【Objective】 This study was aimed to investigate the role of miR-449a/b in the growth, development and reproductive performance of sheep, and provide reference for further studies on the role of miR-449a/b in reproduction regulation of sheep.【Method】 The relative expression of miR-449a/b in hypothalamus, pituitary and ovary of Hu sheep was studied by Real-time quantitative PCR.TargetScan, miRWalk and miRDB softwares were used to predict the target gene of miR-449a/b precursor sequences in sheep.GO function and KEGG pathway enrichment analysis were performed using bioinformatics and KOBAS websites.【Result】 The comparison result of the precursor sequence obtained from the test was consistent with the precursor sequence information of sheep in NCBI database.Real-time quantitative PCR results showed that the relative expression of miR-449a in pituitary was significantly higher than that of miR-449b (P<0.05), the relative expression of miR-449b in ovary was extremely significantly higher than that of miR-449a (P<0.01).The number of target genes of miR-449a and miR-449b were 299 and 23 by Venn diagram analysis using three target gene prediction softwares, respectively.The results of GO function enrichment showed that miR-449a/b were mainly enriched into biological processes.The results of KEGG pathway enrichment were analyzed by the top 20 pathways, including oxytocin signaling pathway and MAPK signaling pathway and other signaling pathways related to reproductive regulation.【Conclusion】 The expression of miR-449a in pituitary was high, and the expression of miR-449b in ovary was high.The number of target genes of miR-449a/b were 299 and 23, respectively, and its gene function was mainly concentrated in biological process, which might be involved in the reproductive regulation in Hu sheep.

【Objective】 The purpose of this study was to explore the difference in the expression profile of porcine Leydig cells under different oxygen concentrations and screen the key genes of hypoxia leading to the damage of porcine Leydig cells.【Method】 Porcine Leydig cells were divided into hypoxia group (1% oxygen, H24 group) and normal group (15.75% oxygen, N24 group) with 3 replicates in each group.The cells were treated for 24 h under corresponding conditions, and the cell survival rate was detected by CCK8 method.Transcriptome sequencing technology was used to screen out the differentially expressed genes in Leydig cells of hypoxia and normal oxygen groups.DESeq 2.0 software was used to analyze the differentially expressed genes, and GO function and KEGG pathway enrichment analysis were performed on the differentially expressed genes, so as to further screen the regulatory genes related to oxygen content.【Result】 After 24 h hypoxia stimulation, the survival rate of porcine Leydig cells in hypoxia group was extremely significantly decreased compared with normal group (P<0.01).A total of 1 654 differentially expressed genes were obtained by transcriptome sequencing, among which 1 242 genes were up-regulated and 412 genes were down-regulated.GO function enrichment analysis showed that 8 enrichment items were obtained, including 1 biological process and 7 molecular functions.KEGG pathway enrichment analysis showed that 10 signaling pathways were significantly enriched, including HIF-1 signaling pathway, JAK-STAT signaling pathway and AGE-RAGE signaling pathway in diabetic complications, etc.Six genes related to hypoxia stress were obtained by enrichment analysis of GO function and KEGG pathway, including signal transducer and activator of transcription 5(STAT5), interleukin-8 (IL-8), C-C motif chemokine ligand 2 (CCL2), interferon alpha and beta receptor subunit 1 (IFNAR1), suppressor of cytokine signaling 1 (SOCS1) and erythropoietin (EPO).【Conclusion】 In this study, six genes related to hypoxia injury and stress repair in porcine Leydig cells (SOCS1, IFNAR1, EPO, STAT5, IL-8 and CCL2 genes) were obtained through transcriptomic analysis of porcine Leydig cells under different oxygen concentrations, providing reference for the study on the low reproductive ability of boar in plateau area.

【Objective】 The purpose of this study was to explore the role of cyclin-dependent kinase inhibitor 1B (CDKN1B) gene in hypoxia adaptation.【Method】 The CDS region of CDKN1B gene of Tibetan pigs was cloned and sequenced, and its sequence was compared and phylogenetic tree was constructed.The bioinformatics analysis of CDKN1B protein was carried out using online software.The relative expression of CDKN1B gene mRNA and protein in heart, kidney and lung tissues of Tibetan pigs and Yorkshire pigs were detected by Real-time quantitative PCR and Western blotting.【Result】 The CDS region of CDKN1B gene in Tibetan pigs was 597 bp, and was 100% similar to Sus scrofa.It was the closest to Sus scrofa and the farthest to Barchydanioreriovar.CDKN1B protein was composed of 198 amino acids and had 30 phosphorylation sites, including 17 serine sites, 9 threonine sites and 4 tyrosine sites, and the amino acid instability index (Ⅱ) was 66.53.The protein was hydrophilic with a maximum hydrophobicity of 1.089 and a minimum of -2.867. The secondary structure of CDKN1B protein in Tibetan pigs was composed of random coil (73.23%), alpha helix (15.66%), extended chain (10.10%) and beta turn (1.01%).The prediction of the tertiary structure was consistent with that of the secondary structure.The protein was a non-transmembrane protein and non-secretory protein, which was mainly located in the nucleus, accounting for about 82.6%, 8.7% in the cytoplasm, and 4.3% in the mitochondria and cytoskeleton, respectively.The correlation between CDKN1B protein and CDK2, CCND1 and CDK4 was as high as 99.9%.The expression of CDKN1B mRNA in Tibetan pigs and Yorkshire pigs was the same as that of protein, which was the highest in heart, followed by kidney and lung.The mRNA expression of CDKN1B gene in heart, kidney and lung of Tibetan pigs were extremely significantly or significantly higher than that of Yorkshire pigs (P<0.01 or P<0.05), and the expression of protein only in kidney tissue was significantly higher than that in Yorkshire pigs (P<0.05).【Conclusion】 The CDS sequence of CDKN1B gene in Tibetan pigs was successfully cloned.The expression of CDKN1B gene was high in heart, kidney and lung, and the expression in Tibetan pigs were higher than that in Yorkshire pigs.These results laid a foundation for further study on the role of CDKN1B gene and its encoded protein in hypoxia adaptation in Tibetan pigs.

【Objective】 The purpose of this study was to understand the causes of the outbreak of Streptococcus agalactis (S.agalactis) disease in high temperature season at the molecular level.【Method】 S.agalactis was cultured at 35 and 28 ℃ respectively, and total RNA was extracted for transcriptome sequencing.The differentially expressed genes(DEGs) were analyzed by Dr Tom's analysis platform of BGI, and the classification and enrichment of KEGG pathway and GO function were carried out.The reliability of transcriptome data was determined by randomly detecting the differential expression of 10 genes by Real-time quantitative PCR.【Result】 A total of 357 DEGs were obtained under 35 and 28 ℃, including 229 up-regulated genes and 128 down-regulated genes.The enrichment of GO database could obtain multiple functional sets with significant, most of which focus on the pathways related to carbon source metabolism, such as carbohydrate catabolic process, glycogen metabolic process, cellular glucan metabolic process, etc.. Many genes related to the pathogenicity of S.agalactis were found among the DEGs, such as the coding genes of alkyl hydroperoxide reductase subunit, CAMP factor, C5a peptidase, etc.. The Pearson correlation coefficient R value between Real-time quantitative PCR and transcriptome data was 0.979 (P<0.001), indicating that the transcriptome data was reliable and effective.【Conclusion】 High temperature could significantly affect the pathway related to the carbon source metabolism of S.agalactis, which might promote the growth and diffusion of the cell by accelerating the use of carbon source.High temperature also induced the up-regulation of several genes related to pathogenicity.Among them, cell membrane lysis caused by cAMP factor and immune escape caused by oxidoreductases might be the main factors that enhance the pathogenicity of S.agalactis under high temperature.

【Objective】 This study was aimed to explore the preparation method of rat hepatocyte (BRL-3A) oxidative stress model induced by sodium hydrosulfite (Na₂S₂O₄), in order to establish a stable oxidative stress model of BRL-3A cells.【Method】 The medium containing Na₂S₂O₄ was used to cause hypoxia of cells, and the normal medium was replaced to reoxygenate BRL-3A cells, thus causing oxidative stress damage of cells.BRL-3A cells were divided into 5 groups, and the control group was cultured normally in a cell incubator (0 mmol/L Na₂S₂O₄), the experiment group was treated with different concentrations (0.1, 1, 5 and 10 mmol/L) of Na₂S₂O₄ for 4 h of hypoxia and 2 h of reoxygenation, then the cell viabiliaty rate was detected by CCK-8 assay.The fluorescent probe DCFH-DA method was used to detect the release of reactive oxygen species (ROS) in cells.The activity of lactate dehydrogenase (LDH) in cell culture medium, the contents of malondialdehyde (MDA) and glutathione (GSH), and the activity of superoxide dismutase (SOD) in BRL-3A cells were detected by kit.【Result】 After hypoxia/reoxygenation treament, compared with blank control group, with the increase of Na₂S₂O₄ concentration, the viability rate of BRL-3A cells was gradually decreased (P<0.01), the release of ROS was gradually increased (P<0.01), the activity of LDH and the content of MDA were increased in a concentration dependent manner (P<0.01), the content of GSH and the activity of SOD were decreased in a concentration dependent manner (P<0.05 or P<0.01).【Conclusion】 This study showed that a stable oxidative stress model of BRL-3A cells could be established which was induced by 5 mmol/L Na₂S₂O₄ for hypoxia of 4 h and reoxygenation of 2 h.

