 3 (PCV3) in Chengdu area.【Method】 A total of 2 113 dead pig tissue samples were collected from 4 harmless disposal sites in Chengdu area from January 2023 to December 2023,and the PCV3 infection was detected by Real-time quantitative PCR.Three pairs of primers were designed for PCR amplification and sequencing of PCV3 genome sequence of positive samples.Through online software splicing PCV3 genome sequence,intercepting ORF2 gene sequence and deducing Cap amino acid sequence,similarity comparison was carried out and genetic evolution tree was constructed.The mutation of Cap amino acid sequence was analyzed.【Result】 The positive rate of PCV3 in 2 113 dead pig tissue samples was 27.21% (575/2 113).According to PCR amplification and sequencing results,the genome sequences of 15 PCV3 strains were obtained by splicing.The similarity analysis showed that the nucleotide sequences similarity among the 15 PCV3 genome was 97.2%-100%,the nucleotide sequences similarity among ORF2 genes was 97.2%-100%,and the amino acid sequences similarity among Cap sequences was 96.7%-100%.The results of genetic evolution tree showed that 12 PCV3 belonged to PCV3a subtype and 3 belonged to PCV3c subtype.A total of 13 of the 15 Cap amino acid sequences were mutated,and there were 13 mutation sites.【Conclusion】 PCV3 in Chengdu area was mainly PCV3a and PCV3c subtypes with high genomic similarity and conservative sequences.The mutation sites of Cap amino acids were mainly concentrated in antigen epitope.The results suggested that scientific prevention and control policies should be formulated in this region to prevent the spread of the virus.

【Objective】 The aim of this study was to understand the biological characteristics and genetic variation of Bovine parvovirus 1 (BPV1) in Guangxi，and provide theoretical basis and support for the prevention and control of BPV1.【Method】 A total of 409 fecal samples of calf diarrhea were collected from cattle farms in Guangxi region，RCR was detected by BPV1 specific primer，and virus was isolated from positive samples by bovine turbinate bone cells (BT). The supernatant of BT cells with cytopathic effect (CPE) was collected，and the virus was identified by PCR amplification，indirect immunofluorescence assay (IFA) and electron microscope observation. The median tissue culture infectious dose (TCID₅₀) was measured at different times after BPV1 isolates infected BT cells，and the multi-step growth curve was drawn. The whole genome sequence of the virus was obtained by high throughput sequencing and its genetic evolution was analyzed.【Result】 One positive BPV1 was detected in 409 fecal samples，with a positive rate of 0.24%. After the blind transfer of BT cells to the third generation，the phenomenon of cell circle shrinkage，bending，formation of cell mass and dissolution was observed. PCR amplification results showed that specific bands could be produced by amplification of CPE supernatant. The round，membraneless virions with a diameter of about 24 nm were observed by electron microscopy. IFA results showed that specific green fluorescence could be observed in BT cells inoculated with the isolate，and a strain of BPV1 named GXBS2209 was successfully isolated. The titer of the 6th generation isolates was 10^5.75 TCID₅₀/mL，and the multi-step growth curve showed that the virus titer peaked at 120 h after the isolate infected BT cells，and then gradually decreased. The sequencing results showed that the full length of the isolated genome was 5 515 bp (GenBank accession No.: PP158225). The sequence similarity between the isolates and the American Bovine parvovirus strain was the highest (98.7%). The genetic and evolutionary analysis showed that the isolate and Bovine parvovirus strain were in the same branch and were closely related. The nucleotide sequence similarities of NS1 and VP2 genes were 98.8%-99.4% and 94.4%-98.1%，and the amino acid sequence similarities were 98.8%-99.5% and 90.3%-99.4%，respectively. There are 2 and 3 amino acid sites substitutions in NS1 and VP2 sequences，respectively.【Conclusion】 A strain of BPV1 was successfully isolated and its whole gene sequence was analyzed，which laid a foundation for the subsequent pathogenesis，vaccine research and prevention and control of BPV1.

