【Objective】 This experiment aimed to explore the effects of mixed lineage kinase domain-like protein (MLKL) on macrophages and fibroblasts during the process of skin wound healing in mice and its regulatory mechanism. 【Method】 The back trauma models of C57BL/6J mice and MLKL gene knockout (MLKL^-/-) mice were established.Immunohistochemistry was used to detect the dynamic expression of MLKL,as well as the recruitment of macrophages and fibroblasts after skin injury in mice.Through in vitro experiments,a co-culture system of fibroblasts and M1/M2 macrophages conditioned medium (M1/M2ø CM) was established.Western blotting was performed to evaluate the protein expression of transforming growth factor-β (TGF-β),fibroblast growth factor-2 (FGF-2),and matrix metalloproteinase-9 (MMP-9) in fibroblasts.M1/M2 macrophages were co-cultured with fibroblast-conditioned medium (FBCM).The secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in M1 macrophages was measured by ELISA.The secretion of IL-10 and the expression of Arginase protein in M2 macrophages were determined by ELISA and Western blotting,respectively. 【Result】 The immunohistochemical staining results showed that positive MLKL signals were detected on the 3rd,5th and 7th days of skin wound healing in C57BL/6J mice.The expression level of MLKL increased with the prolongation of wound healing time.Moreover，the number of CD86-positive M1 macrophages,CD163-positive M2 macrophages and Vimentin-positive fibroblasts around the wound in MLKL^-/- mice was less than that in C57BL/6J mice.In co-culture experiments,compared with the untreated group,the treatment of M1ø CM and M2ø CM derived from C57BL/6J significantly increased the protein expression levels of TGF-β,FGF-2 and MMP-9 in fibroblasts (P＜0.05),whereas after treatment with M1ø CM and M2ø CM derived from MLKL^-/-，there was no significant difference in the protein expression of FGF-2 and MMP-9 in fibroblasts (P＞0.05),while the expression level of TGF-β protein was significantly decreased (P＜0.05).After macrophages were treated with C57BL/6J FBCM,the contents of IL-6 and TNF-α in M1 macrophages,the content of IL-10 and the expression level of Arginase protein in M2 macrophages were significantly increased (P＜0.05).After macrophages were treated with MLKL^-/- FBCM,the contents of related cytokines and protein expression levels were both lower than those in C57BL/6J FBCM treatment group (P＜0.05). 【Conclusion】 MLKL could promote skin wound healing by regulating the interaction between macrophages and fibroblasts,influencing the recruitment of macrophages，thereby promoting the healing of skin wounds.

【Objective】 This study aimed to explore the biological functions of miR-200b and investigate the effect on follicle development in mice. 【Method】 In this study,the mature sequences of miR-200b in human,mice,rat,zebrafish,pig and sheep were obtained from the NCBI database,the sequence analysis was carried out by the online software WebLogo,the miR-200b target genes prediction was carried out by TargetScan and miRDB,and the GO function and KEGG pathway enrichment analysis of these target genes were performed.The miR-200b antagonistic model was constructed by intraperitoneal injection of miRNA antagomir,and blood and samples from various tissues and organs of the mice were collected.The expression level of miR-200b in ovaries was detected by Real-time quantitative PCR. The organ coefficient was counted,the morphological changes of ovarian tissues were observed by HE staining,the follicles were counted,and the serum E₂ level was detected by ELISA. 【Result】 The seed sequences of miR-200b were completely identical across different species,and there were differences in the last 1-2 bases of the mature sequence.A total of 607 target genes were predicted by TargetScan and miRDB software,which were mainly enriched in GO entries such as transcriptional regulation,DNA and protein binding,as well as KEGG signaling pathways such as PI3K-Akt,MAPK,longevity regulation,and estrogen.Compared with control and antagomir NC groups,the expression of miR-200b in ovary of mice in miR-200b antagonist group was extremely significantly downregulated (P＜0.01),the ovarian index and the number of pre-antral follicles were extremely significantly decreased (P＜0.01),and the number of antral follicles was extremely significantly increased (P＜0.01).There was also an increase in follicle volume and granulosa cell number.The serum E₂ level was extremely significantly increased (P＜0.01). 【Conclusion】 miR-200b negatively regulated the growth of granulosa cells and follicle development in mice,which provided new evidence for an in-depth analysis of the regulatory function of miR-200b on follicle development.

【Objective】 Glycerol monolaurate (GML) is a monoglyceride formed by esterification of lauric acid and glycerol.It possesses antimicrobial activity and haspotential as a feed additive to improve animal feed efficiency.This study aimed to investigate the effects of different dosages of GML on in vitro rumen fermentation parameters and methane production in dairy cows. 【Method】 An in vitro fermentation technique was employed using a total mixed ration (TMR) with a concentrate-to-forage ratio of 4∶6 as the substrate.Four GML supplementation levels (0 (control group),0.6%,1.2%,and 1.8%) were tested,with five replicates per treatment.The gas production was measured at 0,6,12,24 and 48 h of fermentation.Rumen fluid and gas samples was collected at 6,12,24 and 48 h for analysis gas production,gas component proportions and yields,and rumen fluid fermentation parameters. 【Result】 ①Compared with control group,the gas production in all GML-supplemented groups at 6 h of fermentation was significantly decreased (P＜0.05).The gas production at 6 and 48 h of fermentation showed a significant linear change with the increase in GML addition (P＜0.05).②Compared with control group,the proportions of methane,hydrogen and carbon dioxide were not affected by GML addition at any time point (P＞0.05).The methane yield in 0.6% and 1.2% GML groups at 6 h of fermentation was significantly decreased (P＜0.05).Regarding gas yields,methane,hydrogen and carbon dioxide at 6 h of fermentation,and carbon dioxide yield at 48 h of fermentation,showed a significant quadratic change with the increase in GML addition (P＜0.05).③Compared with control group,the proportions of total volatile fatty acid (TVFA),acetic acid and propionic acid and A/P in rumen fluid of all GML-supplemented groups showed no significant difference at different time points (P＞0.05).The proportion of butyric acid at 6 h of fermentation exhibited a significant quadratic change (P＜0.05).④Compared with control group,the ammonia nitrogen (NH₃-N) concentration at 6 h of fermentation in 1.2% GML group was significantly increased (P＜0.05).The NH₃-N concentration at 12 h of fermentation in 1.2% GML group was significantly decreased (P＜0.05),while in 1.8% GML group was significantly increased (P＜0.05).The NH₃-N concentration at 24 h of fermentation in all GML-supplemented group was significantly increased (P＜0.05).There was no significant difference of pH among all groups at any time point (P＞0.05).The NH₃-N concentration at 12 h of fermentation exhibited a significant quadratic change (P＜0.05). 【Conclusion】 Under the conditions of this study,supplementation with 0.6% GML significantly reduced cumulative gas production,methane yield,and NH₃-N concentration during 6-12 h of fermentation,without significantly affecting VFA composition and pH,indicating that GML had the potential to modulate rumen fermentation processes and inhibit methane production at appropriate supplementation level,providing a theoretical basis for the development of novel rumen methane-reducing additives.