【Objective】 This study was aimed to establish the isolation and culture technology of goose embryo-derived hepatocytes, explore the mechanism of lipopolysaccharide (LPS) affecting the apoptosis of goose embryo-derived hepatocytes, and provide a reference basis for the study of liver function in goose.【Method】 16-18 days old goose embryos without specific pathogens were selected, and the livers of goose embryos were taken under sterile conditions and digested with 2% collagenase Ⅳ.After passing through the cell sieve, the goose embryo-derived hepatocytes were isolated, cultured and identified by PAS staining.Different concentrations (0 (control group), 0.1, 1.0 and 10 μg/mL) of LPS were added, and the cells were collected at 12, 24 and 36 h, respectively.The level of reactive oxygen species (ROS) and apoptosis in goose embryo-derived hepatocytes were detected by a kit and flow cytometry, respectively.The expression of key apoptosis genes caspase3, caspase9 and p38 mRNA in goose embryo-derived hepatocytes were detected using Real-time quantitative PCR.【Result】 Hepatocytes from goose embryo were successfully isolated and cultured, which had complete morphology, high adhesion rate, high survival rate and stable growth.The results of PAS staining showed that the goose embryo-derived hepatocytes was purplish red in various shades and the nucleus of liver was blue.After adding different concentrations of LPS, oxidative damage was found in goose embryo-derived hepatocytes.When LPS level was 1.0 μg/mL, the level of ROS in goose embryo-derived hepatocytes increased.The late apoptosis of goose embryo-derived hepatocytes was inhibited after the addition of LPS.Except for 36 h, the expression of the key apoptosis genes caspase3, caspase9 and p38 mRNA in goose embryo-derived hepatocytes were significantly decreased (P<0.05).【Conclusion】 In this study, a relatively stable method for isolation and culture of goose embryo-derived hepatocytes was established.After adding different concentrations of LPS, the level of ROS in goose embryo-derived hepatocytes was increased, p38/MAPK pathway was activated, and apoptosis was inhibited.

As the main bioactive compound of saffron, crocin has the ability to scavenge free radicals and relieve oxidative stress, and has the potential to become a green and safe antioxidant.Crocin is mainly catabolized in the intestine and transported to various parts of the body through the blood.The heart, spleen, kidneys and lungs have higher saffron metabolites, exert their biological role at this site.Crocin is low-toxicity substances that can be used to alleviate oxidative damage in the body.The current research showed that the antioxidative role of crocin benefited to alleviate neurodegenerative diseases, inhibit tumor growth, and protect the reproductive and cardiac systems as well.Alleviating oxidative stress is one of the main ways in which saffron glycosides regulate the body's adaptation to abnormal environments, and reducing the production of oxidation products and improving antioxidant enzyme activity are its main regulatory means.Crocin is involving in multiple signaling pathways (TLR2/NF-κB, JAK2-STAT3-ERK and Nrf2 pathways) to alleviate the imbalances between pro-oxidants and antioxidants in the body caused by external material stimulation.The authors reviewed the application of crocin antioxidant action in regulating animal health and its mechanism in different regulatory systems, which provided a theoretical basis for further research on the antioxidant mechanism of saffron glycoside and its regulation of animal health.It provides guidance for the development of novel natural antioxidants for the treatment of diseases or abnormalities.

【Objective】 This experiment was aimed to study the effects of hypoxia on the proliferation and oxidative stress injury of pulmonary artery smooth muscle cells (PASMCs) in yaks(Bos grunniens), and preliminarily explore the adaptive characteristics of pulmonary artery smooth muscle in yaks to hypoxia.【Method】 PASMCs of yaks were isolated and purified using α-smooth muscle actin (α-SMA).The test was divided into normoxia and hypoxia groups, and the proliferation efficiency of PASMCs in yaks was measured by CCK-8 method at 0, 2, 4, 6, 12, 24, 48, 72, 96 and 120 h under hypoxia and normoxia conditions.The protein concentrations of lactate dehydrogenase (LDH), superoxide dismutase (SOD), creatine kinase (CK) and malondialdehyde (MDA) in the cell culture supernatants of normoxia and hypoxia groups at 24, 48, 72 and 96 h of culture were measured by ELISA method.【Result】 The result of cell identification showed that more than 95% of cells could express α-SMA, which indicated that the isolated and purified cells were PASMCs in yaks.CCK-8 results showed that the cell proliferation folds in hypoxia group at 72, 96 and 120 h were extremely significantly lower than that in normoxia group (P<0.01), and the cell proliferation folds in both hypoxia and normoxia groups at 72 h were extremely significantly higher than 48 h (P<0.01), which indicated that the proliferation peak of PASMCs in yaks occurred at 72 h, and the prolonged hypoxia environment could reduce the proliferation efficiency of PASMCs in yaks.ELISA assay results were showed that compared with normoxia group, the LDH protein concentration of PASMCs culture supernatant of yaks in hypoxia group at 72 h was extremely significantly upregulated (P<0.01), the SOD protein concentration at 72 and 96 h were extremely significantly or significantly upregulated (P<0.01 or P<0.05), the CK protein concentration at 48 and 72 h were significantly or extremely significantly upregulated (P<0.05 or P<0.01), and the MDA protein concentrations at 72 h was significantly upregulated and at 96 h was significantly downregulated (P<0.05).Under hypoxia conditions, there was no significant difference of LDH and SOD protein concentrations in supernatant at different time (P>0.05), the CK and MDA protein concentrations at 24, 48 and 72 h were significantly higher than 96 h (P<0.05).Under normoxia conditions, the LDH protein concentration in supernatant at 48 h was significantly higher than 24 and 72 h (P<0.05), the SOD protein concentration at 24 and 48 h were significantly higher than 72 and 96 h (P<0.05), the CK protein concentration at 24 h was significantly higher than 72 and 96 h (P<0.05), the MDA protein concentration at 48 h was significantly higher than 72 h (P<0.05).【Conclusion】 Prolonged hypoxia could enhance oxidative stress and cell damage of PASMCs in yaks, weaken antioxidant effects, and significantly reduced cell proliferation efficiency.Four proteins of LDH, SOD, CK and MDA were played important regulatory roles in damage and anti-damage as well as antioxidant effects of PASMCs in yaks under hypoxia conditions, which might be related to the hypoxia adaptation of pulmonary vasculature in yaks.

【Objective】 This study was aimed to construct a stable expression of adenosine 5'-monophosphate-activated protein kinase (AMPK)/AMPK-T172D NIH3T3 cell lines, and investigate the effect of AMPK on hydrogen peroxide (H₂O₂)-induced cell senescence.【Method】 AMPK gene and its mutant form AMPK-T172D were amplified by PCR and cloned into lentiviral vector pLVX-IRES-Puro.The pLVX-IRES-AMPK/AMPK-T172D recombinant plasmids were constructed and double digestion was verified.The constructed recombinant plasmid was packaged into Lentivirus, infected with NIH3T3 cells, and puromycin screening was used to obtain stable cell lines.Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of AMPK in stable cell lines, respectively.The AMPK/AMPK-T172D overexpression of NIH3T3 cell lines was treated with 8.8 mmol/L H₂O₂, and after 2 days of culture, cellular senescence was detected by senescence-associated-β-galactosidase (SA-β-Gal) and the expression of p53, p21 and IL-6 genes were detected by Real-time quantitative PCR.【Result】 The results of double digestion verification showed that compared with control group, the pLVX-IRES-AMPK/AMPK-T172D expression plasmid was successfully constructed.Real-time quantitative PCR and Western blotting results showed that the expressions of mRNA and protein of AMPK and AMPK-T172D were extremely significantly or significantly increased (P<0.01 or P<0.05).The mRNA expression of carnitine palmitoyltransferase-1 (CPT-1) and fatty acid synthase (FAS) in AMPK/AMPK-T172D overexpressing NIH3T3 cells were significantly or extremely significantly increased (P<0.05 or P<0.01).The cell positive rate of SA-β-Gal was extremely significantly decreased (P<0.01). The mRNA expression of p53, p21 and IL-6 genes were significantly or extremely significantly decreased in AMPK/AMPK-T172D overexpressed NIH3T3 cells(P<0.05 or P<0.01).【Conclusion】 The stable expression of AMPK/AMPK-T172D overexpression NIH3T3 stable cell lines was successfully obtained, and AMPK activation could reduce the cell positive rate of SA-β-Gal and the expression of aging-related factors (p53, p21 and IL-6) in H₂O₂-induced senescent cells.The results would provide a basis for the research on the role of AMPK in aging, possible molecular mechanisms and anti-aging treatment strategies.

Albumin is an important component of serum.It is a highly water-soluble globular protein, which can maintain the balance of colloid osmotic pressure and has both anticoagulant and antioxidant effect.Serum albumin (SA) chemical structure and conformation allows interaction with many different drugs, affecting drugs transport and metabolic processes.As a protein carrier for endogenous and exogenous ligands, SA is of great clinical significance to be used to treat a variety of diseases, including hemorrhagic shock, hypoalbuminemia, chronic liver disease, etc.At the same time, it plays an important role in animal production by acting as cell culture agent, influencing the in vitro maturation of oocytes and the viability of frozen sperm.Therefore, the demand for SA increased annually worldwide, and it is crucial to obtain SA with high purity, high activity and high recovery.Currently, the purification process of SA includes ethyl alcohol precipitation, affinity precipitation, trichloroacetic acid/acetone precipitation method, ammonium sulfate precipitation combined with liquid chromatography, Cohn method combined with liquid chromatography, and chromatography.In order to clarify the extraction and purification process of SA, the structure, physiological functions, applications and purification processes of SA were reviewed in this paper, and the principles, purification effect, advantages, disadvantages of different purification processes were summarized.It was expected to provide a reference for the in-depth research, development, and utilization of SA, and further promotes the application of SA in the fields of medicine, molecular biology research and immuno-diagnostics.

【Objective】 This experiment was designed to study the effect of zinc and Lactobacillus on the prevention and control of Salmonella infection in squabs.【Method】 Ninety White feather King pigeons of 10 days of age were selected and randomly divided into 5 groups, with 18 pigeons in each group.They were control group (C group), infection group (S group, Salmonella Typhimurium), zinc group (Zn group, zinc sulfate+Salmonella Typhimurium), Lactobacillus group (LAB group, Lactobacillus), and combined treatment group (Zn+LAB group, zinc sulfate+Lactobacillus+Salmonella Typhimurium).On the 5th day of the test, the model of Salmonella Typhimurium challenge was established.The pigeons in group C was fed 1 mL PBS as the control, and other groups were fed 1 mL Salmonella Typhimurium suspension (1×10⁹ CFU/mL) for 3 consecutive days.Blood samples and livers were collected for the determination of immune and antioxidant indexes.Spleen and bursae of Fabricius were collected and weighed to calculate spleen index and bursae index of Fabricius.Jejunum and ileum tissues were collected for the preparation of tissue sections.【Result】 Compared with the control group, the ratio of ileum villus height to crypt depth (V/C) in infected group was significantly decreased (P<0.05).Compared with infection group, the monocyte ratio of squab were significantly decreased in zinc group and Lactobacillus group (P<0.05), and jejunum V/C, ileum V/C and serum T-SOD activities were significantly increased in combined treatment group (P<0.05).【Conclusion】 The systemic infection of Salmonella in squabs could be inhibited by increasing spleen organ index and antioxidant capacity, and improving intestinal morphology by instillation of zinc or combined administration of low dose zinc and Lactobacillus, and the combined administration effects were better.

