【Objective】 Pigs play a crucial role in agriculture,and the analysis of their immune traits holds significant importance for enhancing production efficiency and disease resistance.The objective of this study was to identify potential candidate genes associated with immune traits in Duroc×Erhualian F2 generation hybrid pigs,thereby providing valuable gene information for breeding programs aimed at improving porcine disease resistance.【Method】 A total of 294 Duroc×Erhualian F2 generation hybrid pigs were collected from ear and tail tissue samples within 24 hours after birth,and genomic DNA was extracted.Blood samples were collected at 35 days of age to detect cytokines.Genome-wide association study (GWAS) was performed on the immune traits related to cytokines (IFN-α,IFN-γ,IGG,IL8 and IL10) in Duroc×Erhualian F2 generation hybrid pig resource population to identify single nucleotide polymorphism (SNP) site related to the traits.On this basis,the IFN-γ/IL10 value (IFNGIL10) was utilized for further identification of the related SNP and candidate gene influencing the immune traits of pigs.Subsequently,GO function and KEGG pathway enrichment analysis were performed on the identified related genes,and protein-protein interaction (PPI) analysis was performed on the proteins encoded by the candidate genes.【Result】 A total of 10 SNPs were identified through GWAS analysis,which specifically targeted 21 candidate genes associated with immune traits (CH25H,LIPA,SIGMAR1,PIP5KIC,PLCZ1,PIK3C2G,CNTFR,IL11RA,SAP30,HMGB2,CCL21,CCL19,CCL27,GNA11,GNA15,DSCC1,TERF2IP,FZR1,ENPP2,CACTIN and FAS).GO function enrichment analysis results indicated that these candidate genes were significantly enriched in terms such as chemokine activity,cellular response to interleukin-1 and inflammatory response.KEGG enrichment analysis results demonstrated that the candidate genes were significantly enriched in pathways such as cytokine-cytokine receptor interaction,inositol phosphate metabolism and calcium signaling pathway.Furthermore,the PPI analysis constructed a total of 12 subnetworks,suggesting potential interactions among these candidate genes,which were notably enriched within the same pathway,collaboratively participating in biological processes to fulfill their respective functions.【Conclusion】 The present study identified 10 SNPs associated with porcine immune traits through GWAS,and further selected 21 candidate genes for target traits by integrating enrichment analysis and PPI analysis.Overall,the findings from this study not only deepened our understanding of the molecular basis underlying porcine immune traits,but also provided a theoretical foundation for porcine disease resistance breeding.

【Objective】 The purpose of this experiment was to use the genome resequencing data,transcriptome data,and plasma biochemical indicators of Pekin ducks and Mallard ducks to screen for candidate genes related to fat deposition in Pekin ducks.【Method】 Three 42-day-old Pekin ducks and Mallard ducks were selected to collect and separate the blood plasma from wings,and 12 biochemical indexes such as triglyceride (TG) and phospholipid (PLIP) were determined.Based on the genome resequencing data of 30 Pekin ducks and 41 Mallard ducks,various methods such as fixation index (F_ST),cross population extended haplotype homozygosity (XP-EHH),and Tajima’s D test were used to detect potential selective intervals in Pekin ducks.Using three sliding window/step size models,it was ensured that at least one method could be used to filter out the domestication genes of Pekin ducks.Total RNA was extracted from duck liver samples and transcriptome sequencing was performed.Differential expression genes in liver tissues of Pekin ducks and Mallard ducks were screened by differential expression gene analysis and the KEGG pathway enrichment analysis was performed.The intersection of differentially expressed genes and domestication genes of Pekin ducks was selected to screen out candidate genes regulating duck fat deposition.【Result】 The results of plasma biochemical indices showed that the contents of low density lipoprotein cholesterol (LDL-C) and glucose (GLU) in plasma of Pekin ducks were significantly lower than those of Mallard ducks (P＜0.05),and the content of TG was significantly higher than that of Mallard ducks (P＜0.05).A total of 2 681 domestication genes of Pekin ducks and 455 differentially expressed genes in liver were screened.Differentially expressed genes were significantly enriched in metabolic pathways such as glycerolipid metabolism and glycerophospholipid metabolism.Two differentially expressed genes in liver that were strongly selected during the domestication process of Pekin ducks were identified:ACSL5 and ELOVL3.【Conclusion】 This study identified two differentially expressed genes in liver of Pekin ducks and Mallard ducks,namely ELOVL3 and ACSL5.These two genes underwent strong positive selection during the domestication process of Pekin ducks and might be key candidate genes regulating fat deposition in ducks.The results of this study provided a theoretical basis for elucidating the regulatory mechanism of lipid deposition in ducks and precisely regulating the lipid deposition levels in Pekin ducks,offering a reference for further research on duck fat deposition.

【Objective】 TIR domain-containing adaptor protein inducing interferon β (TRIF) gene played the important roles in host resistance and immunity.This study was aimed to study the sequence characteristics and tissue expression of CDS region of TRIF gene in Hezuo pigs.【Method】 Gansu Hezuo pigs was taken as the research object,the full-length CDS region sequence of TRIF gene was cloned and subjected to bioinformatics analysis.The expression levels of TRIF gene were detected from 8 tissues including lung,kidney,spleen,longissimus dorsi muscle,cecum,ileum,rectum and colon through Real-time quantitative PCR.【Result】 The full length of TRIF gene CDS region was 2 142 bp and encoded 713 amino acids,which was 99.81% similarity to TRIF gene sequence of Sus scrofa in NCBI database (accession No.:NM_001315738.2).Phylogenetic tree results showed that Hezuo pigs were closest to Sus scrofa and furthest to Danio rerio.TRIF protein of Hezuo pigs had hydrophilicity,no transmembrane region,no signal peptide and no N-glycosylation modification sites,which was mainly located in the nucleus.TRIF protein was mainly composed by random coils of secondary structure.The tertiary structure prediction was basically consistent with the secondary structure.The protein had six low-complexity domains and a winding coil region.Real-time quantitative PCR results showed that TRIF gene of Hezuo pigs was expressed in eight tissues including lung,kidney,spleen,longissimus dorsi muscle,cecum,ileum,rectum and colon,with the highest expression in longissimus dorsi muscle.【Conclusion】 This study had successfully cloned the CDS region sequence of TRIF gene in Hezuo pigs.The highest expression was found in longissimus dorsi muscle.The results could provide a valuable reference for the further study of TRIF gene in Hezuo pigs.

【Objective】 The aim of this study was to explore the effects of polystyrene nanoparticles (NPs) on reproductive system damage and testosterone synthesis in male mice.【Method】 A total of 24 clean grade male C56 mice were randomly divided into 4 groups:Control group and low (0.1 mg/L),medium (1 mg/L) and high (10 mg/L) dose polystyrene NPs groups,with 6 mice in each group.The experiment lasted for 90 d.After the test,blood was collected from eyeballs of mice,epididymis and testicles were removed after neck removal,and sperm was collected.Testis were weighed,testicular index was counted,testicular histopathological changes were observed by HE staining,total sperm motility and total sperm viability were analyzed by comprehensive semen analysis system.The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in testicular tissue, and the content of serum testosterone were detected by ELISA kit.Western blotting was used to detect the expression of oxidative stress and apoptosis-related proteins.The mRNA and protein expression of genes related to testosterone synthesis were detected by Real-time quantitative PCR and Western blotting,respectively.【Result】 Compared with control group,the testicular index of mice in medium and high dose polystyrene NPs groups was significantly or extremely significantly increased (P＜0.05 or P＜0.01),the total motility and total viability of sperm and serum testosterone content of mice were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The ELISA results showed that compared with control group,the MDA content in testis tissue of mice in polystyrene NPs treated groups was extremely significantly increased (P＜0.01),and the activities of SOD，CAT and GSH-Px were significantly decreased or extremely significantly decreased (P＜0.05 or P＜0.01).Western blotting results showed that compared with control group,the expression of nuclear factor E2 related factor 2 (Nrf2) protein in testicular tissue of mice in medium and high dose polystyrene NPs groups was extremely significantly increased (P＜0.01),and the expression of 3β-hydroxysteroid dehydrogenase (HSD-3β) protein was extremely significantly decreased (P＜0.01).The expression of heme oxygenase-1 (HO-1) protein in testicular tissue of mice in polystyrene NPs treated groups was extremely significantly decreased (P＜0.01).The expression of aspartic cysteine-specific protease 9 (Caspase9),Caspase3,Bcl2-associated X protein (Bax)/B lymphoblastoma-2 (Bcl-2) protein in testicular tissue of mice in high dose polystyrene NPs group were extremely significantly increased (P＜0.01)，and the expression of cholesterol side chain lyase (CYP11A) protein was significantly decreased (P＜0.05).Real-time quantitative PCR results showed that compared with control group, the expression of HSD-17β,cytochrome P450 cholesterol side chain lyase (P450 scc) and steroid-hormone acute regulatory protein (StAR) genes in testicular tissue of mice in polystyrene NPs treated groups were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The expression of HSD-3β and P450c17 genes in testicular tissue of mice in medium and high dose polystyrene NPs groups were extremely significantly or significantly decreased (P＜0.01 or P＜0.05). 【Conclusion】 Polystyrene NPs could induce oxidative stress in testicular tissue of mice,which led to cytogenesis,and eventually led to testicular injury and testosterone synthesis disorder.

Histone acetylation,as a delicate epigenetic modification mechanism,plays a crucial regulatory role at many levels of life.This modification not only regulates the expression level of genes and the three-dimensional spatial structure of chromatin,but also plays an indispensable role in DNA damage repair,a core biological process.The DNA damage repair process is a complex biological response that cells use to remove harmful errors or damage within the DNA molecule,ensuring the integrity and functionality of the genome.The repair process involves a variety of different pathways,such as direct repair (DR),base excision repair (BER),nucleotide excision repair (NER),mismatch repair (MMR),and double strand break repair (DSBR),etc.DNA double-strand breaks (DSBs) are the most common and have the greatest impact on genome stability.With the deepening of research,it has been found that histone acetylation can participate in multiple repair pathways by affecting the recruitment and activity of enzymes related to DNA damage repair,thereby dynamically maintaining genome stability at the epigenetic level.In order to better understand the role of histone acetylation in DNA damage repair,the types of DNA damage were classified,and the histone acetylation sites related to them were sorted out,focusing on the regulatory role of histone H3 and H4 acetylation sites in DNA damage repair,which provided a reference for the regulation and research of related cell phenotypes.

N6-methyladenosine (m⁶A),the RNA methylation of the 6th nitrogen atom of the adenine A base group,is the most common and abundant RNA modification in eukaryotic cells.The dynamic and reversible m⁶A modification is catalyzed by a methyltransferase complex composed of methyltransferase-like 3(METTL3)and its cofactors.The RNA methylation modification signal is eliminated by demethylases such as fat mass and obesity-associated protein (FTO) and ALKB homolog 5 (ALKBH5),and the m⁶A modification site is recognized by m⁶A binding proteins.m⁶A modification has become a research hotspot in the field of epigenetics,as it can regulate molecular regulatory effects such as RNA splicing,enucleation,translation and stability.Skeletal muscle is an important component of the animal body,and its development involves multiple processes such as proliferation,migration,differentiation and fusion of myoblasts to form myotubes,which in turn form muscle fibers,it is regulated by various transcription factors and epigenetics.Among them,m⁶A methylation modification plays a role in regulating the maintenance,proliferation,and differentiation of muscle stem cells.Recent studies have shown that transcription and post-transcriptional regulation mediated by m⁶A methylation play a crucial role in the growth and development of skeletal muscle.The authors introduce the types and functions of m⁶A methylation modification enzymes,and with a focus on summarizing the regulatory effects and mechanisms of m⁶A methylation modification in skeletal muscle growth,development and related diseases,so as to provide new ideas for analyzing the epigenetic regulation mechanism of muscle development and discovering potential therapeutic targets for skeletal muscle diseases.