【Objective】 Nod-like receptor family member C5 (NLRC5) is widely expressed in mammals and is involved in the regulation of immune response and antigen presentation processes. The objective of this experiment was to express the recombinant expression products of porcine NLRC5 gene through eukaryotic expression system，and prepare porcine NLRC5 polyclonal antibody，so as to provide basic support for the subsequent study of the molecular mechanism of porcine NLRC5.【Method】 The transmembrane structure and signal peptide of NLRC5 protein was analyzed by bioinformatics online. The porcine NLRC5 gene was amplified by PCR，and the eukaryotic expression vector pCAGGS-HA-NLRC5 was constructed and transfected into HEK-293F cells. Western blotting was used to detect the expression of recombinant protein，and polyclonal antibodies were prepared by immunizing New Zealand White rabbits. The titer of polyclonal antibody was determined by indirect ELISA，and the specificity of the antibody was detected. Indirect immunofluorescence (IFA) and Western blotting were used to detect the reactivity of NLRC5 polyclonal antibody with different cells. Using NLRC5 polyclonal antibody as the primary antibody，the expression of NLRC5 protein in ST cells treated with siRNA was detected by Western blotting. 【Result】 The results of bioinformatics analysis showed that NLRC5 protein had no transmembrane domain and contained 1 signal peptide. The eukaryotic expression plasmid pCAGGS-HA-NLRC5 was successfully constructed. Western blotting results showed that NLRC5 protein was successfully expressed with molecular weight of about 203 ku. The prepared NLRC5 polyclonal antibody had a titer of 1∶25 600 and good specificity. IFA and Western blotting results showed that the antibody could react with a variety of cell lines. siRNA validation showed that it could be used to detect the expression of NLRC5 in cells.【Conclusion】 The recombinant porcine NLRC5 protein was successfully obtained by eukaryotic expression system and rabbit derived polyclonal antibody was prepared，which provided experimental materials for further investigation of the defense effect of NLRC5 on foreign infection in innate immune response.

【Objective】 This study was aimed to screen a lactic acid bacteria producing bacteriocin resistant to Staphylococcus aureus,analyze the physicochemical characteristics of the bacteriocin produced by the strain,and analyze the whole genome sequencing of the strain.【Method】 In this study,lactic acid bacterum producing antibacterial substances was screened from oranges in Yanbian city,Jilin province,using Staphylococcus aureus as an indicator strain.Morphological observation and similarity identification were performed on the selected strain,and a phylogenetic tree was constructed.Preliminary analysis of the antibacterial substances was carried out by acid excretion,hydrogen peroxide excretion and protease sensitivity tests,and the tolerance of the antibacterial substances to temperature and ultraviolet light was determined.Whole genome sequencing and function was performed on the strains.【Result】 One strain of lactic acid bacteria producing antibacterial substances against Staphylococcus aureus was obtained named as LMM-1,which showed milky white,smooth and moist round colonies on MRS solid medium.After Gram staining,the bacteria were short rod positive under the oil microscope.The nucleic acid sequence was identified by 16S rRNA to be 99.86% similar to the Weissella confuse,it was confirmed that LMM-1 was Weissella confuse.The antibacterial activity of the fermentation supernatant was significantly higher than that of control group when the organic acid was excluded,and the antibacterial activity of antibacterial products of the isolate was almost unchanged when the hydrogen peroxide was excluded.The interference of organic acid and hydrogen peroxide could be excluded.And the inhibitory effect of protease treatment of antibacterial products of the isolate on Staphylococcus aureus was reduced.It still had good antibacterial activity under high temperature of 100 ℃,pH 2.0-8.0 and ultraviolet irradiation for 2 h.The length of LMM-1 genome was 2 194 218 bp,2 045 genes were predicted,and the average GC content of the genome was 44.97%.GO functional enrichment analysis results showed that the catalytic activity in molecular functions and the metabolic processes in biological processes obtained the most genomic functional annotations,with 905 and 801,respectively.KEGG pathway enrichment analysis results showed that there were 49 metabolic pathways in genetic metabolism,genetic information processing and environmental information processing,and a total of 1 050 genes were functionally annotated.