【Objective】 The aim of this study was to investigate the effects of compound Chinese herbal medicine feed additives on the intestinal microbiota and muscle metabolism of pigs,and explore the mechanism of compound Chinese herbal feed additives on the meat quality of fattening pigs. 【Method】 Forty healthy 70-day-old Duroc×Landrace×Yorkshire (DLY) crossbred growing-finishing pigs with an average initial body weight of 30 kg were selected,and randomly divided into two groups,with 4 replicates in each group and 5 pigs in each replicate.Pigs in control group were fed with basic feed,while in Chinese herbal medicine (CHM) group were fed with basic feed containing 0.5% compound Chinese herbal medicine ultrafine powder.The daily feeding amount and remaining feed amount were recorded,and the health status of pigs was monitored in real time.The experimental period was 120 d.Gut microbiota diversity was analyzed via 16S rRNA high-throughput sequencing.Concurrently,untargeted metabolomics techniques were employed to screen for differential metabolites in muscle tissue.An integrated microbiota-metabolite correlation analysis was then applied to elucidate the underlying interaction mechanisms. 【Result】 Based on 16S rRNA sequencing analysis,a total of 18 461 operational taxonomic units (OTUs) were identified,with 5 288 OTUs unique in CHM group.The results of gut microbial community analysis revealed that compared with control group,at the phylum level,the relative abundance of Firmicutes in the intestine of pigs in GHM group was extremely significantly decreased (P＜0.01),while the relative abundance of Bacteroidetes was extremely significantly increased (P＜0.01),and the Firmicutes/Bacteroidetes ratio was 5.03.At the genus level,Streptococcus was the dominant genus between the two groups,the relative abundance of Prevotella in CHM group was extremely significantly higher than that in control group (P＜0.01).Through LDA and LEfSe analysis,a total of 36 differential bacterial communities were screened between the two groups.Compared with control group,the relative abundance of Prevotella,unclassified_Bacteroidales,Oscillospira,Phascolarctobacteria,and CF231 in the intestine of pigs in CHM group was significantly increased.Untargeted metabolomics analysis identified 48 differential metabolites between the two groups,among which 14 metabolites including alanyl-lysine,hippuric acid,and indoxyl sulfate were significantly upregulated,while 34 metabolites including linoleic acid,DL-a-hydroxybutyric acid,and eicosenoic acid were significantly downregulated.Integrated microbiome-metabolome analysis revealed that there was a strong correlation between differential bacterial genera and muscle differential metabolites.Among them,there was a positive correlation between Prevotella and hippuric acid in CHM group,and a negative correlation between Prevotella and DL-a-hydroxybutyric acid. 【Conclusion】 The compound Chinese herbal medicine feed additive indirectly regulated meat quality traits in finishing pigs by regulating intestinal microbiota structure and improving muscle metabolism.Prevotella and Oscillospira were identified as key specific genera influencing meat quality in pigs,while hippuric acid and DL-a-hydroxybutyric acid emerged as important specific metabolites involved in its regulation.

Against the backdrop of the comprehensive implementation of the antibiotic-free feed policy,non-nutritive additives have garnered increasing attention as alternatives to antibiotics in quail farming.This article systematically reviews the research progress on the application of five categories of non-nutritional additives in quail production,including plant extracts and their derivatives,probiotics,enzymes,organic acids,and antimicrobial peptides.Plant extracts and their derivatives improve production performance of quail through multiple pathways,such as regulating digestion and absorption,immune function,and antioxidant capacity.Probiotics exert their effects by modulating gut microbiota balance,enhancing intestinal mucosal barrier function,and boosting immunity.Enzymes primarily improve production performance of quail by degrading anti-nutritional factors and increasing nutrient utilization.Organic acids positively influence quail health by reducing gastrointestinal pH and inhibiting pathogen proliferation.Antimicrobial peptides exhibit dual functionality,combining direct antimicrobial activity with immune regulation.The application of these additives can significantly enhance growth performance,egg production rate,and feed conversion ratio,improve egg and meat quality,and maintain intestinal health of quail.This review also discusses the mechanism,optimal dosage,and challenge associated with different additives,and provides future research directions to offer theoretical references for healthy quail farming and feed additive development.

【Objective】 This study aimed to explore the effect of adding selenomethionine (SeMet) to the rabbit diet on the oxidative damage and inflammatory damage of the ileum caused by deoxynivalenol (DON),providing a new idea for the treatment of DON poisoning diseases. 【Method】 Forty 35-day-old IRA rabbits were randomly divided into 4 groups：The blank control (CON),model (DON),SeMet and DON＋SeMet groups,with 10 rabbits in each group.From the first day of the experiment,rabbits in SeMet and DON＋SeMet groups were fed 0.2 mg/kg SeMet every day for 21 days.On the 15th day,rabbits in DON and DON＋SeMet groups were given 0.5 mg/kg BW of DON by gavage every day for 7 days.On the 21 st day of the experiment,after 24 h of challenge,the ileal tissues of each group were collected.Through HE and PAS staining,as well as scanning electron microscopy,the morphology of the intestinal villi,crypts,the number of goblet cells,and the ultrastructure changes were observed.The levels of oxidative stress-related indicators and inflammatory factors in ileum were detected by ELISA.The expression levels of inflammatory-related genes in ileum were detected by Real-time quantitative PCR. 【Result】 Compared with CON group,a small number of intestinal villi in ileum of DON group were broken,and the villus height and the ratio of villus height to crypt depth were significantly decreased (P＜0.05),the crypt depth was significantly increased (P＜0.05),and the number of goblet cells was significantly reduced (P＜0.05).The results of scanning electron microscopy showed that in DON group,the surface of the ileal villi was atrophied,presenting a granular appearance,and the microvilli were detached and broken.Compared with CON group,the content of interleukin-6 (IL-6) in ileum of DON group was significantly increased (P＜0.05),and the activity of glutathione peroxidase (GSH-Px) was significantly decreased (P＜0.05).The other inflammatory factors (IL-10,IL-1β and tumor necrosis factor-α (TNF-α)) and oxidative stress-related indicators (malondialdehyde (MDA),total superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC)) showed no significant differences (P＞0.05).Compared with DON group,the addition of SeMet to the diet could significantly improve the intestinal pathological damage induced by DON,and the content IL-6 was significantly reduced (P＜0.05). 【Conclusion】 DON could induce inflammatory responses in rabbits and damage the villi structure of the ileum.Meanwhile,SeMet could alleviate the villi rupture and inflammatory damage caused by DON in rabbits.The experimental results provided morphological data and theoretical basis for the intervention of SeMet in the intestinal damage induced by DON,and offered new ideas and methods for the prevention and treatment of DON poisoning diseases.