Broiler ascites syndrome is a nutritional metabolic disease caused by multiple etiology,which is related to heredity,feeding management,nutritional imbalance and environment.Since its first appearance,the disease has caused serious economic losses to the global poultry industry.Research indicates that systemic hypoxia is an important cause of ascites in broilers.Prolonged hypoxia can lead to the injury of pulmonary artery endothelial cells (PAECs),resulting in functional disorders,excessive production of vasoactive factors,abnormal contraction of vascular smooth muscles,and exacerbation of abnormal proliferation,inflammatory responses,and phenotypic transformation of vascular endothelium. Over time,these changes will lead to complex pathological processes such as the thickening of the vascular media and the gradual narrowing of the lumen,which will promote pulmonary artery remodeling and eventually become the direct inducement of ascites formation.As epigenetic regulatory factors,miRNA plays an important role in regulating various signal transduction pathways and thus affecting cell physiological functions.In the formation of pulmonary hypertension,miRNA plays a particularly significant role in regulating the physiological process of pulmonary artery endothelial cells.Consequently,the author discussed the etiology and pathogenesis of broiler ascites syndrome,clarified the functional changes of pulmonary artery endothelial cells and the key role of miRNA in this complex disease process,in order to provide reference for the prevention and control of broiler ascites syndrome.

【Objective】 This experiment was conducted to investigate the effects of low-protein amino acid balanced diets on laying performance,egg quality,serum biochemistry and lipid metabolism of laying ducks,providing a basis for the efficient application of low protein diets with soybean meal free in laying duck production.【Method】 Eighty healthy Longyan Shanma ducks of 21-week-old,with comparable laying performance,were selected and randomly divided into 2 groups with 4 replicates of 10 ducks each,and were housed in individual cages.The ducks from control group were fed a 16.5% crude protein (CP) diet,and ducks from the low-protein group were fed a diet with 14.5% CP,which supplemented with seven kinds of amino acids for the balance of essential amino acids.The experimental period was 12 weeks.The daily egg production,egg weight,and feed intake of each replicate was recorded for evaluating the egg production performance during the experimental period.At weeks 4,8,and 12 of the experiment,6 duck eggs were collected from each replicate,of which 4 were measured for egg quality,2 were measured for the contents of nutrient in yolk and albumin,and yolk lipid metabolism indicators.In the 12th week,two ducks were randomly selected from each replicate to collect plasma and slaughtered.The reproductive organs,abdominal fat index,and plasma and liver lipid metabolism indicators of the ducks were measured.【Result】 Compared with the control group,the average daily feed intake and average egg weight decreased by 10.51% and 2.85% (P＜0.05),respectively,egg production rate and daily egg weight were not significantly different (P＞0.05),and the feed-to-egg ratio decreased by 7.37% (P＝0.06),the relative weight of albumin increased by 3.24% (P＜0.05),and the toughness of eggshells increased by 9.70% (P＝0.06),relative weight of 3-6 mm follicles increased by 34.23% (P＜0.05) of the low-protein group.There were no significant differences between the two groups observed in egg shape index,shell strength and thickness,height of albumin,yolk color,Haugh unit,the relative weights of shell and yolk,the contents of crude protein and crude fat in yolk and albumin,number of dominant follicles,the indices of liver and abdominal lipid, serum biochemical indicators,and the contents of triglyceride and total cholesterol in liver and yolk (P＞0.05).【Conclusion】 Low protein amino acid balanced diet with soybean meal free significantly reduced the average daily feed intake and average egg weight of laying ducks,with a tendency to reduce the feed-to-egg ratio.But it did not affect the daily egg mass,egg quality,reproductive organ development and lipid metabolism of laying ducks.

【Objective】 The experiment was conducted to study the effects of dietary crude protein level on growth performance,blood biochemical indexes and nutrient utilization of Yanjin Black-bone chickens,and to determine the appropriate dietary crude protein level.【Method】 A total of 200 one-day-old Yanjin Black-bone chickens with similar body weight and health were randomly divided into 5 groups with 5 replicates in each group and 8 chickens in each replicate (half male and half female),and the diets were fed with crude protein levels of 17.40%,18.70%,20.00%,21.30% and 22.60%,respectively (17.40% CP,18.70% CP,20.00% CP,21.30% CP and 22.60% CP groups).During the experiment,the average daily gain (ADG),average daily feed intake (ADFI) and feed to gain ratio (F/G) were calculated.Metabolic tests were carried out on days 36-39 to determine total energy,crude protein,and amino acid. On the 43rd day of the experiment,2 chickens in each replicate were selected for blood sampling and slaughter to determine serum biochemical indexes and body composition.【Result】 Dietary crude protein level had no significant effects on initial body weight,final body weight,ADG,ADFI and F/G (P＞0.05),but the final body weight increased first and then decreased with the increase of dietary crude protein level (P=0.095),and the highest final body weight was obtained when the dietary crude protein level was 20.00%.The protein deposition rate of 17.40% CP and 18.70% CP groups was significantly higher than that of 20.00% CP,21.30% CP and 22.60% CP groups (P＜0.05).The deposition rates of 8 essential amino acids except lysine and methionine showed a significant downward trend with the increase of dietary crude protein level (P＜0.05),and all of them decreased linearly and quadratically with the increase of dietary crude protein level (P＜0.05). The nitrogen intake and fecal nitrogen increased linearly and quadratically with the increase of dietary crude protein level (P＜0.05), and nitrogen utilization rate decreased linearly and quadratically with the increase of dietary crude protein level (P＜0.05). The serum uric acid content of chickens in 21.30% CP and 22.60% CP groups were significantly higher than that in 17.40% CP and 18.70% CP groups, and increased linearly and quadratically with the increase of dietary crude protein level (P＜0.05). 【Conclusion】 The recommended dietary crude protein level for Yanjin Black-bone chicken was 20.00%.

With the development of dairy cattle breeding technology,advancements in feed science,and the progress of genetic improvement,the milk yield per cow has continuously increased.Many countries have seen their dairy production reach historical highs.Meanwhile,consumer demand for dairy products has also changed,with a tendency to choose higher-quality products.Rumination is a unique behavior of ruminant animals and is one of the important indicators reflecting their health and production performance.Rumination helps ruminants better digest and absorb nutrients from their food,improving both the quantity and quality of milk.Additionally,monitoring the rumination behavior of ruminants aids producers in timely understanding their health status and production levels,allowing them to adjust breeding strategies promptly and reduce economic losses.In the context of increasingly large-scale and intensive farming models,the time and economic costs of manual monitoring are rising.With technological advancements,intelligent monitoring methods have provided technical support for the automatic monitoring of rumination,leading to the emergence of more intelligent monitoring solutions.In particular,the continuous updates and iterations of hardware technology and software algorithms have begun to showcase machine learning algorithms in both contact-based and non-contact-based monitoring methods for dairy cow rumination.The authors review how these typical factors influence the rumination behavior of dairy cows from multiple aspects,it also organize common contact-based methods for monitoring rumination behavior,as well as non-contact intelligent monitoring methods.The paper elaborates on the algorithm principles and application effects of these relevant methods,listing some common applications of rumination monitoring methods at present and categorizing them.Finally,it discusses the existing issues in intelligent monitoring of rumination behavior and prospects for its future development,aiming to provide references for related research.

【Objective】 This experiment aimed to explore the effects of supplementing rumen-protected methionine (RPM) to diets with different protein levels on the growth performance,rumen fermentation and nutrient apparent digestibility of yaks,so as to provide a reference for dietary protein level and RPM supplementation of house-feeding yaks breeding.【Method】 A 2×3 two-factor experimental design was adopted in this experiment.Two protein levels were low (13%) and high (15%),and three RPM levels were 0,0.05% and 0.10%.A total of thirty-six 4-year-old healthy male yaks with similar body weight (225.29 kg±30.59 kg) and body size were selected and randomly divided into six treatment groups.Each treatment group had six replicates,and each replicate had one yak.Single pen and single feeding were adopted.The pre-trial period was 10 d,followed by a formal trial period of 60 d.Samples of feed,faeces and rumen fluid were collected on 66-69 and 70 d of experiment for determining the growth performance,apparent digestibility of nutrients and rumen fermentation parameters of yaks.【Result】 ①Dietary protein levels had no significant effect on average daily dry matter intake (ADMI) of yaks (P＞0.05),but compared with 13% protein group,average daily gain (ADG) of yaks in 15% protein group was significantly higher (P＜0.05), and feed to gain ratio (F/G) was significantly lower (P＜0.05).The RPM supplementation had no significant effect on ADMI and F/G (P＞0.05),but tended to increase ADG (P＝0.051).There was a trend of interaction between dietary protein levels and RPM levels on ADMI in yaks (P＝0.058).②Dietary protein levels had no significant effect on rumen pH and microbiological proteins (MCP) (P＞0.05),but had a tendency to increase the content of NH₃-N (P＝0.091).There was no significant effect of dietary RPM levels on rumen pH,MCP and NH₃-N (P＞0.05).There was a trend of interaction between dietary protein levels and RPM levels on rumen NH₃-N content in yak (P＝0.092).Compared with 13% protein group, content of propionic acid was higher (P＜0.05),the content of butyric acid (P＝0.066),isobutyric acid (P＝0.073),valeric acid (P＝0.053) and isovaleric acid (P＝0.056) had a tendency to increase in 15% protein group.There was no significant effect of dietary RPM levels on rumen volatile fatty acids (P＞0.05).There was no significant interaction to volatile fatty acid between dietary protein levels and RPM levels (P＞0.05).③Compared with 13% protein group,apparent digestibility of crude protein,calcium and acid detergent fiber in 15% protein group was significantly increased (P＜0.05).There was no significant effect of dietary RPM levels on nutrient apparent digestibility (P＞0.05).There was no significant interaction to nutrients apparent digestibility between dietary protein levels and dietary RPM levels (P＞0.05).【Conclusion】 Under the conditions of this experiment,adding 0.05% rumen protected methionine to the diet with a 15% protein level for 4-year-old house-fed yaks could improve their rumen fermentation,increase their dry matter intake,nutrient apparent digestibility and growth performance.