In recent years, more and more attention has been paid to the interaction between gut microbiota and the whole life process of the host.Gut microbiota plays an irreplaceable role in maintaining the health of the host mainly through the production of bioactive absorbable metabolites.Short chain fatty acids (SCFAs) are the main metabolites produced by gut microbiota fermentation of dietary fiber, mainly including acetate, propionate and butyrate.As a medium mediating the interaction between gut microbiota and various organs of the host, SCFAs are widely involved in the physiological activities of the host, including skeletal muscle.As one of the largest and most important organs of the body, skeletal muscle is very important for the health of the body to maintain its normal physiological function.At present, the studies on the regulation of host by SCFAs, mainly focus on other tissues and organs, while there are still few studies on the regulation of skeletal muscle.In view of the extensive physiological effects of SCFAs and the importance of skeletal muscle itself, the current research on the gut-muscle axis theory, the production pathway and transport of SCFAs, and the regulation of skeletal muscle (mass, endurance, fiber properties) as metabolites of intestinal flora were reviewed, and the current research status and future research directions were prospected.This study aims to deepen the understanding of the physiological process of SCFAs in regulating skeletal muscle, and provide new ideas for exploring biological targets of skeletal muscle-related diseases.

【Objective】 The experiment was designed to study the effects of different ratios of cumin straw replacing wheat straw on growth performance, slaughter performance and plasma biochemical parameters in lambs, in order to improve the utilization of cumin straw in Xinjiang, Gansu and other regions.【Method】 Using a one-factor design, twenty-seven healthy male lambs with similar body weight were selected and randomly divided into three groups:0 (control group), 30% (CS1 group) and 60% (CS2 group) of wheat straw were replaced with cumin straw respectively, and the rest components of the ration were the same.Nine lambs per group were fed individually, with 15 d of pre-test period and 60 d formal test period.During the experiment, the growth performance was measured. And after the trial period, the lambs were slaughtered after blood collection to determine the slaughter performance and plasma biochemical parameters.【Result】 ①The average daily feed intake (ADFI) of lambs in CS1 and CS2 groups were significantly higher than that of control group (P<0.05).There were no significant differences in the initial weight, final weight, average daily gain (ADG) and feed-to-weight ratio (F/G) among three groups of test lambs (P>0.05).②Slaughtering rate, live weight before slaughtering, carcass weight and eye muscle area were not significantly different among three groups (P>0.05).Heart weight of lambs in CS1 group was significantly higher than that in control group (P<0.05).There were no significant differences in weights and indices of liver, spleen, lung and kidney among three groups (P>0.05).③ No significant differences existed in the concentrations of plasma glucose(GLU), triglyceride(TG), total cholesterol(TC), total protein(TP), globulin(GLB), albumin(ALB), growth hormone(GH) and insulin-like growth factor-1(IGF-1) among three groups of test lambs (P>0.05).④ There were no significant differences in the parameters of plasma glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) among three groups of test lambs (P>0.05).【Conclusion】 Cumin straw replacing some of wheat straw could increase ADFI of lambs, with no adverse effects on growth, slaughter performance, plasma biochemical parameters, immune and antioxidant function.Cumin straw could be popularized and used in sheep production.

【Objective】 The purpose of this experiment was to explore the effect of translucent on egg quality and the relationship between the formation of translucent eggs and the intestinal function of laying hens.【Method】 A total of 100 Jining Bairi chickens aged 276 days with the same feeding environment were selected.Eggs were collected at 09:00 every day and translucent production of each hen was counted.The experiment lasted for 42 days.Ten laying hens with high translucent rate of grade 1 were selected as the control group, and ten laying hens with high translucent rate of grade 4 were selected as the translucent group.60 translucent eggs and 60 normal eggs were selected to determine the egg quality.Jejunum and ileum tissues were collected from laying hens on day 42.The morphology of intestinal tissues was observed, and the activities of digestive enzymes, ATPase and antioxidant factors in jejunum and ileum tissues were determined.【Result】 The results showed that, compared with the control group, the eggshell thickness and eggshell proportion in translucent group were extremely significantly increased (P<0.01), while the egg yolk color was significantly decreased (P<0.05).The activities of lipase, sodium-potassium ATPase, glutathione peroxidase and total antioxidant capacity of jejunum of laying hens in translucent group were significantly or extremely significantly decreased (P<0.05 or P<0.01).The activities of amylase and calcium and magnesium ATPase of ileal in translucent group were significantly decreased (P<0.05).There were no significant differences in the villus height, crypt depth and ratio of villus to crypt of jejunum and ileum between translucent and control group (P>0.05).【Conclusion】 Eggshell translucent affected egg quality, resulting in eggshell thickness, eggshell ratio increased and yolk color decreased.Some intestinal digestive enzymes and antioxidant capacity of laying hens with eggshell translucent were decreased, so it was speculated that the production of translucent eggs was related to intestinal digestion, absorption and antioxidant capacity.

【Objective】 The double-layer coated microcapsules were prepared using pectin and chitosan as wall materials using microencapsulation technology.The optimal preparation conditions were determined by morphological characteristics, mechanical strength and embedding rate analysis, and the artificial gastrointestinal tolerance of the capsules was studied in vitro.【Method】 The first layer of microcapsule coating was composed of 8% pectin, different concentrations of curcumin (3.5, 4.0, 4.5, 5.0 and 5.5 mg/mL) and calcium chloride (CaCl₂) solution (0.3, 0.4, 0.5 and 0.6 mol/L), and curcumin and pectin solution of different proportions (3:7, 2:8 and 1:9 (V/V)).Screening the best preparation conditions of pectin curcumin microcapsules.Using 0.8% chitosan at different pH (4.2, 4.6, 5.0, 5.4 and 5.8) as the second layer of microcapsule coating, then screened the optimum conditions for preparation of pectin/chitosan curcumin microcapsule.Three groups of microcapsules (T1, T2 and T3) were prepared under the optimal screening conditions, and their appearance, mechanical strength, embedding rate, drug loading and gastrointestinal artificial tolerance in vitro were evaluated.【Result】 The results showed that when 8% pectin, 4.5 mg/mL curcumin, the ratio of curcumin to pectin was 1:9, and 0.5 mol/L CaCl₂ as the first layer of microcapsule coating, the embedding rate of pectin curcumin microcapsules was 98%.When 0.8% chitosan at pH 4.6, 5.0 and 5.4 was used as the second layer of microcapsule coating, the microcapsules showed uniform size and regular shape.Three groups of microcapsules were prepared under the optimal screening conditions, among which the mechanical strength, drug loading, protection rate in artificial gastric fluid and release rate in intestinal fluid in group T1 were the highest.【Conclusion】 In this study, with pectin and chitosan as wall materials and curcumin as core materials, the pectin/chitosan curcumin microcapsules were prepared under the optimal screening conditions, which had good morphology, mechanical strength, drug loading and gastrointestinal tolerance in vitro.The results could provide reference for the development of curcumin microcapsules with pectin and chitosan as wall materials.

【Objective】 This study was aimed to determine the combined effects of Clostridium perfringens (C.perfringens) phage and Lactobacillus on the production performance, intestinal structure and microbial diversity of broilers, and provide the prevention and control technique for C.perfringens infection.【Method】 240 1-day-old Blue-footed partridge chickens were divided into 4 groups:HN02 phage group, Lactobacillus group, HN02 phage+Lactobacillus combination group and blank control group, with 6 replicates (10 per replicate) fed 14 days by drinking phage and/or Lactobacillus in water continuously every day during the period.The average daily food intake (ADFI), average daily gain (ADG) and feed-to-weight ratio (F/G) were counted, the organ index were measured.The number of C.perfringens of cecal content was determined and analyzed by 16S rRNA gene amplification for the structure and diversity of intestinal microbial community.【Result】 Compared with blank control group, in HN02 phage+Lactobacillus combination group, the ADG and villus height/crypt depth were significantly increased (P<0.05), the F/G and crypts depth of jejunal and ileum were significantly decreased (P<0.05), and the number of C.perfringens decreased 1.17 lg CFU/mL on 14 days with the best bacteriostatic effect.The results of microbial diversity showed that the relative abundance of Proteobacteria, Firmicutes, Actinobacteriota and Bacteroidota in the cecal content flora of broilers was higher after drinking HN02 phage+Lactobacillus, and the relative abundance of Lactobacillus was higher at the genus level.Compared with blank control group, the relative abundance of Staphylococcus was lower after drinking HN02 bacteriophage+Lactobacillus for 14 days.【Conclusion】 The combination of C.perfringens phage and Lactobacillus could improve the growth performance and intestinal development of broilers, and maintain the diversity of intestinal microbial community, which showed great potential in the prevention of C.perfringens infection in poultry industry.

With the rapid development of animal husbandry, the shortage of feed ingredients is becoming increasingly obvious, especially, for protein feed ingredients rapeseed meal (RSM), whose main nutrient is protein, is one of the main sources of low-cost protein feed raw materials.Fermented rapeseed meal (FRSM) has higher nutritional value, which can balance the amino acid level in feed, reduce feed cost and ensure animal health.First, the authors compared the differences between RSM and FRSM in the nutrient, found that FRSM had significantly lower levels of anti-nutritional factors, including glucosinolates, tannins, and phytic acids, but increased levels of crude fat, crude protein, and various amino acids.Second, the anti-nutritional factors of RSM and their treatment were briefly described.At present, fermentation treatment of RSM had been found to be one of the most effective methods of removing anti-nutritional factors from feed.Finally, a review of the research progress and future prospects of FRSM in pigs, poultry, ruminants, meat rabbits and red sea bream was presented to provide a reference for the practical application of FRSM in animal diets.