With the continuous improvement of living standards,consumers’ concern regarding beef quality has been increasing.Transcriptomics and metabolomics,with their advantages of high-throughput,high sensitivity,and systematicity,have become powerful tools for studying beef quality traits.Transcriptomics,through RNA-sequencing (RNA-Seq) technology combined with bioinformatics methods,has revealed the relationship between gene expression patterns and meat quality traits.Metabolomics focuses on endogenous small molecules with molecular weights below 1 500 u within biological systems.It employs techniques such as nuclear magnetic resonance (NMR),gas chromatography-mass spectrometry (GC-MS),and liquid chromatography-mass spectrometry (LC-MS) to detect metabolites,and utilizes multivariate analysis,machine learning (ML),and functional analysis to investigate the correlation between metabolite abundance and meat quality traits.The integrated analysis of transcriptomics and metabolomics can study the relationship between gene expression patterns and metabolite abundance by integrating differentially expressed genes and differentially abundant metabolites.Methods such as gene-metabolite networks and shared KEGG pathway enrichment between the two can comprehensively elucidate the intrinsic differences in meat quality formation and the molecular mechanisms underlying complex traits.This review systematically summarizes the technical principles and workflows of transcriptomics and metabolomics,as well as their current applications in beef quality research.It also discusses the challenges faced by current research and the directions for future development,aiming to provide scientific references for screening molecular breeding markers for high-quality beef cattle and formulating precise nutritional intervention strategies.

【Objective】 This study aimed to investigate the composition and distribution characteristics of cecal microbiota in Black-feathered Muscovy ducks (Cairina moschata) during growth and development at different weeks of age. 【Method】 Experimental subjects included Black-feathered Muscovy ducks at 3,5,7,9,11,13,and 43 weeks of age,with 5 males and 5 females randomly selected for each age of groups.Cecal content samples of Black-feathered Muscovy ducks were subjected to put on 16S rRNA high-throughput sequencing,with analysis performed through QIIME2 and EasyAmplicon software.Microbial community characteristics were evaluated using LEfSe,STEM,PICRUSt2,and Bugbase platforms for microbiota classification and functional enrichment analysis. 【Result】 The diversity analysis results showed that there were significant differences in the cecal microbiota of Black-feathered Muscovy ducks among 3,5,7,13,and 43 weeks of age (P＜0.05).However,a significant difference in Ace index was only observed between male and female Black-feathered Muscovy ducks at 13 weeks of age (P＜0.05).The analysis results of the composition of cecal contents showed that the core microbiota at the phylum level in Black-feathered Muscovy ducks at different weeks of age included Bacteroidota (53.77%),Firmicutes (26.92%),and Desulfobacterota (5.38%).The known core microbiota at the genus level included Bacteroides (27.47%),Rikenellaceae_RC9_gut_group (5.88%),Desulfovibrio (5.21%),and Methanobrevibacter (3.46%).Further heat map analysis of the microbial communities with the top 30 average abundances at the genus level showed significant differences of cecal microbiomes in Black-feathered Muscovy ducks between 3 and 13-43 weeks of age.COG and KEGG functional enrichment analysis indicated that the cecal microbiota of Black-feathered Muscovy ducks during growth and development stages were predominantly associated with metabolic processes and digestive system functions. 【Conclusion】 The cecal microbiotas of Black-feathered Muscovy ducks were predominantly composed of Bacteroidota,Firmicutes,and Desulfobacterota,with dynamic structural and abundance changes occurring at developmental stages to meet physiological demands.Alloprevotella and UCG-008 of the Ruminococcaceae family were candidate key regulatory bacterial genera for the growth and development regulation process of Black-feathered Muscovy ducks at 3-13 weeks of age.The results provided new insights into the effect of gut microbiota on the growth and development characteristics of Black-feathered Muscovy ducks.

Short-chain fatty acids (SCFAs) are metabolic byproducts produced by gut microbiota through the fermentation of dietary fiber and other indigestible carbohydrates,which possess a variety of biological functions,including antimicrobial,anti-inflammatory,and anticancer activities,as well as the regulation of gut microbial composition.SCFAs not only interact with gut microbiota,facilitating inter-microbial communication and modulating the intestinal immune system to enhance barrier function and promote intestinal homeostasis,but can also act as microbial metabolites that cross the gut barrier and exert systemic effects on the host organism.Pork accounts for approximately 30% of global meat sales.With growing consumer demand for high-quality pork,producing premium pork products with distinctive flavor and superior meat quality has become a major focus in the swine industry.In animal husbandry,SCFAs have mainly emphasized their roles in promoting intestinal health and regulating immune function.As the understanding of the relationship between gut microbiota and body metabolism deepens,the mechanisms by which SCFAs,as an important medium of the gut-muscle axis,influences meat quality in pigs through regulating muscle fiber development and intramuscular deposition are becoming increasingly clear.This review summarizes the biological functions of SCFAs,and their relationship with the characteristics of meat quality in pigs,and current research on how SCFAs regulate muscle fiber transformation and intramuscular fat deposition through gut-muscle axis，so as to provide insights for studies and applications of SCFAs regulating meat quality in pigs.

【Objective】 This experiment aimed to investigate the effects of different dietary nutrient levels on the growth performance of double-lamb Tan sheep and their lambs,as well as on the ewe’s nutrient apparent digestibility,serum biochemical parameters,milk composition,and rumen microbiota during the peripartum period (from 60 days prepartum to lamb weaning).The aim was to identify the optimal dietary regimen for an integrated ewe-lamb high-efficiency feeding system. 【Method】 Sixteen healthy,late-pregnancy Tan ewes (2-3 years old) with similar body weight (40.00 kg±2.00 kg) were selected and randomly divided into four groups using a single-factor randomized design,with four ewes per group.Each group was fed one of four pelleted complete diets with different nutrient levels:Group Ⅰ received a basal diet at 100% level (ME 8.00 MJ/kg,CP 13.10%),group Ⅱ at 115% level (ME 9.20 MJ/kg,CP 15.06%),group Ⅲ at 130% level (ME 10.40 MJ/kg,CP 16.93%),and group Ⅳ at 145% level (ME 11.60 MJ/kg,CP 19.00%).The experiment included a 10-day adaptation period followed by a 125-day formal trial.At the end of the experiment,the ewes were weighed.After lambing,the birth weight and average daily gain of the lambs were recorded,and the estrus time of the ewes was also documented.A digestibility and metabolism trial was conducted 15 days before parturition,during which feed,feces,and urine samples were collected to determine the apparent digestibility of nutrients.Venous blood samples were obtained from the ewes at 30 days prepartum,on the day of parturition,and at 30 days postpartum for biochemical analysis.Milk samples were collected on days 1,10,20,30 and 45 postpartum for milk composition determination.Rumen fluid was collected from the ewes 7 days postpartum for metagenomic sequencing. 【Result】 ① Compared with group Ⅰ,final ewe body weight and lamb birth weight were significantly higher in groups Ⅲ and Ⅳ (P＜0.05),while lamb weaning age was significantly lower (P＜0.05).No significant differences were observed between groups Ⅲ and Ⅳ for these indices (P＜0.05).② Apparent digestibility of dry matter,crude protein,organic matter,crude fat,neutral detergent fiber,and acid detergent fiber in ewes showed an increasing trend with rising dietary nutrient levels.Digestibility of dry matter,organic matter,and crude fat in group Ⅳ was significantly higher than that in group Ⅰ (P＜0.05),and crude protein digestibility was significantly higher than that in groups Ⅰ and Ⅱ (P＜0.05).③ On the day of lambing,serum total cholesterol content in group Ⅲ was significantly higher than in group Ⅱ (P＜0.05).At 30 days prepartum,serum triglyceride content in group Ⅲ was significantly higher than in group Ⅰ (P＜0.05),serum glucose content in group Ⅳ was significantly higher than in group Ⅰ (P＜0.05),and serum total protein content in group Ⅳ was significantly higher than in group Ⅲ (P＜0.05).All serum parameters under the four diets remained within normal physiological ranges for ewes.④ No significant differences were observed in colostrum and mature milk contents of milk protein,lactose,total solids,or milk urea nitrogen among the groups (P＞0.05).Milk fat content showed an increasing trend with higher nutrient levels.⑤ Rumen microbiota analysis:The Shannon index in group Ⅳ was significantly higher than that in group Ⅲ (P＜0.05).Principal coordinate analysis revealed significant differences in microbial community structure among groups (P＜0.05).At the phylum level,group Ⅲ exhibited a significantly higher abundance of Bacteroidetes and a lower abundance of Firmicutes compared to groups Ⅰ and Ⅳ (P＜0.05).At the genus level,the abundances of unclassified_o_Bacteroidales,Prevotella,and Ruminococcus were significantly higher in group Ⅲ than in groups Ⅰ and Ⅳ (P＜0.05).KEGG (level 3) pathway analysis indicated that group Ⅲ had significantly enriched pathways compared to groups Ⅰ and Ⅳ,including amino acid and nucleotide sugar metabolism,citrate cycle (TCA cycle),other glycan degradation,sphingolipid metabolism,folate biosynthesis,glutathione metabolism,valine/leucine/isoleucine degradation,vitamin B₆ metabolism,and glycosaminoglycan degradation (P＜0.05).Furthermore,compared to groups Ⅰ and Ⅱ,group Ⅲ showed a significant increase in the relative abundance of glycosyltransferases,glycoside hydrolases,and carbohydrate-active enzymes involved in energy metabolism (P＜0.05). 【Conclusion】 Under the conditions of this experiment,the optimal dietary nutrient levels of metabolizable energy and crude protein for double-lamb Tan sheep,during the period from 60 days prepartum to lamb weaning,were 10.40 MJ/kg and 16.93%,respectively.