Isoorientin is a natural flavonoid plant compound with a wide range of biological activities and pharmacological effects.In nature,isoorientin mainly exists in many plants,such as Fagopyrum esculentum Moench,Gentiana straminea Maxim,Kummerowia striata (Thunb.) Schindl, Lythrum salicaria L and Polygonum orientale L.Previous studies have shown that isoorientin has multiple effects such as improving osteoporosis,treatment of diabetes,antidepressant,anti-tumor,and reducing organ damage.Its mechanism of action is mainly achieved through the biological activities of antioxidant stress,anti-inflammatory,and regulation of cell apoptosis.The key proteins involved in this process include nuclear factor E2-related factor 2 (Nrf2),nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB),mitogen-activated protein kinase (MAPK),tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β),phosphatidylinositide 3-kinases (PI3K),protein kinase B (Akt),heme oxygenase-1 (HO-1),etc.In addition,current research on isoorientin mainly focuses on acute and subacute toxicity tests on rats,mice and related cell lines,lacking basic experiments on other animals or cells such as livestock and poultry,which to some extent affects the product development and clinical application of isoorientin.This article reviewed the functional activity of isoorientin,and proposes prospects for its clinical application,in order to provide new ideas and theoretical support for further research on the biological activity of isoorientin and expand its clinical application.

【Objective】 This study aimed to screen Bacillus subtilis with good probiotic properties.【Method】 Ten fecal samples and six environmental samples were collected from large-scale sheep farms,and the bacteria were isolated and purified by LB solid medium.The strains were identified by morphological observation,biochemical identification and 16S rRNA gene PCR amplification.Then,the probiotic characteristics of the strains were comprehensively evaluated by hemolysis test,drug sensitivity test,drug resistance and virulence gene analysis,in vitro antibacterial test,acid-base and bile salt tolerance test,and mouse safety evaluation.【Result】 The two isolated bacteria formed irregular white protruding colonies on the blood plate,the surface was rough and mountain-like,and there were obvious hemolytic rings around them.The results of biochemical identification showed that the isolated two strains were positive in catalase,VP,glucose,xylose,L-arabinose,mannitol,sucrose,citrate,nitrate reduction,starch hydrolysis,gelatin liquefaction,maltose and mushroom sugar tests,and negative in hydrogen sulfide,citrate and galactose tests.They were preliminarily identified as Bacillus subtilis and named K1 and K2,respectively.The results of 16S rRNA gene PCR amplification and sequencing showed that the similarity between the isolated strains and the reference strains of Bacillus subtilis was 98%-100%,which was in the same branch of the phylogenetic tree,but in different branches with Bacillus velezensis,Salmonella and Enterococcus faecium.The results of bacteriostatic test showed that the supernatant of isolated strains K1 and K2 had good bacteriostatic effect on Staphylococcus aureus,but had no bacteriostatic effect on Escherichia coli and Salmonella.The results of drug sensitivity test showed that the isolated strains K1 and K2 had high sensitivity to penicillin,amoxicillin,cefotaxime,enrofloxacin,ofloxacin,cotrimoxazole,florfenicol and vancomycin,and were intermediate to gentamicin,spectinomycin and roxithromycin,and resistant to tetracycline,polymyxin B and lincomycin.The results of virulence gene detection showed that the isolates K1 and K2 did not carry virulence genes such as nheA,bceT,entFM,ces,cytK,hblA,hblC and hblD.The results of drug resistance gene detection showed that the isolates K1 carried aph(3')-Ⅰa resistance gene and did not carry MCR-1,bcr,floR,rpsl,strB,aadB,aac(6')-Ⅰb,bla_TEM,bla_CTX,ermA,ermC,qnrA,qnrB,sul1,sul2,tetC,tetA,tetM and catA2 resistance genes,while K2 strain carried ermA resistance gene and did not carry other verified resistance genes.The two isolates had good growth activity in LB liquid medium with pH of 4.0,5.0,6.0,8.0,9.0 and 10.0.Among them,strain K2 still had good growth activity in LB liquid medium with pH of 3.0.In LB liquid medium containing 0.3%,0.5%,0.7% and 1.0% bovine bile salts,the two isolates could still survive and maintain relatively good growth activity.The safety test in mice showed that there was no obvious pathological change in mice.【Conclusion】 The two strains isolated in this study were Bacillus subtilis, which had hemolytic properties, antibacterial activity against Staphylococcus aureus and excellent growth activity.In the simulated gastrointestinal environment,it showed diversity in acid,alkali and bile salt tolerance,and remained sensitive to most antibiotics.No major virulence genes were detected.It had good safety to mice and had the potential as a probiotic.which could further explore its application in animal husbandry and human health.

【Objective】 This experiment was aimed at studying the effects of licorice,Picrasma quassioides extract (hereinafter referred to as licorice extract) and montmorillonite on the digestion and metabolism,rumen fermentation parameters and microbial composition of lactating calves.【Method】 45 newborn Chinese Holstein male calves were selected and subjected to a single factor randomized experimental design.The calves were divided into three treatment groups (5 replicates per treatment,3 calves per replicate):The control group (CON group),the licorice extract group (CPE group),and the compound preparation group (MMT group).The calves in CON group were fed with milk replacer powder and basic feed,the calves in CPE and MMT groups were fed with milk replacers supplemented with 1 g/kg licorice extract and 1 g/kg licorice extract+5 g/kg montmorillonite,respectively.The basic feed and other feeding management were consistent with CON group.The experimental period was 67 days,including a pre-trial period of 7 days and a main trial period of 60 days.Ruminal fluid was collected at 67 days of age to measure rumen fermentation indicators and microbial composition.At 68 days of age,a 7-day digestion and metabolism test was conducted to determine the apparent digestibility of nutrients and nitrogen metabolism indicators.【Result】 ①Compared with CON group,the apparent digestibility of organic matter,crude fat,and neutral detergent fiber in MMT and CPE groups of calves were significantly increased (P＜0.05),and there was no significant difference between the two additive groups (P＞0.05).② Compared with CON group,there were no significant differences in total energy digestibility,total energy and digestible energy metabolism,nitrogen apparent digestibility and deposition rate,pH of rumen fluid,rumen total volatile fatty acid content,and various fatty acid contents between MMT and CPE groups of calves (P＞0.05).The levels of NH₃-N and microbial protein in MMT group of lactating calves were significantly higher than those in CON and CPE groups (P＜0.05),while there was no significant difference between CPE and CON groups (P＞0.05).③ The Simpson index of rumen microbiota in lactating calves in MMT group showed an increasing trend compared to the CON and CPE groups (0.05＜P＜0.10).PCoA analysis revealed that the community composition differences between samples in MMT group were relatively small,and the sample similarity was high.The relative abundance of Firmicutes,Trichomonas,Bacteroides,and Vibrio butyricum in the rumen of lactating calves in MMT group were significantly higher than those in CPE group (P＜0.05),while there was no significant difference compared to CON group (P＞0.05).【Conclusion】 Adding 1 g of licorice extract and a composite preparation (1 g of licorice extract and 5 g of montmorillonite) to every kilogram of milk powder during lactation had an improving effect on the digestion and metabolism of nutrients in calves.The apparent digestibility of organic matter,crude fat,and neutral detergent fiber reached a significant level,and the addition of a composite preparation could increase the synthesis efficiency of rumen microbial proteins and the relative abundance of Helicobacter,Pseudomonas,and Vibrio butyricum genera.

Cold stress is a common and inevitable stress factor in the development of animal husbandry,which can cause animal temperature drop,appetite loss,growth rate slowdown,immune function decline, disease susceptibility, etc.,resulting in huge economic losses for breeding production.When animals suffer from cold stress,the body accelerates the metabolism of energy substances such as lipids and sugars to enhance the ability to adapt to cold.Therefore,the authors reviewed the research progress on the effects of cold stress on lipid metabolism and glucose metabolism indexes and found that different animals had different abilities to mobilize energy metabolism and produce heat under cold stress.By further exploring the functional mechanisms of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor (PPAR), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), hormone-sensitive lipase (HSL) and cold-inducible RNA-binding protein (CIRP) in regulating mitochondrial biogenesis,glycogenolysis and fatty acid metabolism,the adaptive mechanism of animal body regulating energy metabolism was revealed from the molecular level.This paper aimed to provide theoretical basis for understanding the metabolic mechanism of animals in cold environment and improving the production performance and breeding benefits of livestock and poultry in cold areas in China.

Fat deposition ability is one of the important factors affecting the lean meat rate,slaughter rate and meat quality of pigs,and the regulatory mechanism of fat deposition involves complex biological processes,among which epigenetic regulators such as non-coding RNA (ncRNA) play a key role.Circular RNA (circRNA) is a covalently closed circular ncRNA existing in eukaryotes,it is relatively conservative and spatiotemporally specific,with rich and diverse action pathways,and is widely involved in various biological processes such as cell proliferation and apoptosis,gene transcription,cancer occurrence and nervous system diseases.In recent years,more and more studies have shown that circRNA plays an important regulatory role in the deposition of subcutaneous,intramuscular and visceral fat in pigs,but most studies are still in the initial stage of discovery and identification,and the specific mechanisms of many upstream and downstream pathways of circRNA are still unclear.At present,circRNA mainly controls the expression of fat deposition-related genes through the molecular sponge mechanism of miRNA,affects lipid metabolism,promotes or inhibits the proliferation and differentiation of adipocytes,and regulates fat deposition in pigs.Based on an overview of the classification and function of adipose tissue,key genes regulating fat deposition,and the biogenesis and function of circRNA,this paper systematically reviews and summarizes the research progress on circRNA regulating fat deposition of pigs at home and abroad,in order to provide a reference for further exploration of the mechanism of pig fat deposition.

Aflatoxin B₁(AFB₁) is a kind of natural toxin,widely found in food and animal feed,with strong acute toxicity,carcinogenicity,teratogenicity and mutagenicity,posing a huge risk to human and animal health.Animal ingestion of AFB₁-contaminated feed causes immunosuppression,reproductive disorders,organ damage and even large-scale death,causing great economic losses to the animal husbandry industry.In addition,AFB₁ is transferred to animal-derived foods such as meat,eggs and milk through feed,and eventually enters the human body through the food chain,causing acute or chronic poisoning and endangering human health.Bioinformatics is an interdisciplinary discipline that integrates computer science,biology and statistics,and obtains,processes,analyzes and interprets biological information through a combination of multi-field research methods.Bioinformatics analysis methods,including network toxicology,transcriptomics,proteomics and metabolomics are the cutting-edge technologies in the field of life sciences and an important tool and technique to elucidate the mechanism of toxicity.The authors focuse on the application of bioinformatics in the mechanism of AFB₁ toxicity,aiming to provide an effective approach and theoretical basis for the prevention and treatment of AFB₁ poisoning and the application of bioinformatics.

Whole cottonseed (WCS) is recognized as a high-protein,high-energy and physically effective fiber feed ingredient and is also processed into cottonseed cake or cottonseed meal (CSM) for use in production.The protein and energy of WCS increases the nutrient content of the diet and improves the digestibility of hay.Addition of moderate amounts of WCS can improve rumen fermentation,dietary digestibility,growth and developmental performance,and milk and meat quality in ruminants.In order to rationalize the use of WCS and develop new protein feeds in actual production,it is necessary to clarify that the appropriate additive amount of WCS is a practical problem that needs to be solved urgently in ruminant farming.At the same time,it is necessary to focus on how to maintain the normal function of the rumen,reduce the feeding cost of the farming industry,improve the feed utilization and economic benefits,and promote the healthy development of the animal husbandry industry.Therefore,the applications of WCS in ruminant production at home and abroad in recent years are summarized,and the effects of WCS on growth and development,slaughter performance,meat quality,lactation performance,milk quality,nutrient metabolism,and rumen fermentation of ruminants are described in relation to the additive amount of different forms of WCS,so as to provide a reference for the improvement of ruminant breeding efficiency.