【Conclusion】 The bacteriocin produced by the Weissella confusa LMM-1 had a good antibacterial activity against Staphylococcus aureus,and the bacteriocin had a certain ability of acid and alkali resistance,high temperature and ultraviolet light,and had a good adaptability to the environment.The isolated strain contained many genes related to catalytic activity and metabolism.The result of this study laid a good foundation for the development of anti-Staphylococcus aureus replacement antibody products.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis.The HEV genome consists of a 5' non-coding region,three open reading frames (ORF1,ORF2 and ORF3),and a 3' non-coding region,with ORF4 found only in HEV-1 and overlapping with ORF1.Various coding proteins play different roles in HEV replication and infection,and HEV replication is mediated by RNA-dependent RNA polymerase (RdRp) encoded by ORF1.HEV RdRp is a complex enzyme composed of multiple protein subunits,with 7 conserved motifs.These conserved motifs play roles in nucleotide recognition,addition,extension,modification,and stabilization during RNA synthesis,ensuring the functionality of RdRp,which plays a crucial role in HEV replication and transcription.Therefore,RdRp as the action target of anti-HEV drug has a good application prospect and is a mainstream idea in drug development.Currently,nucleoside RdRp inhibitors like ribavirin,sofosbuvir and 2CMC,and non-nucleoside RdRp inhibitors like zinc salts,GPC-N114 have shown strong inhibitory effects on HEV and can be potential anti-HEV drugs for further research.The author elaborates on the structure and function of HEV encoding protein and HEV RdRp,and a summary of the RdRp inhibitors that have been discovered to inhibit HEV is provided in order to offer new insights for the development of HEV drugs.

【Objective】 The aim of this study was to isolate and screen potential probiotic strain from the intestinal tract of Zi geese to improve their health status，and analyze its probiotic properties. 【Method】 The contents of different intestinal tract of healthy Zi geese were collected，diluted in gradient and coated on (CaCO₃-MRS) solid medium containing calcium carbonate. After anaerobiccultivation，the isolates were identified through morphology，biochemical tests，16S rRNA gene PCR amplification，respectively. The probiotic properties of the isolates were screened by acid resistance and antibacterial tests. The antibacterial ability，antibiotic sensitivity，acid and bile salt tolerance，gastric and intestinal fluid tolerance were evaluated，and the growth curve and acid production curve were measured. 【Result】 4 suspected lactic acid bacteria strains were isolated from the intestinal tract of healthy Zi geese，named RS-1，RS-2，RS-3 and RS-4，of which RS-3 had good probiotic properties. The colony morphology of RS-3 in CaCO₃-MRS medium was smooth，light yellow and round colony with obvious calcium-soluble ring，Gram-positive Bacillus. The results of biochemical tests showed that the isolates could ferment maltose，cellobiose，sucrose，raffinose，inulin and lactose，as well as mannitol，sorbitol and salicin. The results of esculin test and 1% sodium hippurate test were positive，and the isolates were initially determined to be lactic acid bacteria. 16S rRNA gene sequence analysis showed that 4 isolates were all Streptococcus alactolyticus，and the tolerance of RS-3 was better than that of the other three strains. The fermentation broth of RS-3 had a good inhibitory effect on Staphylococcus aureus. The diameter of inhibitory zone was more than 10 mm，and it was resistant to kanamycin，amicacin，roxithromycin，sulfadiazine，lincomycin，polycolistin B and norfloxacin. Moreover，RS-3 had tolerance to acid，bile salts artificial gastric and intestinal fluids. The mice grew well after administration of RS-3. 【Conclusion】 4 isolates enterogenic Streptococcus alactolyticus of Zi geese were isolated in this experiment，of which RS-3 had good antibacterial and probiotic properties with acid and bile salt tolerance，and could be used as a reliable strain source for the subsequent development of probiotic preparations for geese.