【Objective】 This study aimed to isolate and identify peptides with multiple inhibitory activities against angiotensin-converting enzyme (ACE),dipeptidyl peptidase-Ⅳ (DPP-Ⅳ),and α-glucosidase (AG) from hydrolyzed mare whey protein. 【Method】 Fifteen raw mare milk samples were collected from Nanshan Pasture in Urumqi,Xinjiang.Using mare whey protein as the substrate,hydrolysis was performed with single enzymes (trypsin,neutral protease,and alkaline protease),a two-enzyme combination (trypsin + neutral protease),and a three-enzyme combination (trypsin＋neutral protease＋alkaline protease).The hydrolyzed products were separated by ultrafiltration.The antihypertensive activity of the separated products was evaluated by determining ACE inhibitory activity,while their antidiabetic activity was assessed by measuring AG inhibitory activity and DPP-Ⅳ inhibitory activity.The amino acid composition of the peptides was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).Based on the identification results,peptides with better activity were chemically synthesized,and their inhibitory activity was determined in vitro. 【Result】 Compared with the products of single-enzyme and two-enzyme hydrolysis,the three-enzyme hydrolyzates showed significantly higher performance (P<0.05),with a hydrolysis degree of 74.33%,ACE inhibitory activity of 22.81%,DPP-Ⅳ inhibitory activity of 23.46%,and AG inhibitory activity of 20.77%.From the three-enzyme hydrolyzates with the optimal activity,a series of novel peptides with multiple inhibitory activities were isolated and identified,including HSVAMAASDISLLDSESAPLR,TQMVDEEIMEK,VQIVPDLTR,RALQPLPGRVQIVPDLTR,TELTEEEKNYLK,and LRPTPEDNLEIILR.These peptides were chemically synthesized in vitro,and their activities were determined.The results showed that TQMVDEEIMEK exhibited an ACE inhibition rate of 99.89%,VQIVPDLTR had a DPP-Ⅳ inhibition rate of 90.85%,and TELTEEEKNYLK showed an AG inhibition rate of 97.87%. 【Conclusion】 In this study, six novel peptides with multiple inhibitory activities against ACE,DPP-Ⅳ,and AG from mare whey protein hydrolyzates were successfully isolated and identified,laying a theoretical foundation for the development of functional mare milk products.

【Objective】 This study was conducted to determine the effect of compound Chinese herb ultrafine powder on the immune function of laying hens during late laying period. 【Method】 A total of 216 Xinyang Black-feathered laying hens at 307-day-old were selected and randomly divided into three groups,with eight replicates per group and nine hens per replicate.The birds in control group was fed a basal diet,and the birds in experimental groups were supplemented with 0.5% Salvia miltiorrhiza＋0.25% Ligustrum lucidum＋0.25% Taraxacum (compound 1) and 0.3% motherwort＋0.2% Salvia miltiorrhiza＋0.25% Ligustrum lucidum＋0.25% Taraxacum (compound 2) ultrafine powder,respectively.Samples of plasma，fallopian tube and liver tissue were collected on days 120 of the trial to measure immune ability indexes and related gene expression. 【Result】 Compared with control group,the levels of tubal interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and the mRNA expression of tubal interferon-γ (IFN-γ) were significantly decreased in compound 1 group (P＜0.05);the mRNA expression of tubal IL-2 and splenic TNF-α was significantly decreased in compound 2 group (P＜0.05). Plasma level of IL-6 and TNF-α and the mRNA expression of tubal IL-1β and TNF-α and splenic IL-1β and NF-κB were significantly decreased (P＜0.05);and plasma content of IL-10 and the mRNA expression of tubal IL-10 were significantly increased in both compound 1 and 2 groups (P＜0.05). 【Conclusion】 Dietary addition with the compound Chinese herb ultrafine powder composed of motherwort,Salvia miltiorrhiza,Ligustrum lucidum and Taraxacum could enhance the immune ability of laying hens during late laying period by increasing plasma,tubal and splenic immune cytokine activity and activating tubal and splenic related immune signaling.In addition,these two compounds presented target-specific in the regulation of the immune function of the body.