【Objective】 The purpose of this experiment was to investigate the effect of dietary quercetin on prolonging storage time of eggs.【Method】 A total of 270 healthy, 380-day-old Yukou Jingfen No.8 laying hens with similar body weight and laying rate were randomly divided into 5 groups with 6 replicates per group and 9 hens per replicate.The blank control group was fed with corn-soybean meal basal diet, the positive control group was fed the basal diet with 200 mg/kg vitamin E.The three experimental groups were supplemented with 0.03%, 0.06% and 0.09% quercetin in the basal diet, respectively.The experiment lasted for 70 d.In the end of the experiment, eggs were collected and stored in 4 ℃ for 0, 7, 14 and 28 d.The egg quality, content of MDA, lipids and fatty acids and bacterial colony of eggs were analyzed at different storage times.【Result】 Compared with the blank control group, on day 0 after storage, the thick albumen content and albumen pH of eggs in positive control and quercetin groups were significantly increased (P<0.05), the phospholipid content of egg yolk in 0.09% quercetin group was significantly increased (P<0.05), the palmitic acid content of egg yolk in 0.03% quercetin group was significantly increased (P<0.05), and the arachidonic acid methyl ester content was significantly decreased (P<0.05).On day 14th of storage, the triglyceride content of egg yolk in 0.09% quercetin group was significantly decreased (P<0.05).On day 28th of storage, the alpha linolenic methyl ester content of egg yolk in 0.06% quercetin group was significantly decreased (P<0.05), and the content of gamma linoleate methyl ester and arachidonic acid methyl ester of egg yolk in 0.09% quercetin group was significantly decreased (P<0.05).Compared with the positive control group, the phospholipid content of egg yolk in 0.03% and 0.09% quercetin groups was significantly increased on day 0 after storage (P<0.05).On day 7th of storage, the phospholipid content of egg yolk in quercetin group was significantly increased (P<0.05).On day 28 of storage, the triglyceride content of egg yolk in quercetin group at was significantly decreased (P<0.05).In blank control group, the total number of colonies detected on the surface of eggshell stored for 28 days was 2.4×10³ CFU/g, while no bacterial colonies were detected in eggshell, albumen and yolk in other gropus.【Conclusion】 Dietary quercetin could improve egg quality, increase the contents of phospholipid, saturated fatty acids and antibacterial ability, so as to prolong the storage time of eggs.The results of this study provided a reference for the application of quercetin feed additive in prolonging the storage time of eggs.

【Objective】 The aim of this study was to investigate the effect of BMP/Smad signaling pathway on the growth and steroid hormone synthesis of buffalo ovarian granulosa cells.【Method】 Three pairs siRNAs of Smad4 gene (Smad4-siRNA1, Smad4-siRNA2 and Smad4-siRNA3) and NC-siRNA (control group) were transfected into buffalo granulosa cells by lipofection.The expression level of Smad4 gene was detected by Real-time quantitative PCR to compare the interference efficiency of different siRNAs.Then the siRNA with the highest interference efficiency was transfected into buffalo ovarian granulosa cells.The cell proliferation in the control group and interference group was detected by CCK-8, and the expression of the apoptosis-related genes Bax and Bcl2, cell cycle-related regulation genes CyclinD2 and CDK4 and the steroid hormone synthesis related genes Cyp19A1 and Cyp11A1 were also detected by Real-time quantitative PCR, and the contents of estradiol (E₂) and progesterone (P₄) were detected by ELISA.【Result】 The results of gene interference test showed that the three pairs of siRNAs had extremely significant interference effects on the expression of Smad4 gene (P<0.01), and the interference efficiency of Smad4-siRNA1 was the highest, reaching 64%.The CCK-8 test results showed that compared with control group, the cell proliferation rate of the interference group was significantly decreased (P<0.05).The results of Real-time quantitative PCR showed that compared with the control group, the expression of Bcl2, CyclinD2, CDK4 and Cyp19A1 genes in the interference group were significantly or extremely significantly down-regulated (P<0.05 or P<0.01), while the expression of Cyp11A1 gene was extremely significantly up-regulated (P<0.01), and the expression of Bax gene was not affected.The results of ELISA showed that compared with the control group, the estradiol secretion in the interference group decreased by 10.2%, and the progesterone secretion increased by 24.7%, and the difference was significant (P<0.05).【Conclusion】 The disruption of endogenous BMP/Smad signaling pathway by RNAi-mediated Smad4 gene silencing could not only significantly inhibit the growth of buffalo granulosa cells, but also induce the apoptosis of buffalo granulosa cells.Furthermore, inhibiting BMP/Smad signal would change the production of steroid hormones. BMP/Smad signal pathway played an important role in regulating the growth of buffalo granulosa cells and the production of steroids.

【Objective】 The aim of this study was to explore the effects of sodium pyruvate on the quality of frozen sperm of goat.【Method】 Semen of 6 Yunshang Black goats was collected by pseudoginosis method, and diluted with Optidyl diluent containing 0, 1, 5, 10 and 20 μmol/L sodium pyruvate.The total motility (TM) and progressive motility (PM) were measured by SCA automatic semen analyzer, the plasma membrane integrity was measured by hypotonic tolerance test (HOST).Acrosomal integrity, phosphatidylserine (PS) distribution and reactive oxygen species (ROS) were measured by flow cytometry.【Result】 The results showed that compared with 0 μmol/L sodium pyruvate group, the TM and PM indices of sperm in 1 and 5 μmol/L sodium pyruvate groups were extremely significantly improved (P<0.01), but there was no significant difference among other treatment groups (P>0.05). Curvilineal velocity and average velocity, slide swing, whip times, plasma membrane integrity, acrosome integrity rate, ROS level of sperm in 1 and 5 μmol/L sodium pyruvate groups were extremely significantly or significantly increased (P<0.01 or P<0.05). Flow analysis results showed that the proportion of normal sperm was significantly higher than that in 0 μmol/L sodium pyruvate group when the concentration of sodium pyruvate was 1, 5 and 10 μmol/L (P<0.05).【Conclusion】 In this study, adding sodium pyruvate into diluent could effectively reduce the damage to goat sperms during freezing and thawing, and significantly improve the locomotor ability, structural integrity and antioxidant activity of frozen sperms.A small amount of sodium pyruvate showed an improvement effect on sperm, while an excessive amount of sodium pyruvate showed a negative effect.The optimum concentration was 1 μmol/L in this experiment.

【Objective】 The genetic diversity of single nucleotide polymorphism (SNP) of heparin-binding EGF-like growth factor (HBEGF) gene in Songliao Black pigs and its correlation with reproductive traits were analyzed.【Method】 The SNP of HBEGF gene were screened by Sanger direct sequencing in 90 healthy Songliao Black sows, and the correlation between SNP of HBEGF gene and reproductive traits (total number born, number born alive, birth weight, 3-week weight, weaning weight, weaning piglets and nipple number) were analyzed using SPSS 26.0 software.【Result】 A total of 22 SNPs were found in exon 5 and introns 2, 3, 4 and 5 of HBEGF gene in Songliao Black pigs, and a total of 8 SNPs were found to be significantly correlated with reproductive traits.Three genotypes of AA, AG and GG were contained in g.142114384 A>G and g.142104389_142104388ins A>G, three genotypes of CC, CA and AA were contained in g.142114375 C>A, two genotypes of CC and CT were contained in g.142111433 C>T, three genotypes of GG, GA and AA were contained in g.142106003 G>A and g.142105707_142105706ins G>A, three genotypes of CC, CG and GG were contained in g.142105965 C>G, and three genotypes of CC, CT and TT were contained in g.142105878_142105877ins C>T.The chi-square fitness test showed that g.142114375 C>A, g.142111433 C>T and g.142104389_142104388ins A>G were in Hardy-Weinberg equilibrium (P>0.05).g.142114384 A>G, g.142114375 C>A, g.142106003 G>A, g.142105878_142105877ins C>T and g.142104389_142104388ins A>G were moderate polymorphism (0.25<PIC<0.5), g.142111433 C>T, g.142105965 C>G and g.142105707_142105706ins G>A were low polymorphism (PIC<0.25).The results of association analysis showed that the total number born, number born alive and weaning piglets of AA genotype in g.142114384 A>G were significantly higher than those of GG genotype, the birth weight of GG genotype in g.142114384 A>G was significantly higher than that of AG genotype, and the nipple number of GG genotype in g.142114384 A>G was significantly higher than that of AA and AG genotypes (P<0.05).The total number born and number born alive of AA genotype in g.142114375 C>A were significantly higher than those of CC genotype, and the weaning piglets of AA genotype in g.142114375 C>A was significantly higher than that of CC and CA genotypes (P<0.05).The nipple number of CC genotype in g.142111433 C>T was significantly higher than that of CT genotype (P<0.05).The nipple number of GG genotype in g.142106003 G>A was significantly higher than that of AA genotype (P<0.05).The total number born, number born alive and weaning piglets of CC and GG genotypes in g.142105965 C>G were significantly higher than those of CG genotype (P<0.05).The weaning piglets of CC and TT genotypes in g.142105878_1421 05877ins C>T was significantly higher than that of CT genotype, and the nipple number of TT genotype in g.142105878_142105877 ins C>T was significantly higher than that of CC genotype (P<0.05).The 3-week weight and weaning weight of AA genotype in g.142105707_142105706ins G>A were significantly higher than that of GG genotype (P<0.05).The total number born and number born alive of AG genotype in g.142104389_142104388ins A>G was significantly higher than those of AA genotype (P<0.05).【Conclusion】 There were 8 SNPs of HBEGF gene that had significant correlation with total number born, number born alive, birth weight, 3-week weight, weaning weight, weaning piglets and nipple number of Songliao Black pigs.