【Objective】 The objective of this study was to isolate lactic acid bacteria with probiotic effect from healthy falcon feces and to provide candidate strains for the development and research of probiotic preparations.【Method】 Six falcon fecal samples were collected from Akqi county,and bacterial isolation and cultivation were carried out using selective culture medium.The isolated bacteria were identified through Gram staining,biochemical identification,16S rDNA PCR amplification,and sequence analysis.Biological characteristics such as growth curve,acid production curve,stress resistance,drug sensitivity,in vitro bacteriostasis,self-aggregation ability,surface hydrophobicity and animal safety test were measured.【Result】 The fecal samples from the falcon yielded three isolates,which were identified as blue-purple positive cocci through Gram staining microscopy.The biochemical analysis demonstrated positive results for raffinose,glucose and methyl red,while the touch enzyme test,hydrogen sulfide test,indole test and V-P test all exhibited negative outcomes.16S rDNA sequencing revealed that the isolates shared over 99% similarity with Enterococcus hirae identified by NCBI.Consequently,the three isolates were classified as Enterococcus hirae and designated EH1,EH2 and EH3,respectively.EH1,EH2 and EH3 strains had logarithmic growth periods of 8-16,8-19 and 8-14 h,respectively.The acid production pH of the bacterial solution was stable at 4.5,indicating good acid production performance.Three strains of Enterococcus hirae were able to survive under pH 3.0 and 0.5% bile salt treatment,demonstrating good stress resistance.The cell-free supernatants of three strains of Enterococcus hirae all showed good antibacterial effects on Escherichia coli and Staphylococcus aureus.They were all mediated by streptomycin and cefotaxifur,and were susceptible to ciprofloxacin,gentamicin,erythromycin,tilmicosin,penicillin,ampicillin and chloramphenicol.The self-agglutination capacity of EH1 strain was higher (28.00%),and the hydrophobic capacity of EH2 strain was higher (52.10%).All of three strains of Enterococcus hirae were non-pathogenic to Kunming mice.【Conclusion】 Three strains of Enterococcus hirae were isolated from falcon feces,which had good probiotics and could be used as candidate strains for probiotic preparations.

【Objective】 This study was aimed to investigated the protective effect of potato starch and corn starch on the activity of microcapsulated Pediococcus acidilactici in the process of freeze-drying,as well as the effect on the acid and bile salts resistance of Pediococcus acidilactici and the storage stability of Pediococcus acidilactici,so as to explore ideas and provide theoretical basis for the development and utilization of resistant starch and probiotic protection technology. 【Method】 Three groups of microcapsules were prepared with 3.0% sodium alginate and 0.8%chitosan concentration:Control group,test group 1 (T1) and test group 2 (T2).In control group, 2% glycerol was used as lyophilizing agent,in group T1, 2% glycerol and 10% potato starch was used as lyophilizing agent,and in T2 group, 2% glycerol and 10% corn starch was used as lyophilizing agent.The morphology,mechanical strength,embedding rate,bacterial load,tolerance of artificial gastrointestinal tract in vitro and storage performance of the microcapsules were evaluated.【Result】 ①The embedding rate of microcapsule in groups T1 and T2 was significantly higher than that in control group (P＜0.05).②The lyophilization protection rate of groups T1 and T2 reached more than 70%,which were both significantly higher than that of control group (P＜0.05),and with a higher lyophilization protection rate of 77.66% in group T2.③The particle size,mechanical strength,bacterial load of microcapsules in groups T1 and T2 were significantly higher than those in control group (P＜0.05).