【Objective】 This study aimed to investigate the effects of dietary supplementation with compound preparation of Chinese herbal aqueous extract of consisting of Astragalus membranaceus,Glycyrrhiza uralensis,and Sophora alopecuroides on the activities of carbohydrate-degrading enzymes,microbial composition in rumen of fattening Tan lambs. 【Method】 A single factor design was used in the experiment，and thirty two 3-month-old healthy fattening male Tan lambs weighing around 25 kg were randomly divided into four groups,with 8 lambs in each group.The lambs in control group (k0),experimental group 1 (s1),experimental group 2 (s2) and experimental group 3 (s3) were fed with diets containing 0,0.50%,1.00% and 1.50% compound preparation of Chinese herbal aqueous extract.The pre trial period for the experiment was 10 days,and the main trial period was 60 days.The rumen fluids were collected to measure carbohydrate-degrading enzymes,and analyze rumen species composition and functional genes through microbial metagenomic sequencing at the end of the experiment. 【Result】 There were no significant change in the activities of carboxymethyl-cellulase,pectinase,xylanase,and avicelase in rumen of lambs among the groups (P＞0.05).The amylase and cellobiase activities in s3 group were significantly increased compared with k0 group (P＜0.05). The rumen species composition analysis showed that Pseudomonas,Bacteroidota,and Bacillota were the main dominant bacterial phyla,while Succinivibrio,Prevotella,and Xylanibacter were the main dominant bacterial genera.Through KEGG functional annotation of species genes,the carbohydrate metabolism pathway in the rumen of fattening Tan lambs was found to be active.Through differential analysis of CAZy gene expression profiles among species,it was found that there were significant differences in the overall abundance of CBMs at the level 1 (P＜0.05),while the abundance of carbohydrate-binding modules family genes (CBM14,CBM77 and CBM13),glycoside hydrolase family genes (GH31),and carbohydrate esterase family genes (CE0) at the level 2 were showed significant changes (P＜0.05).The family abundance genes of CBM77,CBM13,and CE0 in s3 group were significantly increased compared to k0 group (P＜0.05).The correlation analysis between ruminal carbohydrate-degrading enzyme activity and rumen CAZy level 2 differential genes showed that xylanase activity was significantly negatively correlated with CE0 family genes abundance (P＜0.05),amylase activity was significantly positively correlated with CBM13 family genes abundance (P＜0.05),and significantly positively correlated with CE0 family genes abundance (P＜0.01).Cellobiase activity was highly positively correlated with CBM77 family genes abundance (P＜0.01). 【Conclusion】 Adding 1.50% compound of Chinese herbal aqueous extract consisting of Astragalus membranaceus,Glycyrrhiza uralensis,and Sophora alopecuroides to the feed could increase the activities of amylase and cellulase in fattening Tan lambs,as well as the abundance of carbohydrate related functional genes (CBM13,CE0,and CBM77) that were positively correlated with them.It would promote the components degradation of starch and cellulose in the rumnal feed and improved the ruminal fermentation characteristics to a certain extent.

Cystine is an amino acid formed by the oxidative linkage of two cysteine molecules via a disulfide bond.It serves not only as a critical building block for protein synthesis but also as an essential nutritional sulfur source for livestock and poultry,playing a vital role in maintaining organismal homeostasis and physiological functions.An appropriate level of cystine is beneficial for the growth and development of livestock and poultry,and helps improve the quality of their products.Previous studies demonstrated that the function of cystine is highly dependent on its efficient transport and dynamic balance within and outside the cells.Once the transport is disrupted,it may lead to a series of metabolic problems such as organ damage in the body.The transport of cystine depends on the solute carrier family 7 member 11 (SLC7A11) protein on the cell membrane and the lysosomal membrane transport carrier protein Cystinosin.The transport process is closely related to the cellular oxidative-antioxidant balance and ferroptosis.Nevertheless,the current research still lacks sufficient exploration of the process by which cells sense changes in cysteine concentration,the mechanism of action of cysteine transporters inside and outside the cells,and the nutritional intervention strategies.Against the backdrop of the global promotion of low-protein diets,the efficient utilization of amino acid resources,including cystine,to reduce nitrogen emissions is of great significance.Therefore,the author systematically sorted out the application effect and transport mechanism of cystine in livestock and poultry production,summarized the regulatory role of cystine in the nutritional metabolism of livestock and poultry,and explored the mechanism of cystine transport and the nutritional metabolism problems caused by transport disorders,with the aim of providing reference for clarifying the mechanism of cystine action and improving the precise nutrition plan of cystine.

【Objective】 This experiment was aimed to investigate the characteristics of calcified shells in eggs with different textures. 【Method】 Pink normal eggs,sand-shelled eggs with their entire body covered with calcium particles (sand-shelled eggs A),sand-shelled eggs with calcium particles concentrated at the blunt end (sand-shelled eggs B),and ring-shaped eggs with multiple dark and light spiral stripes at the blunt end laid by 420-day-old Hy-line Sonia (with 36 eggs of each group) were used for the determination of eggshell quality,crude ash,calcium and phosphorus contents,as well as for the observation of eggshell ultrastructure. 【Result】 ① The eggshell strength of sand-shelled eggs and ring-shaped eggs was significantly lower than that of normal eggs (P＜0.05).There was a significant difference in eggshell strength between sand-shelled eggs and ring-shaped eggs (P＜0.05),which was in the order of sand-shelled eggs A＞ ring-shaped egg＞sand-shelled eggs B.② The inorganic matter content of calcified shells of sand-shelled and ring-shaped eggs was significantly lower than that of normal eggs (P＜0.05).There was a significant difference between the inorganic matter content of calcified shells of sand-shelled and ring-shaped eggs (P＜0.05),which was shown as sand-shelled eggs A＞sand-shelled eggs B＞ring-shaped eggs.The organic matter content of calcified shells with different textures showed an opposite trend to that of inorganic matter content.③ Calcium content of ring-shaped eggs shell was significantly lower than that of sand-shelled eggs and normal eggs (P＜0.05),and there was no significant difference between sand-shelled eggs and normal eggs (P＞0.05).④ The content of phosphorus in eggshell was normal eggs＞sand-shelled eggs A＞ring-shaped eggs＞sand-shelled eggs B.⑤ The mammillary cone layer thickness of eggshell in ring-shaped eggs was lower than that in sand-shelled eggs and normal eggs (P＜0.05).⑥ The effective layer strength of normal eggshells was significantly higher than that of sand-shelled and ring-shaped eggshells (P＜0.05),and the effective layer thickness of sand-shelled eggs B and ring-shaped eggs was significantly lower than that of sand-shell eggs A (P＜0.05).There was no significant difference in the effective layer thickness between sand-shelled eggs B and ring-shaped eggs (P＞0.05).⑦ Integration of principal component analysis and correlation analysis revealed that the critical determinants of eggshell strength were mammillary core layer thickness and calcium content. 【Conclusion】 The shell strength of sand-shelled eggs and ring-shaped eggs was significantly lower than that of normal eggs.Under the condition of similar shell thickness and effective layer thickness,the shell strength of sand-shelled eggs with concentrated calcium particles at the blunt end was the poorest.The thickness of the mammillary cone layer and the calcium content were the main factors affecting eggshell strength.