【Objective】 The experiment was to study the effect of α-linolenic acid(ALA) on cell viability, cell apoptosis, cell cycle-related gene expression and hormone secretion function of buffalo ovarian granulosa cells during in vitro culture.【Method】 In order to screen the optimal concentration of ALA for in vitro cultivation of buffalo ovarian granulosa cells, 1×10⁴/mL of buffalo ovarian granulosa cells with an initial cell count were added to a 96 well plate.After 12 h of cell adhesion, the cells replaced the culture medium with 0 (control group), 10, 50, 100 and 200 μmol/L ALA and treated under the same conditions for 24 h, and cell viability was detected using CCK-8 to screen the optimal treatment concentration and use it for subsequent experiments.Subsequent experiments were divided into two groups:The control group (0 μmol/L) and the treatment group(50 μmol/L).Real-time quantitative PCR was used to detect the relative expression of B-cell lymphoma 2 (Bcl-2), cysteinyl aspartate-specific proteinase-3 (Caspase-3), cyclin-dependent kinase inhibitor 1 (p21), and proliferating cell nuclear antigen (PCNA) genes.Immunofluorescence was used to detect and quantitatively analyze the apoptosis related protein Caspase-3 and cell cycle related protein p21.ELISA was used to detect the levels of estradiol (E₂) and progesterone (P₄) secreted by buffalo granulosa cells in the culture medium.【Result】 Compared with the control group, the cell viability of buffalo ovarian granulosa cells increased with the increase of ALA concentration within the range of 10 to 50 μmol/L after 24 hours of ALA treatment, the cell viability was extremely significantly higher at a concentration of 50 μmol/L compared to the control group (P<0.01).However, as the concentration continued to increase, the activity of buffalo ovarian granulosa cells decreased, and at a concentration of 200 μmol/L, the cell viability was extremely significantly lower than that of the control group (P<0.01).Therefore, a concentration of 50 μmol/L ALA was selected for subsequent experiments.Compared to the control group, after 50 μmol/L ALA treatment, there was no significant difference in the relative expression of anti apoptotic gene Bcl-2 and proliferating cell nuclear antigen gene PCNA (P>0.05), but the expression of pro apoptotic gene Caspase-3 and cell cycle gene p21 were extremely significantly reduced at both mRNA and protein levels (P<0.01), while E₂ and P₄ levels were significantly or extremely significantly increased (P<0.05 or P<0.01).【Conclusion】 Adding 50 μmol/L ALA to the culture medium could enhance the proliferation activity, anti apoptotic ability, and hormone secretion ability of buffalo ovarian granulosa cells, and provided a theoretical reference for regulating the development of buffalo follicles.

【Objective】 The purpose of this study was to study the effect of glutathione (GSH) at different concentrations on cryopreservation of sheep semen.【Method】 The semen were collected from 8 Hu sheeps and diluted with 0, 2, 4, 8 and 16 μmol/L GSH and stored at 5 ℃.Sperm motility parameters including sperm viability, sperm motility, average path velocity (VAP), curvilinear velocity (VCL) and straight line velocity (VSL), sperm quality index including plasma membrane integrity, acrosome integrity, DNA integrity and mitochondrial activity, total antioxidant capacity(T-AOC), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the seminal plasma were measured at 24 and 120 h, respectively.【Result】 After 24 h cryoppreservation of sheep semen, sperm motility, DNA integrity, mitochondrial activity and SOD activity of 4 μmol/L GSH group were significantly higher than those of 0 and 16 μmol/L GSH groups (P<0.05).The sperm motility, sperm viability, VAP, VCL, VSL, plasma membrane integrity, acrosome integrity, DNA integrity, mitochondrial activity and T-AOC levels of 4 μmol/L GSH group were significantly higher than those of 0 and 16 μmol/L GSH groups after 120 h (P<0.05).In addition, MDA content in 4 μmol/L GSH group was significantly lower than that in 8 and 16 μmol/L GSH groups after 24 and 120 h cryopreservation (P<0.05).【Conclusion】 The addition of GSH in semen dilution could improve sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial activity and seminal plasma antioxidant capacity, and significantly improve the cryoppreservation effect of sheep semen.The optimal concentration of GSH was 4 μmol/L.

【Objective】 The purpose of this study was to express the Capsid protein of Duck Tembusu virus (DTMUV) by prokaryotic expression system and prepare its polyclonal antibody to lay a foundation for the study of the molecular mechanism of DTMUV.【Method】 According to the gene sequence of DTMUV-201909 strain, the Capsid gene was cloned into the prokaryotic expression vector pET-30a(+) by one-step cloning technique.The recombinant plasmid was transformed into E.coli BL21(DE3) competent cells.The recombinant protein was induced by IPTG.The recombinant protein was identified by SDS-PAGE and Western blotting.The purified recombinant protein was emulsified with ISA206 adjuvant at equal volume and immunized BALB/c mice to obtain polyclonal antibodies.The titer of the polyclonal antibody obtained was measured by indirect ELISA, and the specificity of the polyclonal antibody was identified by Western blotting and indirect immunofluorescence assay (IFA).【Result】 The recombinant plasmid pET-30a-Capsid was successfully obtained, SDS-PAGE showed that the molecular weight of the expressed recombinant protein was about 18 ku, mainly in the form of inclusion body.Western blotting results showed that the protein could react specifically with anti-His labeled mouse monoclonal antibody and had good reactivity.The results of indirect ELISA showed that the titer of the prepared polyclonal antibody against Capisd protein reached 1:256 000.Western blotting and IFA results showed that the polyclonal antibody could react specifically with DTMUV.【Conclusion】 The mouse anti-Capsid protein polyclonal antibody was successfully prepared.It provided materials for studying the structure and function of Capsid protein, and laid a foundation for revealing the pathogenesis of DTMUV.

【Objective】 The purpose of this study was to study the differences and influencing factors of N gene codon usage bias of Porcine epidemic diarrhea virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV) and Porcine deltacoronavirus (PDCoV).【Method】 The whole genome sequences of the above three PoCoVs were downloaded from GenBank database.The effective codon number (ENC) and relative synonymous codon usage (RSCU) were calculated through websites and software such as EMBOSS explorer, CALcal, DAMBE and CodonW, and the ENC mapping analysis, parity preference analysis and neutral mapping analysis were performed.【Result】 The ENC of N gene sequences of the three PoCoVs ranged from 48.99 to 54.96.The ENC of PEDV, TGEV and PDCoV were 53.74±0.61, 50.09±0.88 and 50.72±0.65, respectively.RSCU analysis showed that the codons of N gene of the three PoCoVs were different, but they generally preferred to end with U.ENC mapping analysis showed that the N gene values of the three PoCoVs were below the standard curve.The parity bias analysis showed that all scatter points were below y=0.5, and the vector was biased to the lower left.Neutral mapping analysis showed that the slope of regression curves in N gene mapping analysis of the PoCoVs was close to 0.The CAI values of S and N genes of the three PoCoVs in Bos taurus, Sus scrofa, Canis lupus familiaris, Homo sapiens and Gallus gallus increased gradually, and the RCDI values decreased gradually and approached 1.【Conclusion】 The codon usage bias of N gene of PEDV, TGEV and PDCoV were different and weak.The N gene of three PoCoVs showed "CpG di-nucleotide deficiency".Natural selection was the main evolutionary pressure for the formation of codon preference for N genes in PEDV, TGEV and PDCoV.Homo sapiens, Sus scrofa and Gallus gallus were suitable to express the N gene of PEDV, TGEV and PDCoV, and the S and N genes of PDCoV were more easily expressed in Gallus gallus.

【Objective】 This experiment was aimed to explore the biological function and molecular mechanism of recombinant porcine RegⅢγ protein.【Method】 RegⅢγ gene fragment was amplified by PCR and connected to pDCP vector to construct RegⅢγ recombinant expression system.The recombinant protein was expressed efficiently at low temperature induced by IPTG and purified by Ni-NTA and dialysis.The inhibitory effects on different bacteria, as well as its functions of promoting proliferation and wound healing, anti-oxidation, and anti-apoptosis of porcine intestinal epithelial cells (IPEC-J2 cells) were analyzed to further explore its molecular mechanism.【Result】 The purified recombinant protein was identified by Western blotting, and the characteristic band was found at 17 ku, which confirmed the successful expression of recombinant porcine RegⅢγ protein.The recombinant porcine RegⅢγ protein showed obvious bactericidal activities against two Gram-positive bacteria, Staphylococcus aureus and Streptococcus, with MIC₉₀ of 39.1 μg/mL, but no bactericidal zone appeared against two Gram-negative bacteria, Escherichia coli and Salmonella Enteritidis.The cell proliferation and wound healing of IPEC-J2 cells were observed evidently when the protein concentration was 10 μg/mL.mRNA expression levels of epidermal growth factor receptor (EGFR), extracellular regulated protein kinase 1 (ERK1), ERK2, cyclin dependent kinase 2 (CDK2), CDK4, cyclin E (Cyclin E) and Cyclin D genes were increased (P<0.05), while the expression of serine/threonine protein kinase 1 (AKT1) gene was decreased(P<0.05).When the recombinant protein was applied to the oxidation-damaged IPEC-J2 cells, it was observed that the activities of SOD and GSH-Px were extremely significantly increased (P<0.01), while MDA content (P<0.05) and LDH activity (P<0.01) were decreased.It was further found that by reducing the mRNA expression levels of proapoptotic genes Caspase-3, Caspase-8, Caspase-9 and Bax, and increasing the mRNA expression level of anti-apoptotic gene Bcl-2, the recombinant protein played the anti-oxidation and anti-apoptosis effects on the oxygen-damaged IPEC-J2 cells.【Conclusion】 Recombinant porcine RegⅢγ protein could selectively inhibit Gram-positive bacteria, promote proliferation and would healing of IPEC-J2 cells, and had certain antioxidant and anti-apoptotic effects.

The muscle of livestock and poultry is the mainly source of protein required for human life activities.Research on the growth and development of muscle is essential to improve the yield and quality of meat products in livestock and poultry.The muscle growth and development in livestock and poultry is an extremely complex process, which is regulated not only by various myogenic regulators (such as MyoD, MyoG and Myf5, etc.), but also by various non-coding RNAs (ncRNA), such as long non-coding RNA (lncRNA), microRNA (miRNA), circular RNA (circRNA), and so on.lncRNA is a type of functional ncRNA that is more than 200 nucleotides in length and lack an open reading frame (ORF), has biological functions such as interfering with gene expression, interacting with proteins, acting as ceRNA and encoding peptides.In recent years, more and more lncRNAs related to muscle growth and development in livestock and poultry have been found and identified, and the specific mechanism and function of many lncRNAs have been gradually determined.Numerous studies have confirmed that lncRNA is widely involved in many stages of muscle growth and development in livestock and poultry, and affects the yield and quality of meat in livestock and poultry.In this article, the general process of muscle growth and development in livestock and poultry, the discovery, classification, and biological functions of lncRNA were introduced, and the currently discovered lncRNA associated with muscle development in various livestock and poultry such as pigs, cattle, sheep, and chickens and their functions were reviewed.This review provided a reference for in-depth research on lncRNA in the growth and development of muscles in livestock and poultry.