【Objective】 Nuclear mitochondrial DNA segments (NUMTs) are formed by the insertion and integration of mitochondrial DNA into the nuclear genome.This study was aimed to map NUMTs in rabbit genome based on the latest reference genome sequence UM_NZW_1.0,analyze the distribution characteristics and genomic environment preferences,and predict the biological function of NUMTs,so as to decipher the NUMTs information in rabbit genome.【Method】 The nuclear DNA sequence of the latest rabbit reference genome UM_NZW_1.0 was aligned with the mitochondrial genome to map the NUMTs,and the length and chromosomal distribution of NUMTs were analyzed.NUMTs fragments were integrated into NUMTs regions to study insertion events.GC content and repeat sequence ratio of NUMTs in nuclear genome were investigated to understand their insertion preferences in genomic context.The distribution of homologous mitochondrial sequences of NUMTs in mitochondrial genome was explored.Gene annotation was performed to predict the potential biological function of NUMTs.【Result】 Totally of 237 NUMTs sequences were detected in rabbit genome,there were no NUMTs on chromosomes 3,5,6,8 and 10,with a total length of 311 549 bp,accounting for 0.015512% of the total reference genome size.These NUMTs might be formed by 87 NUMTs insertion events.NUMTs were more likely to form in regions with lower GC content and more repetitive sequences in rabbit genome,indicating that the formation of NUMTs in rabbit genome was not random.The entire mitochondrial genome sequence had formed NUMTs,with genes such as COX1,COX2, ND2,tRNA-C,tRNA-A,tRNA-W and tRNA-V covering more than 20 times.By annotating the genes covered by NUMTs and their upstream and downstream 1 000 bp regions,it was speculated that NUMTs might be related to function such as reproduction and tnervous system.【Conclusion】 Segments of the mitochondrial genome had been inserted into chromosome to form NUMTs throughout the evolution process of rabbits,NUMTs were not uniformly distributed across all chromosomes.NUMTs tended to be found in genomic regions characterized by lower GC content and a higher density of repetitive sequences.NUMTs might play roles in crucial biological processes,such as reproduction and nervous system,which offered valuable genetic insights for the molecular breeding of rabbits.

【Objective】 The genotype of ATP-binding cassette G subfamily member 2 (ABCG2) gene was identified and green-shell homozygous M11 Peking ducks (60-week-old) were screened.The effects of different genotypes of ABCG2 gene on eggshell color,biliverdin synthesis ability,egg quality and laying performance were explored.【Method】 60-week-old M11 Peking ducks were used as the experimental population.The genotype of ABCG2 gene was identified by bidirectional allele-specific PCR (Bi-PASA) technology,and were verified by Sanger sequencing.Starting from the first day of 60 weeks of age,duck eggs were collected continuously for 5 days to measure the egg quality,eggshell color grades,red value (R value),green value (G value),blue value (B value) and biliverdin content.Ten ducks were randomly selected from different genotype populations,and collected their livers and eggshell glands.The biliverdin content in the liver and eggshell glands,as well as the expression of genes related to biliverdin synthesis in the eggshell gland were measured.【Result】 Bi-PASA genotyping results were consistent with Sanger sequencing.The frequencies of AA,AG and GG genotypes in the population were 68.97%,26.49% and 4.55%,respectively,and there was a white shell rate of 4.55%.The egg shape index of duck eggs produced by AG genotype female ducks were significantly higher than those of AA genotype,their Haugh unit was significantly smaller than that of AA genotype,and the egg weight was significantly higher than that of AA and GG genotypes (P＜0.05).The eggshell color grades and biliverdin content of duck eggs produced by AA and AG genotype female ducks were significantly higher than that of GG genotype (P＜0.05),and the R and B values of eggshells were significantly lower than those of GG genotype (P＜0.05),but there was no significant difference between AG and AA genotypes (P＞0.05).The biliverdin contents of the liver and eggshell glands of AA and AG genotype female ducks were significantly or extremely significantly higher than those of GG genotype (P＜0.05 or P＜0.01)，the expression levels of 5'-aminolevulinic acid synthase 1 (ALAS1),fecal porphyrinogen oxidase (CPOX) and ABCG2 genes in eggshell gland were significantly or extremely significantly higher than those in GG genotype (P＜0.05 or P＜0.01) and the gene expression level of biliverdin reductase A (BLVRA) was extremely significantly lower than that of GG genotype (P＜0.01),but there was no significant difference between AG and AA genotypes (P＞0.05).【Conclusion】 Bi-PASA technology could accurately determine the ABCG2 genotype.The genotype of ABCG2 gene could significantly affect the color grade,R value,G value,B value and biliverdin content of duck egg shells.Duck eggs produced by AA and AG genotype female ducks were all green.The eggs produced by the GG genotype female ducks were all white.The AA and AG genotype female ducks had stronger biliverdin synthesis and transport abilities,but the eggs produced by the AA and AG genotype female ducks all had very light or extremely green eggs.The results provided theoretical basis and practical reference for breeding white-feathered and green-shelled duck strains.

【Objective】 This study was aimed to explore microRNA (miRNA) related to skeletal muscle growth and development during the embryonic period of Tibetan chickens,analyze their differential expression and construct the miRNA-mRNA regulatory network,so as to provide a theoretical basis for further understanding the regulatory mechanism of miRNA in the growth and development of chicken muscles.【Method】 RNA-Seq was performed on the leg muscle tissues of Tibetan chickens at the 10th (E10),14th (E14) and 18th (E18) days of embryonic stage.The differentially expressed miRNA among three periods was screened through bioinformatics analysis,and the differentially expressed miRNA was enriched by GO function and KEGG pathway,and the mRNA and miRNA related to muscle development were screened to construct the mRNA-miRNA regulatory network.Six differentially expressed miRNAs were randomly selected for Real-time quantitative PCR to verify RNA-Seq results.【Result】 The results showed that 52 miRNAs were differentially expressed between E10 and E14,137 miRNAs were differentially expressed between E10 and E18,33 miRNAs were differentially expressed between E14 and E18,and 8 miRNAs were differentially expressed in leg muscles of Tibetan chickens among three periods.The results of GO function enrichment analysis showed that the items related to skeletal muscle growth and development were actin binding,myofibrillar assembly,actomyosin structure and actin filament-based processes,muscle fiber membrane,etc.KEGG pathway enrichment analysis showed that a large number of genes were significantly enriched in the signaling pathways related to the biological functions of cell adhesion,survival,migration,proliferation,metabolism and differentiation,and immune regulation.The results of miRNA-3P-MYO1D/PDLIM3,miRNA-1662-DMD/SYNM,and miRNA-184-3p-FHOD1 might play an important role in the muscle growth and development of chicken embryos.miRNAs such as miRNA-133c-3p,miRNA-184-3p,miRNA-1662,miRNA-210a-3p and miRNA-1a-3p might be involved in the process of muscle growth and development.Real-time quantitative PCR results showed that the expression of six miRNAs were consistent with RNA-Seq results.【Conclusion】 A total of 222 differentially expressed miRNAs were detected,among which 8 miRNAs were differentially expressed at all three periods,providing theoretical basis for subsequent studies on the regulation of miRNA on muscle growth and development in chicken embryos.

【Objective】 The aim of this study was to compare the slaughter performance and meat quality of Nu-Gui hybrid F1 generation goats (Nubian goats♂×Guizhou Black goats ♀) with purebred Guizhou Black goats,and explore the improvement effect of crossbreeding Nubian and Guizhou Black goats on the production performance of Guizhou Black goats.【Method】 Thirty male lambs of Nu-Gui hybrid F1 generation and Guizhou Black goats born in the same period were selected,respectively,the birth weight was recorded,and the body weight and body size were measured at 3,6,9,and 12 months of age.Seven Nu-Gui hybrid F1 generation and Guizhou Black goats at 14 months of age were randomly selected for slaughter,respectively,the slaughter performance was determined.Meanwhile,the meat quality traits and the contents of amino acids and fatty acids in longissimus dorsi muscle were determined.【Result】 ①The body weight of Nu-Gui hybrid F1 generation goats at 3,6,9,and 12 months of age were significantly higher than those of Guizhou Black goats (P＜0.05)，and all body size traits at 12 months of age were significantly higher than those of Guizhou Black goats (P＜0.05).②The slaughter rate,pure meat percentage,carcass net meat rate and eye muscle area of Nu-Gui hybrid F1 generation goats were significantly higher than those of Guizhou Black goats (P＜0.05).③The muscle shear force and dripping loss of Nu-Gui hybrid F1 generation goats were significantly lower than those of Guizhou Black goats (P＜0.05),and the cooked meat percentage was significantly higher than that of Guizhou Black goats (P＜0.05).④17 kinds of amino acids and 37 kinds of fatty acids were detected in longissimus dorsi muscle of Nu-Gui hybrid F1 generation goats,the contents of lysine,glutamic acid,aspartate,oleic acid,linoleic acid,gamma linolenic acid, and arachidonic acid were significantly higher than those of Guizhou Black goats (P＜0.05),while the contents of arginine,palmitic acid, and stearic acid were significantly lower than those of Guizhou Black goats (P＜0.05).There was no significant difference in the content of saturated fatty acids between two breeds (P＞0.05),but the contents of monounsaturated fatty acids and polyunsaturated fatty acids of Nu-Gui hybrid F1 generation goats were significantly higher than those of Guizhou Black goats (P＜0.05).【Conclusion】 The crossbreeding between Nubian and Guizhou Black goats could significantly improve the growth rate and meat production performance of F1 generation,and improve the muscle quality.

【Objective】 The purpose of this study was to clone and sequence the neuromedin B (NMB) gene from Mulard duck,and to detect the expression of NMB gene in Mulard duck.【Method】 According to the predicted NMB gene sequence of duck in the NCBI database (accession No.：XM_027466185.2),primers were designed to amplify NMB gene fragment in Mulard duck by PCR amplification,and the NMB gene sequence was obtained by cloning and sequencing.The nucleotide and amino acid sequences of NMB in Mulard duck were analyzed using bioinformation analysis software,and the phylogenetic tree was constructed.Real-time quantitative PCR was used to detect the expression of NMB gene mRNA in multiple tissues of Mulard duck.【Result】 In this study,NMB gene of Mulard duck was cloned with 387 bp CDS sequence and encoding 121 amino acids,including 14 amino acids (GNLWATGHFMGKKS) of the conserved domain of bombesin,which was identical to that in NMB of Gallus gallus，Mus musculus，Sus crofa，Bos taurus and Homo sapiens.The similarity analysis showed that the similarity of nucleotide and amino acid sequences of NMB between Mulard duck and Gallus gallus were 87.9% and 89.8%,respectively,and the similarity with other animals were lower.In addition,NMB of Mulard duck was evolutionarily close to Gallus gallus,and far from domestic animals such as Bos taurus, Equus caballus,Sus crofa and Canis lupus familiaris.The results of bioinformatics analysis showed that NMB protein of Mulard duck was a hydrophilic protein,and its N-terminal 22 amino acids were signal peptides.In the secondary and tertiary structures,the proportion of alpha helix,extended chain,beta turn and random coil were 53.91%,14.06%,7.03% and 25.00%,respectively.The NMB protein of Mulard duck mainly binded to NMBR and GRPR proteins.Real-time quantitative PCR results showed that NMB gene mRNA was expressed in the central and peripheral tissues and organs of Mulard duck.Compared with the jejunum,the expression levels of NMB gene were relatively high in the central cerebellum,brain and optic lobe (P＜0.01),and relatively high in the peripheral trachea,lung,pineal gland,thyroid,kidney and uropygial gland (P＜0.01).In addition,the expression trend of NMB gene mRNA in Mulard duck of different genders was basically the same,but there were different expression in female and male ducks in individual tissues.The expression of NMB gene mRNA in female duck was relatively higher than that in male duck in cerebellum and pancreas (P＜0.01),but the opposite was true in pineal gland and kidney (P＜0.01 or P＜0.05).【Conclusion】 NMB protein in Mulard duck had the conserved domain of bombesin.The genetic relationship was the closest between Mulard duck and Gallus gallus,and the protein similarity was high.NMB protein was hydrophilic protein and had signal peptide.NMB gene was highly expressed in the central (cerebellum,brain and optic lobe) and peripheral (pineal gland,kidney,uropygial gland,thyroid gland and trachea,etc.) tissues and organs of Mulard duck.The results could provide reference for further study on the function of NMB gene in Mulard duck.