【Objective】 The study aimed to investigate the effects of supplementing with cationic guanidine acetic acid (CGAA) on the production performance,serum biochemical indices,antioxidant capacity,and immune function of Kazakh ewes in late gestation during cool-season grazing,to provide a reference for the application of CGAA in ruminant farming. 【Method】 A total of 60 healthy grazing Kazakh ewes with similar body weight (47.80 kg±0.30 kg) on the 90-100th day of gestation were randomly divided into 4 treatment groups with 15 replicates in each group.Each sheep was fed 500 g of concentrate mixed with CGAA (0 g in control group,0.6 g in trial group Ⅰ,1.2 g in trial group Ⅱ and 1.8 g in trial group Ⅲ) every day.Feeding was carried out for 37 days under supplementary feeding conditions,including 7 days for pre-feeding and 30 days for the trial period.During the experiment,the production performance of ewes (initial weight,final weight,average daily gain and lamb birth weight) was measured,and 10 ewes were randomly selected from each treatment group for jugular vein blood collection,and their serum biochemical,antioxidant and immune index were measured. 【Result】 The average daily weight gain of ewes in the experimental groups Ⅱ and Ⅲ were extremely significant higher than that in control group and experimental group Ⅰ (P＜0.01).The birth weight and feed intake of ewes in groups Ⅱ and Ⅲ were significantly higher than those in the control group (P＜0.05).Compared with control group,the serum triglyceride content of ewes in group Ⅲ was significantly reduced (P＜0.05).In group Ⅱ,the content of serum urea nitrogen,tumor necrosis factor and aspartate aminotransferase were extremely significantly decreased (P＜0.01),the contents of creatinine and creatine,the activities of superoxide dismutase and glutathione-peroxidase,total antioxidant capacity and insulin growth factor level were extremely significantly increased (P＜0.01),and progesterone and cortisol hormones were significantly decreased (P＜0.05).The levels of L-arginine and nitric oxide in groupsⅠ and Ⅱ were extremely significantly increased (P＜0.01). 【Conclusion】 Under grazing conditions,supplemental feeding with CGAA could increase the average daily gain of Kazakh sheep ewes and the birth weight of lambs in the middle and late stages of gestation.It could enhance the antioxidant capacity and immune function of ewes,and reduce the secretion level of hormones such as cortisol.It was advisable to feed Kazakh sheep ewes with CGAA 1.2 g/d in the middle and late stages of gestation.

Chromium,as one of the essential trace elements in animal organism,mainly exerts its biological function in the form of trivalent chromium.Chromium is divided into two categories:Organic chromium and inorganic chromium.Inorganic chromium has limitations in its application due to its low absorption rate and susceptibility to the influence of other feed ingredients.Organic chromium,as a class of nutritional supplements,has biological functions such as improving insulin sensitivity,regulating blood glucose levels,participating in lipid metabolism,and enhancing the ability to resist oxidative stress.Organic chromium plays an important role in promoting animal growth,improving production performance and reproductive performance.In swine production,organic chromium can improve the growth performance of pigs,reduce the thickness of backfat,increase the rate of lean meat,and improve the pork quality.In the production of ruminants,organic chromium can alleviate the impact of environmental stress on animals,and improve the quality of milk and meat.In chicken production,organic chromium can enhance the production performance,strengthen the body’s antioxidant capacity,and reduce mortality rate.The author outlined the types of organic chromium and their absorption and metabolic processes,and reviewed the biological functions of organic chromium and its application progress in different animal diets.With the aim of providing references for the rational application and in-depth study of organic chromium in livestock production.

【Objective】 This study aimed to reveal the genetic mechanisms of porcine ovarian cysts from the neuroendocrine system perspective,the candidate genes related to porcine ovarian cysts in the hypothalamus-pituitary axis were identified. 【Method】 The hypothalamus and pituitary tissues from three sows with ovarian cysts and three normal estrus sows were subjected to RNA-Seq using Illumina high-throughput sequencing technology.Differentially expressed genes were screened,and subjected to GO function and KEGG pathway enrichment analysis.The expression of 12 differentially expressed genes in hypothalamus and pituitary were validated using Real-time quantitative PCR to verify the reliability of the transcriptome data. 【Result】 Compared with the normal estrus sows,a total of 283 genes were significantly differentially expressed in hypothalamus of sows with ovarian cysts,including 173 down-regulated and 110 up-regulated genes.In the pituitary of sows with ovarian cysts,a total of 1 149 genes were significantly differentially expressed,including 563 down-regulated and 586 up-regulated genes.Based on the expression of the differentially expressed genes,FoldChange,as well as the results of GO function and KEGG pathway enrichment analysis, POMC, KISS1,PMCH,PRLH, VIP, NTRK1,and TAC3 were identified as potential candidate genes related to ovarian cysts in hypothalamus,whereas MC3R,SSTR3,FSHB,OXT,PRL,TSHB, LHB,and TAC1 were proposed as potential candidate genes in pituitary.Key pathways such as G-protein coupled receptor signaling pathway,hormone activity,transmembrane signal receptor activity,neuroactive ligand-receptor interaction,thyroid hormone synthesis,and ECM-receptor interaction might play an important role in the occurrence of ovarian cysts.Real-time quantitative PCR results showed that the expression trends of differentially expressed genes were basically consistent with RNA-Seq data. 【Conclusion】 Genes such as POMC,KISS1,and PMCH in hypothalamus,and MC3R,SSTR3,and FSHB in pituitary,might be candidate genes related to porcine ovarian cysts.Pathways including G-protein coupled receptor signaling pathway,hormone activity,and transmembrane signaling receptor activity likely played important roles in the pathological process of porcine ovarian cysts.The results provided crucial data support for elucidating the genetic mechanisms of porcine ovarian cysts from the perspective of the neuroendocrine system.

【Objective】 Zhangmu cattle is endemic to Tibet but on the brink of extinction.In this study,a rescue conservation was conducted using somatic cell nuclear transfer (SCNT) technology to provide a paradigm for the conservation of endangered species in high-altitude regions. 【Method】 Ear margin fibroblast from Zhangmu cattle were established by enzyme digestion method,reconstructed embryos were generated via SCNT technique and then transferred to surrogate cows for in vivo development.Bisulfite sequencing was performed on imprinting domains,including paternally expressed gene 10-sarcoglycan epsilon (PEG10-SGCE),insulin-like growth factor 2-H19 imprinted maternally expressed transcript (IGF2-H19) and guanine nucleotide-binding protein alpha subunit (GNAS),in tissues from aborted fetuses. 【Result】 SCNT embryos were produced using ear margin fibroblast of Zhangmu cattle with normal karyotype as donor cells.A total of 32 cloned blastocysts were transferred into the uteri of 21 recipient cows,among which 12 were successfully pregnant and 5 gave birth to cloned cattle.Short tandem repeat analysis showed that the nuclear DNA of cloned cattle was completely matched with that of donor fibroblast.The PEG1-SGCE,IGF2-H19 and GNAS imprinted domains were hypermethylated in the placenta of 2 aborted fetuses that occurred abortion at 270 days of gestation,and the GNAS imprinted domain was abnormally methylated at either a low or high manner in different tissues of the aborted fetuses. 【Conclusion】 SCNT technology could be applied to the rescue conservation of the endangered Zhangmu cattle.