【Objective】 The purpose of the test was to understand the infection status of the main bacterial pathogens responsible for porcine respiratory disease complex (PRDC) in large-scale pig farms in China.【Method】 A total of 2 204 samples of lungs, trachea and secretions, spleens and lymph nodes were collected from 647 large-scale pig farms with clinically obvious respiratory symptoms in 26 provinces, cities and autonomous regions of China in 2021.The sample was inoculated into the plate for purification and culture, and then bacteria were identified by morphological observation, Gram staining and PCR identification.In addition, serotype analysis, mixed infection analysis and drug sensitivity test were carried out on isolated Streptococcus suis (SS), Haemophilus parasuis (Hps), Pasteurella multocida (Pm), Bordetella bronchiseptica (Bb) and Actinobacillus pleuropneumoniae (APP).【Result】 The results showed that 647 strains of pathogenic bacteria were isolated and identified.The isolation rate of pathogenic bacteria was 29.35% (647/2 204), including 306 strains of SS (13.88%), 137 strains of Hps (6.22%), 86 strains of Pm (3.90%), 74 strains of Bb (3.35%) and 44 strains of APP (2.00%), respectively.The isolated SS, Hps and APP were chosen to do serotyping tests.The results showed that the major SS was serotype 2, the major Hps was serotype 5, 13 and 4, the major APP was serotype 1.The results of multiple infections showed that the single pathogen infected 407 pig farms, 214 strains of SS, 82 strains of Hps, 38 strains of Pm, 47 strains of Bb and 26 strains of APP were isolated.There were 70 double-mixed infected pig farms, mainly SS+Hps (22 pig farms) and SS+Pm (18 pig farms).There were 6 triple-mixed infected pig farms, mainly SS+Hps+Pm (2 pig farms) and SS+Hps+APP (2 pig farms).The results of drug sensitivity test showed that the isolated bacteria showed different degrees of resistance to 19 kinds of antimicrobial agents, and all the isolated bacteria were resistant to doxycycline and lincomycin, among which Bb showed severe multiple resistance.【Conclusion】 A total of 647 strains of porcine respiratory bacteria pathogens were isolated.SS was mainly serotype 2, Hps was mainly serotype 5, 13 and 4, and APP was mainly serotype 1.The type of multiple infections among different pathogens was complicated, and SS+Hps was the most common type of mixed infection.In this study, different pathogenic bacteria showed different degrees of resistance to 19 kinds of antibiotics.These data could provide a foundation for monitoring epidemiological patterns of bacterial diseases in large-scale pig farms in China, as well as provide reference basis for prevention and control.

【Objective】 The purpose of this study was to obtain the CDS region of macrophage migration inhibitory factor (MIF) gene of Eimeria tenella, and explore its bioinformatics characteristics, so as to provide a theoretical basis for subsequent epitope screening.【Method】 The positive samples suspected of Eimeria tenella infection in Jinzhong area were collected for PCR identification.According to the MIF gene sequence of Eimeria tenella published in GenBank, the CDS region of MIF gene was amplified by RT-PCR, and the recombinant plasmid pET28a-EtMIF was constructed.PCR and double enzyme digestion were used for identification, sequencing and sequence analysis.Bioinformatics software was used to predict the physical and chemical properties, structure and function of MIF protein.【Result】 The CDS region of MIF gene in Eimeria tenella was successfully cloned, with a total length of 348 bp, encoding 115 amino acids, and a relative molecular weight was 1.203×10⁴.The phylogenetic tree analysis showed that the amino acid sequences of MIF gene of Eimeria tenella and Eimeria necatrix had the closest relationship, with the similarity of 98.3%.MIF protein was a non-transmembrane, soluble, non-secretory protein with subcellular localization mainly in the cytoplasm and contains 10 phosphorylation sites and a conserved domain.The secondary structure was composed of random coil (35.65%), extended chain (30.43%), alpha helix (27.83%) and beta turn (6.09%).The tertiary structure was predicted to be consistent with the secondary structure.There were three best potential epitopes.【Conclusion】 MIF gene of Eimeria tenella was successfully cloned, and the bioinformatics analysis of this gene was carried out.The results provided a reference for future research on the function of MIF gene and the screening of genetic engineering vaccine candidates.

Adjuvants are immune stimulators that enhance and regulate the immune response of vaccine antigens.They can produce stronger immune response when used together with antigens.Adjuvants can activate the host's innate immune system.When adjuvants and antigens are injected into the body at the same time or in advance, they can change the shape of antigens and prolong their retention in the body, promote the presentation ability of mononuclear phagocytes to antigens and stimulate the proliferation and differentiation of lymphocytes, thereby enhancing the host's immune response to antigens or changing the type of immune response.Adjuvants have a history of more than 100 years.At present, the most widely used vaccine adjuvant is aluminum adjuvant.Traditional inactivated vaccines rely largely on aluminum salt based compounds as the main adjuvant.However, in recent years, with the rapid development of genetic engineering technology and biotechnology, recombinant vaccines, genetic engineering vector vaccines, nucleic acid vaccines and other novel vaccines have been gradually developed.Compared with traditional vaccines, these novel vaccines often have problems such as poor immune response.Due to the early antigen impurity, the traditional aluminum adjuvant can no longer meet the needs.In order to resolve the problem of poor immunogenicity of novel vaccines, researchers are committed to developing novel broad-spectrum and efficient vaccine adjuvants to enhance the efficacy of novel vaccines.Novel vaccine adjuvants have various types, different functions and complex mechanisms.They can be divided into novel oil emulsion adjuvants, liposome adjuvants, CpG oligonucleotide adjuvants, cytokine adjuvants, nano adjuvants, polysaccharide adjuvants and complex system adjuvants.Therefore, the development history, mechanism of action and classification of novel vaccine adjuvants were analyzed and summarized in this review, which would provide reference for the development of novel vaccine adjuvants.

【Objective】 This study was aimed to understand the prevalence and genetic variation of Porcine epidemic diarrhea virus (PEDV) from 2020 to 2021.【Method】 In this study, 525 samples of 73 pig farms in some regions of China were tested by RT-PCR.The PEDV S1 gene of the positive samples were amplified and sequenced, and epidemiological statistical analysis was conducted.Nucleotide similarity and amino acid site variation were analyzed by DNAStar software.The genetic evolution of PEDV S1 gene was analyzed by Mega 6.06 software, and the suspected recombinant sequence of S1 gene was analyzed by RDP4 software.【Result】 The PEDV positive rate was 26.29%(138/525) in 525 clinical samples from 2020 to 2021.Epidemiological statistical analysis showed that winter was the high incidence season of PEDV, and the infection rate of 1-day-old piglets was the highest (42.75%, 59/183), which was widely prevalent in 6 regions of China.Genetic evolution analysis of S1 gene showed that 138 PEDV S1 gene sequences were mainly located in two branches, and 131 strains belonged to G2 group, of which 126 strains belonged to G2a subgroup with HBXY2 and SNJ-P, another 5 strains belonged to G2b subgroup with AJ1102, LW/L and S14, and the remaining 7 strains were located between G1 and G2 groups, forming an independent branch.The recombination analysis showed that 7 strains in this branch had the same recombination condition, and the breakpoint was at 59-695 bp, which might be cross-group recombination strains.The nucleotide similarity between 138 S1 gene sequences was 85.7%-100%, and the nucleotide similarity with classic strain CV777 was 85.2%-92.3%.Compared with the classical strain CV777, the nucleotides at 56, 59-62, 138-139 and 159-160 sites of S1 gene of 131 strains isolated from group G2 were similar.The amino acid sequence was the same as that of the reference strains in G2 group, with feature deletion and insertion.【Conclusion】 The PEDV epidemic strains showed a trend of variation from 2020 to 2021, and the main epidemic genotype was G2 group, which was closely related to the variation strains prevalent after 2010, but far related to the classical strains, and there was cross-group recombination, which was of great significance for the continuous monitoring of PEDV epidemic and variability in China.

【Objective】 The purpose of the test was to construct a prokaryotic expression system for the truncated leukotoxin (LktA) of Mannheimia haemolytica, express the fusion protein and purify and identify it, and provide a material basis for the establishment of detection methods for Mannheimia haemolytica.【Method】 The specific primers were designed and synthesized, and the lkta truncated gene of Mannheimia haemolytica was amplified by PCR.The PCR product and pET-28b(+) vector were digested with restriction endonucleases EcoR Ⅰ and Hind Ⅲ, and the lkta truncated gene of Mannheimia haemolytica was cloned into the pET-28b(+) vector using T4 DNA ligase.The expression of fusion protein LktA was induced using IPTG.After purification by Ni²⁺ affinity chromatography, the fusion protein was identified using SDS-PAGE and Western blotting.The polyclonal antibody was prepared by immunizing 4-month-old healthy female New Zealand White rabbits with fusion protein LktA.After four immunizations, the antibody titer was determined.【Result】 The sequencing results showed that the lkta gene sequence of Mannheimia haemolytica in the constructed expression vector was correct.The fusion protein LktA had a molecular weight of about 30 ku and was highly expressed in Escherichia coli.The fusion protein existed in the form of inclusion bodies, and the purified fusion protein LktA electrophoresis pattern under denaturation conditions showed a single band, with a purity of >90%.The purified fusion protein LktA could specifically bind to His antibody.After immunizing New Zealand White rabbits with purified fusion protein LktA, polyclonal antibodies were successfully prepared, with an antibody titer of over 1:256 000.【Conclusion】 In this study, we successfully constructed a prokaryotic expression vector containing truncated LktA from Mannheimia haemolytica and prepared polyclonal antibodies against it, providing a material basis for the establishment of a later detection method for Mannheimia haemolytica.