【Objective】 The aim of this study was to identify candidate gene and molecular marker that affected the comb trait of Nandan-Yao chickens,thus to provide technical support for the selection and breeding of comb traits in Nandan-Yao chickens.【Method】 In this study,phenotypes of comb traits were collected from 464 Nandan-Yao chickens at 7,10,15,18,22 and 26 weeks of age.Meanwhile,DNA were extracted from blood samples and genotyping was conducted using "GXChickChip-I" chip.Genome-wide association study (GWAS) were performed on the comb height,comb length,comb thickness and comb area of Nandan-Yao chickens at different ages by the univariate mixed linear model in GEMMA software.The gene annotated from the associated single nucleotide polymorphism (SNP) were subjected to GO function and KEGG pathway enrichment analysis.【Result】 A total of 7,8,21 and 10 SNPs were detected to be associated with comb area,comb height,comb length and comb thickness in Nandan-Yao chickens,respectively.These SNPs were annotated to 37 genes.Through bioinformatics analysis and gene functional annotation,7 genes of CELF2,MAP7,MAP3K5,AKT3,IFNGR1,IL20RA and MANBA were identified as candidate genes related to comb traits in Nandan-Yao chickens.【Conclusion】 This study revealed 39 potential SNPs associated with comb traits in Nandan-Yao chickens,and 7 genes that might influence comb size were identified.The results could provide effective molecular makers for the selection of comb traits of Nandan-Yao chickens and an important theoretical foundation for the breeding of excellent indigenous chickens.

【Objective】 The aim of this study was to identify the single nucleotide polymorphism (SNPs) loci of protein phosphatase 3 catalytic subunit α (PPP3CA) of Zhijin White geese,and explore the effect of PPP3CA gene polymorphism on body size traits.【Method】 The PPP3CA gene SNPs of 144 Zhijin White geese were identified by PCR amplification and Sanger direct sequencing.The gene frequency,genotype frequency,number of effective allele (Ne) and polymorphism information content (PIC) were calculated using Excel 2010 software,and the Hardy-Weinberg equilibrium state was analyzed.The association between SNPs and body size traits of 144 Zhijin White geese were analyzed by One-Way analysis of variance in the general linear model (GLM) of SPSS 25.0 software.【Result】 9 SNPs were found in intron 2 and 4 of PPP3CA gene in Zhijin White geese,namely g.58398 A＞G,g.58504 A＞G,g.58541 A＞G,g.68997 A＞G,g.69001 C＞T,g.69034 C＞T,g.69052 T＞C,g.69070 T＞C,and g.69114 C＞T,respectively.Except for g.69034 C＞T,which was a low polymorphic site (PIC＜0.25),the other SNPs were moderate polymorphic loci (0.25＜PIC＜0.5).The Chi-square test results showed that the SNPs g.58398A ＞G,g.69001 C＞T,g.69070 T＞C and g.69114 C＞T were consistent with Hardy-Weinberg equilibrium (P＞0.05).However,the SNPs g.58504 A＞G,g.58541 A＞G,g.68997 A＞G,g.69034 C＞T and g.69052 T＞C deviated from Hardy-Weinberg equilibrium (P＜0.05).The results of association analysis showed that in the g.58504 A＞G and g.58541 A＞G,the pelvic width of GG genotype individuals were extremely significant or significantly higher than that of AA genotype (P＜0.01 or P＜0.05).【Conclusion】 There were 9 SNPs in PPP3CA gene of Zhijin White geese,among which g.58504 A＞G and g.58541 A＞G were correlated with pelvic width to different degrees,indicating that these SNPs could be used as reference loci for molecular marker-assisted breeding of Zhijin White geese,laying a theoretical foundation for further study of PPP3CA gene in geese.

【Objective】 The purpose of this study was to obtain the HA1 protein,a structural domain of the haemagglutinin (HA) head of Swine influenza virus (SIV) subtype H3N2 with good immunogenicity,and prepare specific polyclonal antibodies for the identification and functional study of the HA1 protein.【Method】 In this study,the HA1 fragment of SIV type A (A/swine/Guangdong/04/2005(H3N2)) strain was amplified,the prokaryotic expression vector pET-28a(＋)-H3N2-HA1 was constructed by homologous recombination,and the recombinant positive plasmid were transformed into Escherichia coli BL21(DE3) competent cells after identification by PCR and sequencing.The conditions of HA1 protein expression (including induction temperature,induction time and IPTG concentration) were optimised,and protein purification was carried out by nickel column affinity chromatography.The recombinant protein obtained was mixed with QuickAntibody-Mouse3W adjuvant and then immunised with BALB/c mice by intramuscular injection to prepare mouse polyclonal antibody against HA1 protein of H3N2 SIV,and the titer,reactivity and specificity of the polyclonal antibody were determined by ELISA,Western blotting and immunofluorescence assay (IFA).【Result】 In this study,the prokaryotic expression vector pET-28a(＋)-H3N2-HA1 was successfully constructed.The highest expression of HA1 recombinant protein of H3N2 SIV was induced by 1 mmol/L IPTG at 26 ℃ for 10 h,and it was mainly expressed in the form of inclusion body,with the protein molecular mass size of about 39.5 ku.Five polyclonal antibodies against HA1 protein of H3N2 SIV were successfully prepared,with a titer of 1∶1 638 400.Western blotting and IFA identification results showed that the prepared polyclonal antibodies could specifically recognize HA1 protein of H3N2 SIV eukaryotic expressed,and did not react with the HA1 protein of other subtypes of influenza viruses,such as H1N1 SIV and H7N9 Avian influenza virus (AIV),with a good reactivity and specificity.【Conclusion】 In this study, HA1 protein of H3N2 SIV with good immunogenicity and was successfully expressed and purified,and mouse derived polyclonal antibodies with good reactivity and specificity were prepared,which provided an important tool for further study of the biological function of HA1 protein of H3N2 SIV.

【Objective】 This study was aimed to understand the genomic variation of Cluster 3 goose Tembusu virus (TMUV) and its pathogenicity to geese.【Method】 BHK-21 cells were used to isolate the liver tissue samples of geese infected with TMUV,and identified by RT-PCR,indirect immunofluorescence assay (IFA),transmission electron microscope observation,and the growth curve of isolated viruses were detected.After the whole genome amplification of TMUV isolated strains,the genetic evolution was analyzed by ModelFinder,MrBayes and other softwares.The amino acid mutation sites of E protein of TMUV isolated strains were analyzed.After detecting the viral titers of TMUV isolates,the isolates were used to challenge 30-day-old geese,respectively.Then the clinical dissection of each tissue and organ,and the histopathological changes were observed.The viral load in each tissue and organ was detected by Real-time PCR.【Result】 Two TMUV nucleic acid positive samples were successfully identified by RT-PCR.After inoculation into BHK-21 cells,significant lesions were observed within 60 h.Significant red fluorescence could be observed by IFA detection of the third-generation virus solution,and virus particles with a diameter of about 50 nm and a capsule could be observed by transmission electron microscopy.Two strains of TMUV were successfully isolated from diseased goose liver tissue and named JM3 and JM1205,respectively.The one-step growth curve of the virus showed that JM3 and JM1205 strains had the highest virus titers after 60 and 48 h of cultivation,respectively.The whole genome amplification results showed that the full-length genomes of JM3 and JM1205 strains were both 10 994 bp.The genetic evolutionary tree showed that both JM3 and JM1205 strains were members of Cluster 3 TMUV,and had the closest genetic distance to the CTLN isolate of Cluster 3 TMUV chicken source.The analysis of amino acid mutation sites showed that compared with the earliest uploaded TMUV strain MM1775 in GenBank,JM3 and JM1205 strains had multiple amino acid site mutations in the E protein,among which the V157A mutation might be related to the increased virulence of TMUV.After 1 d of infection,geese began to show symptoms of green and sparse feces,and after 7 d of infection,neurological symptoms began to appear.JM3 group continued to detoxify 14 d after challenge,while the JM1205 group continued to detoxify until 11 d after challenge.After 6 d of infection,the geese showed a slowdown and decrease in weight growth,and began to recover slowly and increase by 10 d.Autopsy revealed varying degrees of splenomegaly,pancreatic necrosis,liver whitening,and cerebral congestion in the infected geese.In addition,JM3 strain infected geese showed ovarian bleeding and pericardial effusion.Geese infected with JM1205 strain showed cardiac bleeding.The viral load in the spleen was highest at all time points after infection,reaching its peak at 3 d after infection and gradually decreasing thereafter.【Conclusion】 This study isolated two strains of Cluster 3 TMUV-JM3 and JM1205 from goose farms in Guangdong province.Both isolates were pathogenic to geese and could replicate in multiple organs,causing symptoms such as ataxia and weight loss.

Mycoplasmas,prevalent animal pathogens,inflict substantial economic damage on the livestock and poultry sectors.Clinical strategies to combat Mycoplasma diseases encompass prophylactic vaccination and antibiotic therapy.Yet,conventional vaccines-both inactivated and attenuated-pose challenges such as diminished immunogenicity and virulence reversion,while prolonged monotherapy with antibiotics fosters antibiotic resistance.Bacteriophages offer a promising alternative,circumventing these issues and emerging as an effective biological control in veterinary and aquaculture settings.Mycoplasma bacteriophages were discovered and studied as early as the 1980s,and it is expected to achieve the goal of preventing and treating Mycoplasma diseases through the application of bacteriophages.However,the complex cultivation of Mycoplasmas has limited extensive bacteriophage research.Further isolation and in-depth study of Mycoplasma bacteriophages are imperative for advancing practical applications.The author conducted a systematic phylogenetic analysis of the whole genome of Mycoplasma in NCBI database,listed common pathogenic Mycoplasmas in livestock and poultry and the symptoms they caused,elucidated the pathogenesis and conventional treatment methods of Mycoplasma,summarized the research progress of bacteriophages and the practicality of Mycoplasma bacteriophages,and provided important references for the prevention and treatment of Mycoplasma diseases in animal husbandry field.