【Objective】 This experiment aimed to systematically analyze the circadian rhythm-related gene family in chickens,explore the expression patterns of endogenous rhythm genes in the pineal gland of chickens under the constant darkness conditions,and analyze the expression patterns of partial rhythm genes in pineal gland of chickens from late embryonic stage to early chick rearing stage. 【Method】 Various rhythm-related gene family sequences were obtained from InterPro database,the phylogenetic tree was constructed,and the physicochemical properties,conserved domains,and cis-regulatory elements were analyzed.Based on transcriptome data,the RAIN algorithm and PSEA were used to analyze the expression characteristics of endogenous rhythm genes.Finally,Real-time quantitative PCR was used to analyze the expression patterns of rhythm genes in pineal gland of Yanshan Hongyu chickens and Babcock B380 chickens at different stages. 【Result】 Seven rhythm-related gene families (PER,CRY,CLOCK,BMAL,ROR,TIMELESS,and OPN4) were identified.Phylogenetic tree showed that CLOCK and BMAL genes were relatively close in evolution.Chromosome localization analysis revealed that the members of each gene family were dispersed across different chromosomes,and OPN4 gene was tandemly repeated with RRH gene.Protein physicochemical analysis showed that the molecular weight of PER3 and TIMELESS proteins exceeded 130 ku,while the other family members were smaller than 100 ku.OPN4 and its subtype OPN4-1 were hydrophobic and contained 7 transmembrane domain (7tm_GPCRs),while the other proteins were hydrophilic and lack transmembrane domains.RAIN algorithm results showed significant phase differences in the expression of CRY1 and CRY2 genes,with differences in the transcription factor binding sites and density in their promoter regions.PSEA analysis results found that under the constant darkness conditions,the genes CRY2 and PER3,which exhibited high expression during the subjective night,were involved in circadian rhythm regulation and pathways related to DNA damage binding,energy metabolism,and lipid metabolism.The genes CLOCK,ARNTL,and CRY1,which exhibited high expression during the subjective day,participated in circadian gene transcription activation,DNA repair,and damage response pathways.Real-time quantitative PCR results showed that at subjective day 2 h on incubation days 19,and on day 1 and days 10 post-hatching,the expression of CRY2,PER2,and CLOCK genes firstly significantly increased and then significantly decreased (P＜0.05),while the expression of TIMELESS and OPN4 genes continuously increased (P＜0.05). 【Conclusion】 The circadian rhythm-related gene familiy in chickens was widely dispersed on chromosomes,and there was tandem duplication between OPN4 and RRH genes.The phase difference of CRY gene expression might be influenced by regulatory elements in their promoter regions.On day 1 post-hatching,light stimulation might significantly upregulate the expression of core rhythm genes such as CRY2 and PER2 in pineal gland of chickens.By days 10 post-hatching,the circadian regulatory network tended to mature.

As people’s quality of life improves,the demand for meat quality and quantity increases.Local pig breeds in China are rich in resources,and under long-term natural and artificial selection,they have formed excellent characteristics such as strong adaptability,excellent meat quality,roughage tolerance,good resistance to adversity,etc.,which are excellent breeding materials for cultivating new high-quality breeds (lines).Crossbreeding improvement is a common technique used by breeders to increase economic value in pig production,and it has been often used to cross foreign lean pig breeds with local pig breeds,so as to obtain breeds or lines that take into account the excellent meat quality characteristics of local pigs and the high meat yield of foreign pig breeds.The rapid development of omics analysis technology in recent years has facilitated a deeper investigation of meat quality differences,and multi-omics data can be used to comprehensively analyse the complex regulatory networks that causes the differences,thus explaining the regulatory mechanisms more accurately.The authors briefly describe the differences in meat quality traits between local pig breeds and introduce pig breeds before and after crossbreeding,and analyse the regulatory mechanisms of meat quality differences in the light of the progress of multi-omics and gut microbiomics technology in meat quality,in order to provide a reference for the construction of local pig breed improvement system.

【Objective】 This study aimed to analyze the codon usage patterns and characteristics of mitochondrial DNA (mtDNA) gene sequences in Shitan chickens and native chicken breeds of China,and explore the evolutionary relationship between Shitan chickens and main native chicken breeds of China. 【Method】 mtDNA gene sequences from 10 healthy adult Shitan chickens were obtained.CodonW 1.4.2 software was used to analyze the codon usage bias of mtDNA gene in Shitan chickens and 17 native chicken breeds of China,and codon preference parameters were calculated.Subsequently,cluster analysis of preferred codons,amino acid analysis,factor correlation analysis,and cluster analysis of gene sequences were performed. 【Result】 Both Shitan chickens and main native chicken breeds of China exhibited a preference for codons ending with C in mtDNA sequences,while codons ending with G were used less frequently.In Shitan chickens,27 codons including CGA,CUA,ACC,and UUC had a relative synonymous codon usage (RSCU) value greater than 1,indicating they were preferred codons.Shitan chickens showed similar codon usage bias with Taoyuan chickens,Wuhua Sanhuang chickens,Guangxi Sanhuang chickens,Rugao Yellow chickens,Hainan Wenchang chickens,Hetian chickens,and Wannan Sanhuang chickens.Consequently,these seven breeds,along with Shitan chickens,were selected for further analysis to clarify the genetic relationship.Amino acid composition analysis revealed that leucine was the most abundant amino acid in mitochondria of Shitan chickens,followed by isoleucine and threonine.Among these eight chicken breeds, AC- and GU- ending codons showed significant divergence,with AC-ending codons being significantly more prevalent.Cluster analysis based on mtDNA gene sequences indicated that Shitan chickens was phylogenetically closest to Rugao Yellow chickens. 【Conclusion】 Shitan chickens and maind native chicken breeds of China exhibited a significant preference for C-ending codons,with notably higher usage of AC-ending codons.Leucine was the most abundant amino acid in mitochondria of Shitan chickens.Shitan chickens shared the closest genetic relationship with Rugao Yellow chickens.

【Objective】 This study aimed to establish a classification and evaluation system for porcine MⅡ-stage oocytes based on cytoplasmic color and the morphology of the first polar body,and systematically analyze the morphological characteristics of oocytes at different grades and its effect on embryonic developmental competence,with the goal of improving the selection efficiency of high-quality matured oocytes and enhancing the developmental potential of somatic cell nuclear transfer (SCNT) embryos,so as to provide a scientific basis for the application of SCNT technology in livestock production. 【Method】 In vitro matured porcine MⅡ-stage oocytes were collected and categorized into four grades (A,B,C,and D) based on cytoplasmic appearance (dense black or pale granular) and the morphology of the first polar body (intact and round or fragmented and elongated).Cytoplasmic diameters of oocytes were measured under a microscope,the microfilament distribution and expression were assessed using phalloidin-FITC staining,the lipid droplet size and number were evaluated by BODIPY staining,and the spindle morphology and abnormality rates were observed using α-tubulin-TRITC staining.Parthenogenetic activation (PA) was performed by electrical stimulation,followed by assessments of cleavage rate,blastocyst rate,and apoptosis rate.SCNT embryos were generated using inbred porcine fetal fibroblasts as nuclear donors to evaluate developmental competence across the oocyte grades. 【Result】 Grade A porcine oocytes accounted for the highest proportion (28.60%),with the larger cytoplasmic diameter (117.57 μm),which was significantly higher than that of grades C and D oocytes (P＜0.05).The expression of microfilament in grade A oocytes was moderate (85.85 A.U.),and the spindle abnormality rate was the lowest (24.56%).The blastocyst rates of PA and SCNT embryos in grade A oocytes were the highest (52.37% and 30.80%),which were significantly higher than that in grade D oocytes (P＜0.05).The cytoplasmic diameter of grade B porcine oocytes were the highest (117.67 μm),which was significantly higher than that of grades C and D oocytes (P＜0.05).The expression of microfilament and the proportion of small lipid droplets in grade B oocytes were the highest (88.72 A.U.and 44.07%),while the blastocyst apoptosis rate was the lowest (87.12 A.U.).The cleavage rate and blastocyst rate of PA embryos in grade B oocytes were significantly lower than that in grade A oocytes (P＜0.05).In contrast,grades C and D porcine oocytes exhibited paler cytoplasm,larger lipid droplets,higher spindle abnormality rates,and significant lower blastocyst rate of PA and SCNT embryos. 【Conclusion】 Through morphological grading of porcine MⅡ-stage oocytes,combined with multiple cellular quality and developmental indicators evaluations,it was confirmed that grade A oocytes with dense cytoplasm color and intact the first polar body possessed higher developmental potential.The morphological classification system provided a reliable and non-invasive criterion for selecting high-quality porcine oocytes,optimized the efficiency of SCNT cloning technology,and promoted the advancement of porcine cloning technology in livestock breeding.