【Objective】 This study was aimed to truncatly express ORF2 gene of Porcine parvovirus type 6 (PPV6) prokaryotic expression system and prepare its polyclonal antibody, so as to provide materials for the follow-up study of the biological function of PPV6 VP1 protein.【Method】 Using the genome of the PPV6 isolate as a template, a truncated ORF2 gene fragment was obtained by PCR amplification and inserted into the prokaryotic expression vector pET30a(+) to construct the recombinant expression plasmid pET30a-PPV6-ORF2.After enzyme digestion and sequencing, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, then the recombinant protein was induced by IPTG, purified by Ni-NTA resin affinity chromatography, and identified by SDS-PAGE and Western blotting.The purified recombinant protein was emulsified with Freund's adjuvant and immunized with New Zealand White rabbits to prepare polyclonal antibodies.Western blotting, indirect immunofluorescence assay (IFA) and indirect ELISA were used to identify the specificity of the immunized rabbit serum.【Result】 The recombinant expression vector pET30a-PPV6-ORF2 was successfully constructed, and the prokaryotic truncated expression of PPV6 VP1_{(348 aa-675 aa)} protein was obtained.SDS-PAGE results showed that the recombinant PPV6 VP1_{(348 aa-675 aa)} protein was about 40 ku in size and existed mainly in the form of inclusion bodies.Western blotting results showed that the protein could specifically bind to PPV6 positive pig serum, and had good immunogenicity.Western blotting and indirect ELISA results showed that the purified immunized rabbit serum reacted specifically with the PPV6 whole virus protein, and the antibody titer was 1:25 600.The IFA identification results showed that the rabbit derived polyclonal antibody against PPV6 VP1_{(348 aa-675 aa)} protein had good specificity.【Conclusion】 In this study, ORF2 gene of PPV6 was successfully truncately expressed, and the polyclonal antibody of PPV6 VP1_{(348 aa-675 aa)} protein was successfully prepared, laid a foundation for further study of VP1 protein and the establishment of serological detection method for PPV6.

Filamentous phages are widely distributed in Gram-negative bacteria.In contrast to lytic phages, after the filamentous phage infects the host, it integrates its genome into the host chromosome through specific recognition sites, and uses the host DNA replication mechanism to complete its own DNA replication.Finally, the mature filamentous phage is secreted and released through the host cell membrane, while the host bacteria can still proliferate, but the growth rate slows down.Since 1996 Vibrio cholerae filamentous phage CTXφ were found to contain the genes encoding cholera toxin, filamentous phages have attracted considerable attention for their interaction with host bacteria.In recent years, numerous studies have shown that filamentous bacteriophages are not only important in horizontal gene transfer but also endow host bacteria with many characteristics related to pathogenicity and viability.This review presents an overview of the role of filamentous phages in host adaptation, including enhancing bacterial biofilm formation, encoding bacterial toxins, modulating production and release of toxins, helping bacteria to adapt the extreme marine environment, helping bacteria resist repeated infection of bacteriophages, so as to increase understanding of filamentous bacteriophage-host interaction, and provide the guidance and basis for bacteriophage therapy to prevent and control bacterial diseases.

【Objective】 The purpose of this test was to investigate the mechanism of Chinese veterinary medicine compound Rukang granules (RKG) on dairy cow mastitis.【Method】 The effective ingredients and targets of RKG were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database and Encyclopedia of Traditional Chinese Medicine (ETCM) platform.Cytoscape v 3.7.2 was used to construct traditional Chinese medicine-ingredient-target.The targets of dairy cow mastitis were obtained from NCBI, OMIM, GeneCards and MalaCards platform.After comprehensive analysis of the targets of dairy cow mastitis and RKG, protein-protein interaction (PPI) network was constructed, then GO function and KEGG pathway enrichment analysis of core network were performed using DAVID.The molecular docking was used to verify the results of network pharmacology.【Result】 There were 23 active ingredients were found in RKG, such as quercetin, isorhamnetin, kaempferol, etc.49 targets related to dairy cow mastitis, like tumor necrosis factor (TNF), Toll-like receptor 4 (TLR4), interluekin-17A (IL17A), etc.After conducting PPI analysis on Bisogenet plug-in, it was found that there were 77 key targets for the effect of RKG on cow mastitis, involving target tumor protein P53 (TP53) and nuclear factor κB P65 (RELA) and breast cancer gene 1 (BRCA1), etc.GO function enrichment analysis showed that biological process enriched in DNA repair, protein ubiquitination, etc.Molecular function enriched in inubiquitin protein ligase binding and ATP binding.Cell component involved cytosol, cytoplasm and nucleus.KEGG pathway enrichment analysis showed that RKG mainly regulated mitogen-activated protein kinase (MAPK), PI3K-Akt and other signaling pathways.The molecular docking results showed that hydrogen bonds were formed between the ingredients quercetin, kaempferol and isorhamnetin and targets BRCA1, RELA and TP53 with affinity less than -3 kJ/mol, among which RELA had the strongest affinity with the above components.【Conclusion】 Network pharmacological analysis showed that RKG might regulate MAPK signaling pathway, PI3K-Akt signaling pathway by targeting to TP53, MAPK, BRCA1, RELA and other targets through the active ingredients, such as quercetin, kaempferol and isorhamnetin, so as to achieve the treatment of dairy cow mastitis.

【Objective】 The purpose of the test was to explore the optimal extraction conditions of Scutellaria baicalensis polysaccharides by water extraction and alcohol precipitation, improve the extraction rate of Scutellaria baicalensis polysaccharide, and explore the structure of Scutellaria baicalensis polysaccharide, so as to provide the basis for industrial extraction of Scutellaria baicalensis polysaccharide.【Method】 The effects of extraction time, extraction temperature, material-to-liquid ratio and extraction times on the extraction yield of polysaccharide were investigated by single factor test.Box-Behnken central combination experimental design method was used to select the optimal extraction time, extraction temperature, material-to-liquid ratio and extraction times, the extraction rate of polysaccharide was the response value (the design of four factors and three levels test), through the response surface analysis to determine the optimal extraction conditions of Scutellaria baicalensis polysaccharide extraction by water extraction and alcohol precipitation method.The polysaccharide was separated and purified by column chromatography.The group, purity, molecular weight and monosaccharide composition of Scutellaria baicalensis polysaccharide were analyzed by infrared spectrum, ultraviolet spectrum, high performance liquid chromatography (HPLC) and gel chromatography.【Result】 Response surface analysis determined that the extraction temperature was 98 ℃, the material-to-liquid ratio was 1:12.40 (g/mL), the extraction time was 138 min, and the extraction times were 4, the extraction yield of crude polysaccharide was 12.23%.Hq-3-1 was purified by DEAE-52 cellulose column chromatography and dextran G-100 gel column chromatography.Gel permeation chromatography analysis showed that Hq-3-1 was a homogeneous polysaccharide with a molecular weight of 58 794 u.The monosaccharides of Hq-3-1 components were analyzed by pre-column derivatization by HPLC.The results showed that the monosaccharides contained fructose, mannose, rhamnose-glucuronic acid, galactose, arabinose and fucose.The UV absorption map showed that Hq-3-1 had no absorption peak at 260 and 280 nm, indicating that it did not contain nucleic acid and protein.Infrared spectrum analysis showed that Hq-3-1 had a polysaccharide characteristic absorption peak, which was α-D-mannose-pyranoid heteropoly.【Conclusion】 Under the optimal extraction conditions, the extraction rate of Scutellaria baicalensis polysaccharides by water extraction and alcohol precipitation was 12.23%.This method was simple, convenient, and had a high extraction rate.This study proved that Scutellaria baicalensis polysaccharide was a heteropolysaccharide with a molecular weight of 58 794 u.

【Objective】 The purpose of the experiment was to investigate the therapeutic effect of extracts of Physalis Calyx seu Fructus on dextran sodium sulfate (DSS)-induced ulcerative colitis in mouse.【Method】 108 SPF-grade male Kunming mice were randomly divided into 6 groups:Blank control (CON), model control (DSS), positive control (SET), and extracts of Physalis Calyx seu Fructus low (EPCF-L), medium (EPCF-M) and high dose (EPCF-H) groups, with 18 mice in each group.After the experiment started, all mice except those in CON group drank 3.0% DSS solution freely, while mice in CON group drank water normally without any special treatment, from day 8 onwards, mice in SET group were given sulfasalazine enteric-coated tablets (100 mg/kg) by gavage, and mice in EPCF-L, EPCF-M and EPCF-H groups were given gavage with 0.25, 0.5 and 1 g/kg extracts of Physalis Calyx seu Fructus respectively, and distilled water was given to mice in CON and DSS groups in the same way, once daily at 0.2 mL each, for 7 days.During the experiment, mice were weighed individually every day, the feces of mice were observed, the disease activity index (DAI), colonic mucosal injury index (CMDI) and histopathological changes of colon were calculated, and the antioxidant indexes of mouse colon tissues were determined including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and inflammatory factors interleukin-6 (IL-6) and IL-1β mRNA expression levels in the colonic tissues of mice were measured.【Result】 Compared with CON group, the weight of mice in DSS group decreased significantly, and their feces did not form or even showed bloody stools. DAI score and CMDI score increased significantly (P<0.05).A large number of inflammatory cells were seen in the colon tissues, the intestinal mucosal structure was destroyed.The activities of SOD, CAT and GSH-Px were significantly reduced (P<0.05), and the mRNA expression levels of IL-6 and IL-1β gene were significantly increased (P<0.05), indicating that the mouse ulcerative colitis model was successfully established.Compared with DSS group, there was a reduction in inflammatory cell infiltration, mucosal congestion and haemorrhage in the colon tissue, DAI and CMDI scores were significantly reduced (P<0.05), activities of SOD, CAT and GSH-Px were significantly increased (P<0.05) and the mRNA expression levels of IL-6 and IL-1β gene in SET, EPCF-M and EPCF-H groups were significantly reduced (P<0.05).【Conclusion】 The therapeutic effect of Physalis Calyx seu Fructus extract on DSS-induced ulcerative colitis in mice might be achieved by improving the antioxidant function of the body and reducing the production of inflammatory factors.