【Objective】 In this study,the sod gene deletion strain was constructed to elucidate the effect of sod gene on the anti-oxidative stress ability of Listeria monocytogenes 10403S.【Method】 The sod gene deletion strain was constructed by homologous recombination method.The growth ability of parent strain 10403S,deletion strain 10403SΔsod and complemented strain 10403S CΔsod were analyzed by growth test.The effect of sod gene on oxidative stress of Listeria monocytogenes was determined by oxidative stress test.The effect of oxidative stress on SOD protein expression was determined by Western blotting.【Result】 The results of PCR showed that the target fragments of 10403SΔsod and 10403SCΔsod were 1 468 and 1 875 bp,respectively,deletion strain 10403SΔsod and complemented strain 10403S CΔsod were successfully constructed.The results of growth test showed that sod gene deletion did not affect the growth ability of 10403S.The results of anti-oxidative stress test showed that sod gene deletion reduced the anti-oxidative stress ability of 10403S under the condition of 20 mmol/L H₂O₂(P＜0.01).The results of Western blotting showed that the expression level of SOD protein of Listeria monocytogenes was significantly up-regulated under oxidative stress (P＜0.01).【Conclusion】 The deletion of sod gene did not affect the normal growth of Listeria monocytogenes 10403S,but it could lead to a decrease in oxidative stress ability,and the expression level of SOD protein in 10403S was significantly increased under the condition of 20 mmol/L H₂O₂.

【Objective】 The purpose of this study was to clone VP7 gene of genetype G8 Bovine rotavirus (BRV) isolated in Xinjiang,and analyze the molecular genetic characteristics of the VP7 protein,so as to provide a theoretical basis for studying its biological functions and new vaccines.【Method】 The viral genome RNA was extracted from the 6th passages of BRV isolate in Xinjiang,and VP7 gene fragment of BRV was amplified by RT-PCR,cloned into pMD19-T vector,and then transformed into Escherichia coli DH5α competent cells.The plasmid was extracted,digested and sequenced.VP7 protein was analyzed using bioinformatics software,including physicochemical properties,transmembrane structure,hydrophilicity/hydrophobicity analysis,subcellular localization,signal peptides,glycosylation sites,phosphorylation sites,secondary structure,tertiary structure homology modeling,and prediction of B and T cell antigen epitopes.【Result】 VP7 gene nucleotide sequence was successfully obtained by RT-PCR amplification, with a total length of 1 062 bp, and then submitted to NCBI to obtain GenBank accession No.: OR136878.1. This sequence had the highest identity (89.4%) with the nucleotide sequence of RVA/Cow-wt/TUR/Amasya-1/2015/G8P[5] strain (GenBank accession No.:KX212865.1),the two strains belonged to genotype G8 of serotype A.Bioinformatics analysis suggested that the BRV VP7 protein was a hydrophobic unstable protein with two transmembrane regions and without signal peptide.VP7 protein was located in endoplasmic reticulum membrane of the host cell.It contained 2 N-glycosylation sites and 132 phosphorylation sites.The secondary structure of VP7 protein was mainly consisted of alpha helix and random coil,which accounted for 36.20% and 35.28%,respectively.VP7 protein could be homologously modeled with the template (SMTL ID:8bp8.1) in the SWISS-MODEL database,with a sequence identity of 82.82%,which indicated a high coincidence rate between them.The GMQE,QMEAN and Ramachandran favored values of the model were 0.75,0.79 and 95.52%,respectively,indicating that the spatial conformation was reasonable and the model was accurate and reliable.The VP7 protein possessed several B and T cell antigenic epitopes.While the epitope length was 16 amino acids and the score exceeds 0.80,there were 12 B cell epitopes.While the epitope length was 9 amino acids,with 4 alleles selected and a score greater than 0.5,there were 6 cytotoxic T lymphocyte (CTL) epitopes.While the epitope length ranged from 12 to 18 amino acids,with 4 alleles selected and a score greater than 0.8,there were 11 helper T lymphocyte (HTL) epitopes.【Conclusion】 The complete nucleotide sequence of VP7 gene of genetype G8 BRV isolated in Xinjiang was successfully obtained for the first time.VP7 protein was an unstable protein with two transmembrane regions and without signal peptide. It was located in endoplasmic reticulum membrane of the host cell,and possessed several B and T cell antigenic epitopes.The experimental results laid the foundation for the epidemic,diagnosis,virus-host interaction,and the research of genetically engineering vaccine targeting VP7 protein of BRV.

【Objective】 Neosporidiosis is a protozoan disease that causes clinical symptoms such as abortion,stillbirth or movement disorder in pregnant animals.The aim of this experiment was to determine the effect of dihydroartemisinin on sperm quality and spermatogenic function genes of male mice infected with Neosporidium neosporidium, in order to acheive anti-Neosporidium neosporidium effect.【Method】 BALB/c male mice model infected with Neosporidium neosporidium were taken as the study object,and dihydroartemisinin was administered intragastically at a dose of 0.2 g/kg for 7 days.Meanwhile,blank control and model groups were set up.After administration,the reproductive organ index and sperm quality of male mice were measured,and the morphological changes of sperm were observed by modified Pap staining.The expression of spermatogenic function genes C-kit,Plzf,Sycp3 and Stra8 were detected at 7th,14th,21st,35th and 42nd days after administration.On the 7th day of administration,the apoptosis of spermatogenic cells was detected by flow cytometry.【Result】 The male mice model of Neosporidium neosporidium infection were established successfully.Compared with blank control group,the reproductive organ index,sperm density and sperm motility of mice in model group were extremely significantly decreased (P＜0.01),while the sperm malformation rate was extremely significantly increased (P＜0.01).The expressions of C-kit,Plzf,Sycp3 and Stra8 genes were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The apoptosis rate of spermatogenic cells was extremely significantly increased (P＜0.01).In dihydroartemisinin group,the reproductive organ index (excluding the 21st day),sperm density and sperm motility were significantly or extremely significantly decreased (P＜0.05 or P＜0.01),while the sperm malformation rate was significantly increased (P＜0.01).The expression of C-kit,Plzf,Sycp3 and Stra8 genes were decreased,reaching significant levels at some time points (P＜0.05).The apoptosis rate of spermatogenic cells was extremely significantly increased (P＜0.01).Compared with model group,the reproductive organ index of mice in dihydroartemisinin group was significantly or extremely significantly increased (P＜0.05 or P＜0.01),the sperm density and sperm motility were increased,the sperm malformation rate was significantly decreased (P＜0.01).The expression of C-kit,Plzf,Sycp3 and Stra8 genes were increased,reaching significant or extremely significant levels at some time points (P＜0.05 or P＜0.01),and the apoptosis rate of spermatogenic cells was extremely significantly decreased (P＜0.01).【Conclusion】 Dihydroartemisinin could regulate the reproductive organ index of male mice,improve sperm quality,reduce the production of malformed sperm,increase the expression of spermatogenic function genes Sycp3,C-kit,Plzf and Stra8,and improve the damage of Neosporidium neosporidium on spermatogenic sperm quality and spermatogenic function genes of male mice.The results provided a reference for the research of effective anti-Neosporidium neosporidium drugs.

【Objective】 The purpose of this study was to explore the species of Paramphistomum in Ordos fine-wool sheep and screen out ideal anthelmintic drugs.【Method】 Based on the preliminary diagnosis of paramphistomiasis by fecal egg examination,the Paramphistomum in rumen of Ordos fine-wool sheep were collected through necropsy for morphological and molecular biological identification.Three parasites were randomly selected to extract DNA,and then the internal transcribed spacer 2 (ITS-2) of ribosome was amplified by PCR.After sequencing the amplified products,BLAST alignment analysis was performed in NCBI database,and the phylogenetic tree was constructed.Niclosamide,nitroclofene,rafoxanide and 3% trichlorfon combined with nitroclofene were respectively used to conduct a comparative test on the anthelmintic effects in sheep infected with paramphistomiasis.【Result】 The band size of 441 bp was obtained by PCR amplification of ITS-2 gene,which was consistent with the target gene band.The sequence similarity between the three parasites and the ITS-2 (KJ459936.1) from China was 99.3%,99.3% and 99.8%,respectively.The results of phylogenetic analysis showed that this Paramphistomum was in the same large branch as Paramphistomum from China (KP341671),Ireland (AB973398),Argentina (HM209066) and Germany (MZ532797),and was in a small branch with Paramphistomum cervi,indicating that the Paramphistomum obtained in this study was Paramphistomum cervi.In the anthelmintic test,the egg reduction rates in niclosamide,nitroclofene,rafoxanide and 3% trichlorfon combined with nitroclofene groups were 44.00%,26.73%,23.00% and 90.80%,respectively.【Conclusion】 The Paramphistomum parasitizing in Ordos fine-wool sheep in Wushen Banner,Inner Mongolia was Paramphistomum cervi,and the treatment with a combination of 3% trichlorfon and rafoxanide could achieve a better anthelmintic effect.

【Objective】 Classical swine fever virus (CSFV) and porcine Pseudorabies virus (PRV) are two important pathogens affecting the development of current swine industry.Currently,there is no effective drug treatment,and the prevention mainly depends on vaccination.The aim of this study was to construct a recombinant PRV strain containing part of the epitope region of CSFV E2 gene and explore its biological characteristics,so as to provide reference for the development of dual vaccine against CSFV and PRV simultaneously.【Method】 Firstly,a gene segment containing the 4 epitope regions of B/C/D/A of CSFV E2 protein was inserted into the BamHⅠ site of the pG-EGFP recombinant plasmid,and the recombinant plasmid pG-E2AD-EGFP was constructed.The plasmid was then transfected into ST cells inoculated with PRV tri-gene deletion strain rPRV NY-gE^―/gI^―/TK^― and homologous recombination occurred in ST cells.The recombinant virus rPRV-E2AD-EGFP with green fluorescent protein was rescued by the plaque purification.The EGFP gene was knocked out using CRISPR/Cas9 knockout vector,and the non-fluorescent recombinant virus rPRV-E2AD was purified by plaque.The expression of A-D epitopes of E2 of recombinant virus rPRV-E2AD was detected by PCR amplification and Western blotting.The proliferation of the virus after treatment with different physicochemical properties was detected by 50% tissue culture infective dose (TCID₅₀) method,and the culture and physicochemical properties of the recombinant virus were evaluated.【Result】 Recombinant virus rPRV-E2AD-EGFP expressing both CSFV E2AD antigen and EGFP was obtained through intracellular homologous recombination and viral plaque purification.EGFP gene was knocked out by CRISPR/Cas9 technique,and recombinant virus rPRV-E2AD without EGFP gene expression was obtained.Western blotting results showed that the protein size of this strain was about 27 ku,which was consistent with the CSFV E2AD antigen size.The growth characteristics of rPRV-E2AD and parent strains were basically the same in ST,IPEC-J2,PK-15 and Vero cells,both of which caused cell fusion at 12 h after infection and cell loss at 36 h after infection.Physical and chemical characteristics test results showed that the recombinant strain and the parent strain had similar culture characteristics under the same physical and chemical conditions.【Conclusion】 In this study,recombinant virus rPRV-E2AD was successfully obtained,and the foreign gene did not affect the proliferation characteristics of the parent strain.The results provided the candidate strains for the development of recombinant CSFV E2 gene live vector vaccine,and also provided the research basis for the development of PRV live vector vaccine.