④The in vitro tolerance test of artificial gastrointestinal fluids revealed that the survival rate of groups T1 and T2 in simulated gastric fluid containing pepsin was higher than that of control group,and the difference was significant after 2 h (P＜0.05).The Pediococcus acidilactici microcapsules in three groups started to release rapidly in simulated intestinal fluid after 10 min,and at 1 h,they were basically completely disintegrated.Pediococcus acidilactici microcapsules in groups T1 and T2 were stable in bile salt environment.The survival rate of Pediococcus acidilactici in groups T1 and T2 in bile salt environment was significantly higher than that of control group (P＜0.05) after 4 h.⑤Three groups of microcapsules were stored at 25 ℃ for 8 weeks,the survival rate of Pediococcus acidilactici in groups T1 and T2 was significantly higher than that in control group (P＜0.05),and the survival rate of Pediococcus acidilactici in group T2 was higher (52.29%).After 8 weeks of storage at －20 ℃,the survival rate of Pediococcus acidilactici in groups T1 and T2 was higher than that in control group,but the difference was not significant (P＞0.05).【Conclusion】 In summary,through the measures of microencapsulation technology and the addition of plant starch as a protective agent,the freeze-drying protection rate and storage performance of Pediococcus acidilactici microcapsules could be improved,among which corn starch had higher freeze-drying protection and storage performance of Pediococcus acidilactici microcapsules.The results could provide reference for the development of Pediococcus acidilactici microcapsules with sodium alginate and chitosan as wall materials.

【Objective】 The aim of this experiment was to study the preparation of Astragalus polysaccharide liposomes (APSL) and their transport in Caco-2 cell model. 【Method】 APSL was prepared by film dispersion method，the structure of APSL was observed by transmission electron microscopy，and the encapsulation rate and drug loading capacity of liposomes were detected by the method of fisetin. A Caco-2 monolayer cell model was established，and the structure was observed by transmission electron microscopy. The denseness and completeness of the Caco-2 cell model was evaluated by measuring the cell resistance value and apparent permeability coefficient (Papp) of sodium fluorescein，respectively. Transmembrane transport experiments were performed from the apical side (AP) to the basolateral side (BL) and from the BL to the AP side to analyze the effects of drug concentration，and P-gp inhibitor Verapamil (Ver) on the transport. 【Result】 The encapsulation rate of APSL was 71.74%±4.87%，the drug loading was 2.92%，the spherical bilayer structure was visible under transmission electron microscopy，the particle size was (126.6±0.283) nm (n=3)，and the polymer dispersity index (PDI) was 0.190±0.009. Caco-2 cells were observed to be tightly bound under transmission electron microscopy，forming a microvillus structure，and the transmembrane resistivity value reached 650 Ω·cm²，the alkaline phosphatase (ALP) ratio reached 3.05，and the Papp of sodium fluorescein was 1.96×10^-7 cm/s，which fulfilled the requirements of the transport experiments. At different concentrations (75，150 and 300 μg/mL)，the efflux ratio (ER) of APS was higher than 1.5，and the ER of APSL was lower than 1.5. The Papp of the APSL group was extremely significantly higher than that of APS group in the absorption direction of AP-BL at three concentrations (P＜0.01)，and the ER was lower than that of APS group. After adding P-gp protein inhibitors，compared with APS group，the Papp of APS+Ver group was extremely significantly increased in the AP-BL direction (P＜0.01)，while the ER was decreased. However，there was no significant change in Papp on both sides of APSL+Ver group (P＞0.05). Compared with the ER of APS group (3.995)，the ER of APSL group (1.005) was decreased.【Conclusion】 APS was poorly transported on the monolayer model of Caco-2 cells in vitro as a low-permeability compound，and there might be P-gp protein exocytosis，whereas APSL was able to increase the transport and absorption of APS，and improve the permeability of the drug.