【Objective】 The experiment was conducted to explore the effect of fermentation by Bacillus velezensis on the content changes of the main active components of the self-prepared Rukang prescription, including the flavonoid glycosides from Vaccaria segetalis, caffeic acid and rutin.【Method】 The study established high performance liquid chromatography(HPLC) methods and investigation method linear range, precision, stability, repeatability, sample recovery for the determination of flavonoid glycosides from Vaccaria segetalis, caffeic acid and rutin in self-prepared Rukang prescription.The fermentation culture medium was prepared with the main ingredients of self-prepared Rukang prescription, such as the powder of Vaccaria segetalis, Taraxacum mongolicum Hand.-Mazz, Astragalus membranaceus, Angelica sinensis, Glycyrrhiza uralensis Fisch., accounting for 30%, soybean powder accounting for 10%, CaCO₃ accounting for 0.2%, and ultra-pure water accounting for 59.8%.The fermentation group was inoculated with 2% (v/w) of Bacillus velezensis seed liquid, mixed evenly, and then fermented.After fermentation, double amount of water was added, repeatedly simmered, and filtered as samples to be tested.The contents of flavonoids glycosides from Vaccaria segetalis, caffeic acid and rutin in the fermented and non-fermented Rukang prescription were determined by HPLC.【Result】 The established HPLC methods for the determination of flavonoid glycosides from Vaccaria segetalis, caffeic acid and rutin in Rukang prescription, the linear relationship was good within the range of 0.01-0.5, 5-120 and 10-100 μg/mL, respectively, and the methodological investigations were good, the established method had good precision, stability, repeatability and recovery rate.Under the same physical conditions, the contents of flavonoids glycosides from Vaccaria segetalis, caffeic acid and rutin in the extract of self-prepared Rukang prescription fermented by Bacillus velezensis were 4.99, 6.86 and 5.81 mg/mL, respectively.The contents of flavonoids glycosides from Vaccaria segetalis, caffeic acid and rutin in the non-fermented self-prepared Rukang prescription were 3.70, 3.01 and 3.27 mg/mL, respectively.The difference between the two groups was extremely significant (P<0.01).The results showed that Bacillus velezensis was suitable for fermenting self-prepared Rukang prescription, and the extraction effect was better than that of non-fermented group.The fermentation of Bacillus velezensis was beneficial to the release of flavonoids glycosides from Vaccaria segetalis, caffeic acid and rutin in self-prepared Rukang prescription.【Conclusion】 The fermentation of self-prepared Rukang prescription by Bacillus velezensis could significantly increase the content of main effective ingredients, which provided a technical reference for the efficient extraction of self-prepared Rukang prescription.

【Objective】 This study was aimed to analyze and predict the potential active ingredients and targets of Cuscuta chinensis flavonoids in alleviating the reproductive damage of offspring exposed to bisphenol A (BPA) during pregnancy, to explore its possible mechanism of action, and then to protect animal reproductive function.【Method】 The potential targets of BPA were determined by searching the CTD database, and then the main active ingredients and potential targets in Cuscuta chinensis flavonoids were screened through the TCMSP database and TCMIP platform to obtain common targets of drugs and pollutants BPA, and the protein-protein interaction (PPI) network was constructed by using the STRING database, and the DAVID database was used for GO function and KEGG pathway enrichment analysis.AutoDock Tools 1.5.7 software was used to connect the main active ingredients with core targets.In the clinical experiment, 8-week-old SPF mice were used as the BPA exposure model during pregnancy, and the control group (gavage corn oil from 1 to 17 d of pregnancy, 0.2 mL/d), BPA group (gavage corn oil containing 5 mg/kg BW BPA from 1 to 17 d of pregnancy, 0.2 mL/d) and Cuscuta chinensis flavonoids group (gavage corn oil containing 5 mg/kg BW BPA and 40 mg/kg BW Cuscuta chinensis flavonoids by intragastric administration from 1 to 17 d of pregnancy, 0.2 mL/d) were set up.At the age of 21 days, the level of estrogen (E₂) in serum and the contents of ESR1 and ESR2 in ovarian tissue were measured.【Result】 The results suggested that the possible key ingredients of Cuscuta chinensis flavonoids were quercetin, kaempferol and isorhamna.There were 59 intersection targets of Cuscuta chinensis flavonoids and BPA.The five most highly correlated targets in the PPI network of Cuscuta flavonoids interfering with BPA were serine/threonine protein kinase 1 (AKT1), epidermal growth factor receptor (EGFR), estrogen receptor α (ESR1), mitogen-activated protein kinase 1 (MAPK1) and mitogen-activated protein kinase 1 (MAPK3).GO functional analysis showed that potential targets were enriched to a total of 375 items, mainly involving phosphorylation, cellular response to reactive oxygen species, membrane, cytoplasm, protein binding, nucleotide binding, transferase, and kinase activities, etc. KEGG pathway enrichment analysis showed that Cuscuta chinensis flavonoids were more abundant in cancer pathways, followed by estrogen signaling pathways, relaxin signaling pathways, endocrine resistance, and other pathways, which might be the most important signaling pathways of Cuscuta chinensis flavonoids to intervene in BPA.In addition, the intervention process was also related to the PI3K-Akt signaling pathway and GnRH signaling pathway.Molecular docking results showed that quercetin, kaempferol and isorhamnetin had high binding activity to the key targets of AKT1, EGFR, ESR1 and MAPK1, the ligand compounds could be stably placed in the active docking pocket of the receptor protein.Clinical trials had found that BPA extremely significantly reduced serum E₂ level in offspring mice (P<0.01), and extremely significantly increased ESR1 and ESR2 content (P<0.01), and disrupted endocrine homeostasis, while Cuscuta chinensis flavonoids could normalize hormone and receptor levels.【Conclusion】 The mechanism of action of Cuscuta chinensis flavonoids in alleviating reproductive injury caused by exposure to BPA during pregnancy was multi-target and multi-system, which could affect cancer pathways in addition to affecting endocrine related pathways.

【Objective】 This study was amied to study biological characteristics of Avibacterium paragallinarum (Apg) in Guangdong province and its differences with Apg strains in China and abroad.【Method】 Suspected cases of infectious rhinitis of geese were collected from Guangdong from December 2019 to January 2022.After specific PCR amplification, bacterial isolation and purification, 16S rRNA gene sequencing and identification, sequence comparison was conducted and phylogenetic tree was constructed to determine the type of the isolated strain, and drug sensitivity test and animal regression test were conducted on the isolated strain.【Result】 A total of 3 strains from goose derived Apg were isolated and identified, named G-AP1, G-AP2 and G-AP3, respectively, which all belonged to NAD-dependent strains.The isolated strain fermented glucose and sucrose to produce acid but not gas, and the oxidase test was positive.The results of phylogenetic tree showed that the serotypes of the three isolates were all type A and were closely related to each other, which were close to most of the isolates in China and distant to the isolates abroad.Isolated strains infected geese, the geese showed facial swelling and runny nose fluid after 24 h and milky white and flocculent nasal fluid flowed out from the swollen part when squeezed after 48 h.The infected geese showed subcutaneous swelling, edema, bleeding, fibrous exudation and caseous exudate accumulated in the sinus cavity.The drug sensitivity test showed that ceftiofurme, ceftianol, amikacin, quinolones, flufenicol and clindamycin had strong growth inhibition on the three isolates in vitro.【Conclusion】 In this study, three strains of Apg from geese were successfully isolated, which could cause infection and infectivity of geese.The results provided a reference for the prevention and control of infectious rhinitis of geese in Guangdong.

【Objective】 The purpose of the experiment was to predict and analyze the proportion of Bacillus anthracis (B.anthracis) carrying prophage and the situation of prophage carrying antibiotic resistance gene and virulence gene, to understand and analyze the influence of B.anthracis prophage on bacterial resistance and virulence, and to provide reference for the subsequent research and application of B.anthracis prophage.【Method】 The online analysis software PHASTER was used to predict the prophages carried by B.anthracis, the comprehensive antibiotic research database (CARD) and virulence factors database (VFDB) were used to predict the antibiotic resistance genes and virulence factors carried by the prophages.【Result】 1 421 B.anthracis prophages were predicted, of which 267 were intact prophages, 321 were questionable prophages and 833 were incomplete prophages.The percentage of the prophage genome ranged between 3% to 4% in each B.anthracis genome.The 771 prophages carried 1 075 drug resistance genes.There were 29 resistance phenotypes, from 28 different resistance families, covering 6 antibiotic resistance mechanisms of action.The 394 prophages encoded 688 virulence factors, grouped into 71 virulence genes and 52 virulence factors.Analysis of the composition of possible host strain sources for the virulence factor revealed that, in addition to B.anthracis, Legionella pneumophila subsp., Bacillus cereus and Bacillus thuringiensis also had a high structural proportion as possible host sources for the virulence factor.【Conclusion】 B.anthracis commonly carried prophages, but the prophage genome made up a low proportion of the B.anthracis genome.Approximately 54% prophages carried antibiotic resistance genes, dominated by phosphonic acid and β-lactam.About 28% prophages carried virulence genes.Prophages might play a role in the acquisition and transmission of antibiotic resistance genes, virulence genes and the evolution of pathogenicity in B.anthracis.

【Objective】 Based on the method of network pharmacology and molecular docking, the mechanism of scutellarin against follicular atresia was investigated to screen the mechanism of scutellarin against zearalenone (ZEA)-induced granulosa cell damage.【Method】 The databases of PharmMapper, TCMSP, SymMap and GeneCards were used to obtain the target of scutellarin for anti-follicular atresia.The target protein interaction network diagram was constructed using Cytoscape v 3.7.2 software and the key targets were screened.DAVID database was used to analyze GO function and KEGG pathway enrichment of targets, and construct drug-targets-pathway network diagram.AutoDock Tools 1.5.6 software was used for molecular docking verification.【Result】 34 intersection targets were obtained, and 9 core targets were screened, including insulin-like growth factor 1 receptor (IGF1R), mitogen-activated protein kinase 3 (MAPK3), angiotensinogen (AGT), G1/S-specific cyclin-D1 (CCND1), fibronectin 1 (FN1), tumor necrosis factor(TNF), caspase-3 (CASP3), neurogenic locus notch homolog protein 1 (NOTCH1) and interleukin 6 (IL6).GO function enrichment analysis obtained 264 biological processe including regulation of gene expression, positive regulation of cell proliferation, and negative regulation of apoptosis, 25 cellular component including mitochondria, endoplasmic reticulum and cytoplasm, and 23 molecular function including growth factor activity, protein binding and extracellular matrix structure constitute.By KEGG pathway enrichment analysis, 115 related pathways such as cancer pathway, IL17 signaling pathway, PI3K-Akt signaling pathway were obtained.The results of molecular docking verification showed that the affinity of scutellarin with 9 core target proteins was <0 kJ/mol, indicating that the protein could spontaneously bind with scutellarin.【Conclusion】 Scutellarin might antagonize follicular atresia through multiple targets and pathways, which was a candidate research approach for the mechanism of scutellarin's anti ZEA induced granulosa cell injury.