【Objective】 This study was aimed to investigate the species of parasitic argasid ticks on the surface of sheep and the infection status of Anaplasma ovis in certain areas of Southern Xinjiang,China,and provide a scientific basis for the prevention and control of Anaplasma ovis disease in the region.【Method】 A total of 134 samples of hungry argasid ticks from sheep were collected from Yutian,Yingjisha,Jiashi,Qira,Wushi and Awat in Xinjiang from 2022 to 2023.These samples were identified through morphological and molecular biology methods.PCR technique was employed to amplify the 16S rDNA gene of argasid ticks and the major surface protein 4 (MSP4) gene of Anaplasma ovis.Furthermore,sequences with high similarity were retrieved from GenBank for local multiple sequence alignments,and phylogenetic tree was constructed using Mega X.【Result】 After morphological identification,the dorsum of the argasid ticks lacked a shield and featured a scattered distribution of stellate fossae,which were initially identified as Ornithodoros. Through molecular biological identification,its 16S rDNA gene sequence was found to be closest to Ornithodoros lahorensis in Xinjiang strain (accession No.：ON159483) in GenBank,with 100% similarity.The detection of Anaplasma ovis in argasid ticks by PCR had a positive rate of 14.92% (20/134).The positive sequence was compared with the MSP4 gene sequence of Xinjiang (accession No.：OP503167) and Sudan (accession No.：KU497709) strains,and 6 base differences and 6 genotypes of Anaplasma ovis were identified,named as LA1,LA2,LA3,LY1,LY2 and LT1，respectively,the accession No.were as follows:PP997635-PP997640.Genetic evolutionary analysis revealed that the MSP4 gene sequence of Anaplasma ovis was closest to Sudan (accession No.：MF740812) and Pakistan (accession No.：MT311203) strains,with similarities ranging from 98.77% to 99.69%.【Conclusion】 The argasid ticks in Southern Xinjiang were all identified as Ornithodoros lahorensis,and the positive rate of Anaplasma ovis from sheep in these samples was 14.92%.The genotype of Anaplasma ovis from sheep in Southern Xinjiang were predominantly type LT1.The results had enriched the Anaplasma ovis database of Xinjiang，and provided a theoretical reference for the epidemiological study of pathogens carried by argasid ticks,as well as for the prevention and control of tick-borne diseases in the region.

In recent years,respiratory Coronaviruses have become a focus of research.They mainly infect ciliated cells and goblet cells of the host trachea,resulting in loss of tight connections between cells,damage the integrity of the respiratory system barrier,and then cause low respiratory tract infection.The trachea epithelium cells (TECs),as an important barrier against pathogen invasion in the body,are also the target cells of many pathogens,and are an important model for the study of respiratory Coronaviruses.During the host-virus interactions,respiratory Coronaviruses preferentially attack ciliary cells,resulting in ciliary clearance dysfunction and destruction of the defensive barrier of the tracheal epithelium,thus exacerbating secondary infection.The authors summarized the morphological structure and physiological function of tracheal epithelial cells,and focused on the methods of isolation and culture of tracheal epithelial cells in vitro,as well as their advantages and disadvantages.The mechanism of interaction between respiratory pathogens and their hosts was further discussed，in order to provide a reference for the isolation and culture of tracheal epithelial cells,and provide support for the study of the infection mechanism and prevention strategy of respiratory Coronavirus.

【Objective】 The enhancement function and regulatory mechanism of caffeic acid (CA) on immunosuppression of macrophages were studied,in order to provide theoretical support for the development of a new immunoenhancer for immunosuppressive diseases (ISD).【Method】 Transmission electron microscopy and nanoparticle tracking analysis (NTA) were used to determine the morphology and concentration of macrophage-derived exosomes (Exos).Phosphoramide mustard (PM) was used to establish the immunosuppression model,RAW264.7 cells were treated with CA of 6.25,12.5,25,50, 100 and 200 μmol/L for 24 h, respectively,cytotoxicity test and neutral red were used to investigate cell viability and phagocytosis,Exos concentration was determined by NTA,mRNA expression in Exos was tested by transcriptomics,GO function and KEGG pathway were used to analyze the enrichment of mRNA differentially expressed genes,respectively,in order to explore the mechanism of CA enhancing immune function.【Result】 Exos was a complete cup shape with a bilayer membrane structure and a particle size between 30 and 200 nm. Compared with PM model group,CA could prominently enhance the activity and phagocytosis of macrophages (P＜0.01),encourage the secretion of Exos (P＜0.01),and upregulate 1 961 mRNA differentially expressed genes and downregulate 3 274 mRNA differentially expressed genes in Exos.GO function enrichment analysis showed that CA mainly affected cell process,biological regulation and molecular binding,and its function was to positively regulate the chemotaxis of dendritic cells.KEGG pathway enrichment analysis demonstrated that CA played an important role in the immune system,endocrine system,infectious diseases and signal transduction,mainly regulating T cell receptor and B cell receptor related signal pathways,indicating that CA could exert an important influence on immune cells through mediating Exos mRAN expression.【Conclusion】 CA enhanced the viability and phagocytosis of macrophages by promoting the generation of macrophage derived Exos and regulating mRNA gene expression.

【Objective】 The purpose of this experiment was to study the bacteriostatic effect of chicken derived Lactobacillus salivarius on Escherichia coli (CVCC1450) standard strain,and provide probiotic strains for the prevention and treatment of chicken colibacillosis in the future.【Method】 In this study,selective medium combined with molecular biology method was used to isolate probiotic strains from cloaca of healthy White laying hens.Morphological observation and 16S rDNA sequencing were used to identify the isolates,and the antibacterial activity and antibacterial substances of the isolates were analyzed.The growth curve and acid production curve of the isolates were established to explore their tolerance in the digestive tract,and their safety was evaluated by drug sensitivity test and hemolysis test.【Result】 Two strains of probiotics were isolated,all of which were Lactobacillus salivarius,and had good inhibitory effect on Escherichia coli with a diameter of more than 20 mm.After pH of the supernatant was adjusted to 7.0,the antibacterial activity of the isolates decreased significantly,indicating that the main antibacterial substance of the two strains of Lactobacillus salivarius was organic acid.Two strains of Lactobacillus salivarius had good growth characteristics and acid production ability.After 10 h of cultivation,they reached their plateau phase,and after 22 h of cultivation,the pH decreased to 3.75.The tolerance to bile salt solution and artificial gastrointestinal fluid of the two strains was strong,with a survival rate of over 27% in 0.3% bile salt solution and over 55% in artificial gastrointestinal fluid.Two strains of Lactobacillus salivarius were safe and sensitive to most antibiotics such as streptomycin,ampicillin,cefotaxime,ofloxacin,penicillin,cefazolin and doxycycline,and resistant to three antibiotics gentamicin,kanamycin and furazolidone,without hemolytic activity.【Conclusion】 In this study,two strains of Lactobacillus salivarus were isolated,which had good antibacterial activity against Escherichia coli,and laid a foundation for providing safe,efficient and non-residual green microecological preparations for the prevention and treatment of chicken and other poultry diarrhea caused by Escherichia coli.

【Objective】 The aim of this study was to prepare a kind of eprinomectin mixed micelle (EPR-MMs) which could be used for transdermal adminstration.【Method】 EPR-MMs were prepared by membrane hydration method using PEG-40 hydrogenated castor oi and nonyl phenol polyoxyethylene ether 40 as carriers.The particle size,polydispersity index (PDI),encapsulation efficiency,loading efficiency,and stability of EPR-MMs were evaluated through single-factor examination.Parameters such as the surfactant ratio,drug surfactant ratio,hydration rotation speed,and hydration time were screened and optimized to establish the best prescription and process parameters.The critical micelle concentration (CMC) was determined using the pyrene fluorescence probe method.The microstructure was characterized by transmission electron microscopy and Fourier transform infrared spectroscopy (FT-IR).The in vitro transdermal properties of EPR-MMs were investigated by using Franz diffusion cell,with a commercial pour-on agent as the control.【Result】 The optimum process prescription of EPR-MMs was as follows:Surfactant ratio was 20∶1,drug and surfactant ratio was 1∶9,hydration speed was 1 200 r/min and hydration time was 4 h.The CMC of EPR-MMs prepared under the optimal screening conditions was 11.596 μg/mL.EPR-MMs was spheroid under transmission electron microscope,with suitable particle size and good morphology.FT-IR analysis confirmed that the drug was encapsulated in the micelle.Stability studies showed that the prepared EPR-MMs had good stability when placed at room temperature for 7 d.In vitro permeation results demonstrated that the cumulative permeation amount per unit area of EPR-MMs was 44.26 μg/cm²,which was about 3.82 times that of the pour-on,and the penetration rate was 3.90 times that of the pour-on. 【Conclusion】 The stable EPR-MMs with suitable particle size was successfully prepared,and its physicochemical properties were verified by multiple characterization methods,showing good in vitro release characteristics,and higher transdermal rate and permeability than the commercial pour-on.

【Objective】 The purpose of this study was to investigate the epidemiological and antibiotic resistance characteristics of Clostridium perfringens isolates from beef cattle in some areas of Inner Mongolia,and provide scientific basis for the prevention and treatment of Clostridium perfringens disease in beef cattle in these areas.【Method】 The tissue samples of 37 dead beef cattle were collected,and the bacteria were isolated and identified by isolation culture,staining microscopy and biochemical test.The toxin types and drug resistance genes of the isolated bacteria were detected by PCR,and the drug susceptibility test was conducted by K-B disk method.【Result】 The isolated bacteria appeared as black circular colonies on TSC medium and produced cloudy gas on TSA medium.Microscopic examination revealed short and thick Gram positive rods.The isolated bacteria fermented sucrose,glucose,maltose and hydrogen sulfide,causing gelatin to liquefy and undergo "burst" fermentation in an iron containing milk culture medium.Eight strains of Clostridium perfringens were isolated from the samples of 37 dead beef cattle,with an isolation rate of 21.62%.The PCR typing results of toxin genes showed that all the isolated strains only had cpa toxin and were type A Clostridium perfringens.The PCR detection results of resistance genes showed that multiple resistance genes were detected,with bla_TEM,tetM,ermB and sul2 genes having the highest detection rate of 100%.The detection rates of resistance genes aph(3')-Ⅲ-F,tetB(P) and ermC were 87.5%,62.5% and 62.5%,respectively,while the remaining resistance genes were not detected.The results of the drug sensitivity test showed that 8 strains of Clostridium perfringens were resistant to aminoglycoside drugs,with resistance rates of 100% for amikacin and streptomycin,and 87.5% for gentamicin and kanamycin.The resistance rates to compound sulfamethoxazole tetracycline and erythromycin were 100%,75.0% and 50.0%,respectively.And they were sensitive to ampicillin,cefazolin,chloramphenicol and clindamycin.【Conclusion】 There was a prevalence of Clostridium perfringens in cattle herds in some areas of Inner Mongolia,mainly type A,which had developed severe tolerance to antibiotics and multiple drug resistance.The experimental results could provide scientific basis for the epidemiological research,rational clinical drug use,and scientific prevention and control of Clostridium perfringens.