Senecavirus A (SVA),a small RNA virus,has been causing vesicular lesions in pigs in recent years.It belongs to the genus Senecavirus in the family Picornaviridae.So far,research in the fields of genetic evolution,genome structure and function,pathogenesis,and epidemiology of SVA has achieved certain results,and various laboratory diagnostic methods have been established.In China,the range of SVA infection is quite extensive,predominantly presenting as asymptomatic latent infections,and co-infection with other pathogens are relatively common.At present,China has achieved significant technological improvements in the field of SVA detection.These advances include the application of several novel detection methods such as reverse transcription isothermal amplification PCR (RT-iiPCR),recombinase polymerase amplification (RPA),Real-time fluorescent reverse transcription recombinase-aided amplification (rRT-RAA),reverse transcription droplet digital PCR (RT-ddPCR),and Real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP).However,considering that SVA is a novel RNA virus prone to mutation and recombination,there are no effective commercial vaccines or medications available against it currently,making the effective control and prevention of SVA a significant challenge.The current focus of SVA vaccine research is primarily on inactivated vaccines,but it also extends to subunit vaccines,live attenuated vaccines,nucleic acid vaccines,virus-like particle vaccines,and recombinant vector vaccines.For the antiviral treatment of infections caused by SVA,medications such as ribavirin or reboxetine mesylate can be utilized.Furthermore,extracts from traditional Chinese medicine and other natural products are being explored as potential antiviral agents.Here,the author is aimed to summarize the recent advance in researches on the epidemic distribution,etiology,epidemiology,differential diagnosis,as well as prevention and control of SVA,and to provide new perspectives and insights into strategies for SVA prevention and control.

【Objective】 The purpose of this study was to investigate the effects of triclocarban (TCC) on immune function and the expression of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor κB (NF-κB) signaling pathway in spleen of rats.【Method】 A total of 96 5-week-old male SD rats were randomly divided into 4 groups： Corn oil control group (group C) and 0.1，20 and 100 mg/kg TCC groups (TL，TM and TH)，with 24 rats in each group. Different doses of TCC were thoroughly mixed with corn oil and given intragastric administration once a day. Rats in group C were given intragastric administration of the same amount of corn oil without TCC，the experiment lasted for 60 days. After the experiment，blood and spleen tissues of rats were collected，and serum biochemical indexes，splenic lymphocyte proliferation capacity，macrophage phagocytosis index，serum hemolysin level and spleen natural killer cell (NK) activity were detected. The serum immunoglobulin content of rats was detected by ELISA. The lymphocyte subsets in peripheral blood of rats were detected by flow cytometry. Real-time quantitative PCR and Western blotting were used to detect mRNA and protein expression levels of TLR4/MyD88/NF-κB pathway marker genes and downstream factors，respectively.【Result】 Compared with group C，serum glucose (Glu) level in TH group was significantly decreased (P＜0.05), and serum creatinine (CREA) level in TM group was significantly increased (P＜0.05). The splenic lymphocyte proliferation ability and serum hemolysin level of TM group were significantly decreased (P＜0.05). ELISA results showed that the levels of immunoglobulin A (IgA)，IgM and IgG in serum of rats in TH group and IgG in serum in TM group were extremely significantly decreased compared with group C (P＜0.01). Flow cytometry results showed that，compared with group C ，there were no significant differences in the proportion of CD3⁺，CD4⁺ T cells and the ratio of CD4⁺ and CD8⁺ in peripheral blood of rats in different doses of TCC groups (P ＞ 0.05). Real-time quantitative PCR results showed that，compared with group C，the mRNA relative expression levels of TLR4，MyD88 and IL-1β in spleen of rats in different doses of TCC groups were significantly or extremely significantly increased (P ＜ 0.05 or P ＜ 0.01). Western blotting results showed that compared with group C，the expressions of TLR4，MyD88 and NF-κB proteins in spleen of TM group were extremely significantly increased (P ＜ 0.01).【Conclusion】 TCC could produce certain toxic effects on cellular and humoral immunity of rats，and significantly activate the TLR4/MyD88/NF-κB signaling pathway in rat spleen. TCC might damage the immune system of rats by targeting the TLR4/MyD88/NF-κB pathway in the spleen and induce immunotoxicity in rats.