【Objective】 The objective of this experiment was to investigate the effect of cell-free culture supernatant (CFCS) of Lactobacillus salivarius (L.salivarius) on the hemolytic activity of Staphylococcus aureus (S.aureus)and to compare the effect of different culture conditions on the bacteriostatic effect of CFCS of L.salivarius.【Method】 The minimum inhibitory concentration (MIC) and subinhibitory concentration (SIC) of CFCS of L.salivarius on S.aureus were determined by broth dilution method.The influence of CFCS of L.salivarius on the hemolytic activity of S.aureus was detected by semi-quantitative method.The relative expression levels of α hemolysin (hla) and AraC/XylS family transcriptional regulators rbf gene in S.aureus were determined by Real-time quantitative PCR.The effects of CFCS of L.salivarius on mortality,organ bacteria load and histopathological changes of mice were detected by pathogenicity test.The effects of different medium and culture conditions on the antibacterial activity of CFCS of L.salivarius were determined by Oxford Cup double-layer agar diffusion method,respectively.【Result】 The MIC and SIC of CFCS of L.salivarius against S.aureus were 31.25 and 3.906 mg/mL,respectively.After treatment with CFCS of L.salivarius with MIC,compared with positive control group,the hemolytic activity of S.aureus was extremely significantly decreased (P＜0.01),the relative expression level of hla gene was significantly decreased (P ＜ 0.05),and the relative expression level of rbf gene was extremely significantly increased (P＜0.01).The results of pathogenicity test in mice showed that after treatment with MIC of CFCS of L.salivarius,the bacteria load in liver,kidney and spleen of mice infected with S.aureus was extremely significantly decreased (P＜0.01),and the lesions of liver,kidney and spleen tissues were significantly reduced.The fermentation of CFCS of L.salivarius in MRS medium had the best inhibitory effect on S.aureus,and the inhibitory zone diameter was 17.64 mm,followed by TPY medium (12.83 mm).Based on MRS medium,when the initial glucose content was 4%,the initial nitrogen source content was 1.1%,the initial pH was 4.0 and the fermentation time was 48 h,CFCS of L.salivarius had the best bacteriostatic effect on S.aureus.【Conclusion】 CFCS of L.salivarius could inhibit the hemolytic activity of S.aureus and reduce its pathogenicity.The change of culture conditions could significantly affect the antibacterial activity of CFCS of L.salivarius.

【Objective】 This study was aimed to isolate and identify Enterococcus faecalis and determine the status of drug resistance and virulence genes in samples collected from Tibetan pigs in Linzhi,Tibet,and provide a certain reference for the pathogenic diagnosis of enterococcosis in Tibetan pigs.【Method】 Bacterial isolation and culture,Gram staining,biochemical experiments,Ef0027 specific gene PCR identification,16S rRNA identification were performed on samples collected from Tibetan pigs,and the drug resistance and virulence genes of the isolated strains were detected by PCR method.【Result】 The isolated bacteria appeared brown black colonies on PSE agar medium,suspected to be Enterococcus faecalis.Gram staining microscopy showed that the isolated bacteria appeared as single or double oval shaped cocci.The biochemical identification results showed that the isolated bacteria were negative for arabinose, rhamnose and raffinose, while they were positive for glucose, sorbitol, sucrose, lactose, arginine dihydrolase and mannitol. PCR amplification results showed that an Ef0027 gene band with a size of approximately 518 bp was obtained.A band with a size of approximately 1 500 bp was amplified from 16S rRNA gene,and the similarity with the sequence of Enterococcus faecalis registered in NCBI was greater than 98%.Eight strains of Enterococcus faecalis were isolated and identified in this study.tetB,catⅠ,gyrA,bla_TEM-1,aac3 and SUL-Ⅰ resistance genes were detected in all 8 strains,while fosA7 gene was not detected.The results of virulence gene detection showed that Esp,EfaA,GelE and Agg genes were detected in all the 8 strains,the detection rate of CylA gene was 87.5%,and no Ace gene was detected.【Conclusion】 In this study,8 strains of Enterococcus faecalis of Tibetan pig origin were isolated,all of which had multiple drug resistance genes and virulence genes.The results of this study could provide some reference for the etiological diagnosis of Enterococcus faecalis disease from Tibetan pigs.

【Objective】 The experiment was to explore the effects of compound organic acids on growth performance,intestinal morphology and cecal microflora of broilers infected with Clostridium perfringens.【Method】 Sixty one-day-old Sanhuang chickens were randomly divided into 6 treatment groups with 10 chickens in each group,which were blank control group (BC), Clostridium perfringens model group (P),positive drug (lincomycin) treatment group (PT) and 10,20 and 40 mg/kg compound organic acid treatment groups (L,M and H),respectively.At 1 day of age,except BC group,broilers in other groups were gavage 1.5×10⁸ CFU Clostridium perfringens for 7 d.Simultaneously,broilers in PT group was gavage with 1 mL lincomycin for 7 d，and broilers in L,M and H groups were intragastrically given 1 mL of compound organic acids with corresponding concentration for 7 d.Broilers in BC group were given 1 mL normal saline intragastrically,and the experiment lasted for 13 d.The change of body weight and remaining feed amount of broilers were recorded every day during the experiment,and feed to gain ratio was calculated.On the 13th day,duodenum,jejunum and ileum of broilers in each group were collected,intestinal tissue morphology was determined,and cecal contents were collected for cecal microbial 16S rRNA sequencing analysis.【Result】 ①Compared with BC group,the average daily gain and average daily feed intake of broilers in P group were decreased,and the feed to gain ratio was increased (P＞0.05).Compared with P group,the average daily gain of broilers in PT,L,M and H groups was increased and the feed to gain ratio was decreased (P＞0.05).With the increase of compound organic acid dose,the average daily gain of broilers in L,M and H groups showed an increasing trend. ②Compared with BC group,villus height and V/C value of duodenum,jejunum and ileum of broilers in P group were significantly decreased (P＜0.05).Compared with P group,the villus height of duodenum,jejunum and ileum of broilers in PT,L,M and H groups was significantly increased (P＜0.05),and the V/C value of duodenum and jejunum was significantly increased (P＜0.05).The villus height and V/C value of duodenum of broilers in H group were the highest,and significantly higher than those in other groups (P＜0.05).③The results of sequencing analysis showed that 76 OTUs were obtained from cecal contents samples of the 6 groups,including 62 common core OTUs,and the unique OTUs of BC,PT,L,M and H groups were 5,2,1,1 and 5,respectively.The diversity of cecal microflora of broilers in P group was lower,and the diversity and demeanor of cecal microflora of broilers in H group were the highest,and the species abundance was also the highest.④ Compared with BC group,the ratio of Bacteroidetes to Firmicutes of cecal microorganisms in P group was increased (P＞0.05).Compared with P group,the ratio of Bacteroidetes to Firmicutes of cecal microorganisms in H group was increased (P＞0.05).【Conclusion】 Compound organic acids could prevent the development of intestinal lesions in necrotic enteritis caused by Clostridium perfringens infection.Under the conditions of this experiment,40 mg/kg compound organic acids had the best therapeutic effect.

Veterinary drugs have played an irreplaceable role in preventing livestock and poultry diseases,promoting growth and improving feed conversion rate.However,food safety problems caused by veterinary drug residues are common in recent years,and it is urgent to carry out detection of veterinary drug residues in animal-derived food to ensure food safety.The author summarized the application progress of pretreatment methods (liquid-liquid extraction,solid phase extraction,accelerated solvent extraction,QuEChERS,dispersive solid-phase extraction and magnetic solid-phase extraction),instrument detection techniques ((ultra) high performance liquid chromatography,(ultra) high performance liquid chromatography-mass spectrometry,gas chromatography-mass spectrometry and matrix assisted laser desorption ionization time-of-flight mass spectrometry)，and the rapid detection technology (immunological detection technology,biosensor detection technology,surface enhanced Raman spectroscopy and microbial inhibition analysis technology).The advantages and disadvantages of various pretreatment methods and detection technologies were analyzed,and the future development direction was discussed from the perspective of complementary advantages of instrument detection technology and rapid detection technology.In order to achieve rapid,convenient,intelligent and accurate detection of veterinary drug residues,provided strong support for market supervision and escort food safety.

Osteoarthritis (OA) is a chronic degenerative joint disease that causes locomotor dysfunction in horses.In recent years,intra-articular injection of mesenchymal stromal cells (MSCs) and their derivatives have become a potential treatment for equine OA.MSC-derived cell-free therapies use bioactive substances secreted by isolated MSCs as formulations instead of the MSCs themselves.The main formulations currently include mesenchymal stromal cell-conditioned medium (MSC-CM) and extracellular vesicles (EVs),which theoretically have lower immunogenicity and carcinogenic risks,showing great potential for application.Since factors such as tissue source,isolation procedures,culture conditions,and storage conditions during the preparation process can affect the secretome composition and therapeutic effects,it is necessary to further optimize the production protocol to develop a more reliable method specifically for the treatment of OA.The proposed mechanisms of MSC-derived cell-free therapy for treating OA involve various bioactive factors secreted by MSCs.These factors activate or inhibit cellular pathways,leading to immunomodulatory effects,inhibition of chondrocyte apoptosis,promotion of cartilage and matrix repair and synthesis,and reduction of matrix catabolism.The recent research advancements in MSC-derived cell-free therapy for treating equine OA primarily consist of in vitro cell-level studies.These studies demonstrate that MSC-derived cell-free therapy has anti-inflammatory and growth-promoting effects on chondrocytes,indicating its potential application in treating equine OA.The authors reviewed the preparation,therapeutic mechanism,and potential applications of MSC cell-free therapy in order to provide reference for its future research and clinical applications.

After artificial breeding of cows,if it is not possible to timely and accurately determine whether they are pregnant and deal with non-pregnant individuals in a timely manner,it will reduce reproductive efficiency,and also cause prolongation of calving intervals,affecting the lifetime milk production and milk production efficiency.And the prolongation of calving intervals increase the non-breeding feeding period,leading to increased feeding costs.Therefore,pregnancy diagnosis is an important part of dairy cow breeding management.Only by detecting non-pregnant cows as early as possible after mating and minimizing non-breeding feeding costs can improve breeding efficiency.Traditional pregnancy diagnosis methods such as rectal palpation,ultrasound examination and progesterone testing have achieved good diagnostic results,but they require a large amount of manpower and resources,and require professional technicians to conduct tedious examinations,resulting in low efficiency and increased management costs.In recent years,ELISA detection kits and colloidal gold test strips based on pregnancy related glycoproteins have become popular for diagnosing pregnancy in breeding cows.Although they have greatly improved the accuracy and convenience of detection,blood collection,testing,and ear number verification are still cumbersome and not suitable for large-scale promotion and use.The revelation of the patterns of activity and body temperature changes in pregnant cows also promotes the emergence of new technologies for diagnosing cow pregnancy.By developing automated technology for pregnancy diagnosis,collecting and analyzing physiological indicators of cow activity and body temperature,and determining whether cows are pregnant,cost savings and efficiency can be achieved,hoping to provide new ideas for improving the efficiency of cow pregnancy diagnosis.