With the rapid development of China’s animal husbandry industry,livestock with high reproductive rates,high production efficiency,and strong adaptability have significant advantages in breeding economic benefits and variety selection.Artificial insemination technology,as a crucial link in the propagation of livestock,transmit the genetic information of high-quality breeding livestock.Among them,semen preservation technology is the foundation for expanding artificial insemination,by prolonging the survival time of sperm in vitro and enhancing the utilization rate of superior male breeding animals.Currently,there are three commonly employed methods of ultra-low-temperature cryopreservation，normal-temperature preservation,and low-temperature preservation.Ambient temperature preservation facilitates short-term semen transport,whereas cryopreservation focuses on long-term,cross-spatiotemporal storage.The low-temperature preservation technology enables semen to be unaffected by time,geographical location,and the development characteristics of the breeding animals themselves,thereby strengthening genetic exchanges among different breeds.The low-temperature preservation diluent,by adding antioxidant substances such as lecithin,effectively mitigates the oxidative damage to sperm during the low-temperature preservation process and increases the conception rate of female animals after artificial insemination.This article systematically summarizes the main additive components of low-temperature diluents for livestock semen,the common pathways of energy metabolism in livestock spermatozoa,analyzes the metabolic changes of livestock spermatozoa under low-temperature conditions and their underlying mechanisms,aiming to understand the metabolism of livestock spermatozoa,provide theoretical references for formulating more effective diluents,and contribute to the large-scale farming of livestock,breed improvement,the advancement of breeding programs,and the enhancement of economic benefits.

【Objective】 This study aimed to analyze the association between single nucleotide polymorphism (SNP) of taste receptor TAS1R1 gene and growth traits in Guizhou indigenous pigs,providing effective molecular markers and theoretical foundations for pig genetic breeding. 【Method】 Ear tissue samples from 262 Guizhou indigenous pigs (Congjiang Xiang pigs,Jiangkou Luobo pigs,Baixi pigs,Guanling pigs,and Qiandong Hua pigs) were collected for DNA extraction.Nine pairs of TAS1R1 gene specific primers were designed using Primer Premier 6.0 software,PCR amplification products were sequenced,and SNP was identified via SeqMan program in DNAStar.Population genetic characteristics of SNP were analyzed using SHEsis online software.The association between SNP and growth traits in Guizhou indigenous pigs were evaluated via the general linear model (GLM) in SPSS 25.0 software.RNAfold web server was used to predict mRNA secondary structural changes induced by SNP. 【Result】 Two SNPs were identified:g.67401632 G＞C (exon 3) and g.67404545 C＞T (exon 5),both of which were missense mutations,causing valine to leucine and threonine to methionine,respectively.Genotype analysis results showed that CC was the dominant genotype for g.67401632 G＞C and g.67404545 C＞T.Genetic diversity analysis results showed moderate polymorphism at g.67401632 G＞C in Congjiang Xiang pigs,Guanling pigs,and Qiandong Hua pigs (0.25＜PIC＜0.50),but low polymorphism in Jiangkou Luobo pigs and Baixi pigs (PIC＜0.25),and low polymorphism in Baixi pigs at g.67404545 C＞T.Hardy-Weinberg equilibrium was observed for g.67401632 G＞C in Guanling pigs (P＜0.01),while other pig breeds at g.67401632 G＞C and g.67404545 C＞T deviated from Hardy-Weinberg equilibrium (P＞0.05).Association analysis results indicated that CC genotype at g.67401632 G＞C in 8-month-old Congjiang Xiang pigs exhibited significantly greater body length than that of CG genotype (P＜0.05).Conversely,CG genotype in Jiangkou Luobo pigs showed longer body length than that of CC genotype (P＜0.05).No significant associations were found between SNP and growth traits in 1-5-month-old Congjiang Xiang pigs and Qiandong Hua pigs (P＞0.05).The secondary structure prediction results showed that g.67401632 G＞C and g.67404545 C＞T enhanced mRNA secondary structure stability of TAS1R1 gene. 【Conclusion】 Two missense mutations g.67401632 G＞C (exon 3) and g.67404545 C＞T (exon 5) were identified in TAS1R1 gene.g.67401632 G＞C had potential associations with growth traits in Guizhou indigenous pigs,indicating that TAS1R1 could be considered as a candidate gene for growth traits in pig genetic breeding.

【Objective】 This study aimed to analyze the population structure and conservation prioritization of 7 representative indigenous Chinese goat breeds,thereby promoting the conservation of goat genetic resources and the sustainable and healthy development of the goat industry. 【Method】 Genomic data from 63 individuals representing 7 indigenous Chinese goat breeds,including the Tibetan goat,Inner Mongolia cashmere goat,Liaoning cashmere goat,Xiangdong Black goat,Hainan Black goat,Guizhou Black goat and Luoping Yellow goat,were obtained using the Illumina 50K chip.Population structure was characterized through principal component analysis (PCA),phylogenetic tree and Admixture analysis,with PCA and Admixture results visualized using R Studio and the phylogenetic tree annotated and visualized using the online iTOL platform.By integrating the 7 representative breeds into a single metapopulation,the changes in total gene and allelic diversity were analyzed by sequentially removing each breed.Through simulation,the proportional contribution of each breed to the synthetic population in terms of gene and allelic diversity was assessed,thereby determining the conservation priorities among the breeds. 【Result】 In the population structure analysis,both PCA and phylogenetic tree results revealed that the seven breeds were distinctly divided into three branches:The Western & Northern branch,the Southeastern branch and the Southwestern branch.The Western & Northern branch included the Liaoning cashmere goat,Inner Mongolia cashmere goat and Tibetan goat;The Southeastern branch comprised the Xiangdong Black goat and Hainan Black goat;And the Southwestern branch encompassed the Guizhou Black goat and Luoping Yellow goat.Admixture analysis indicated that the optimal population structure was achieved at K=3. In the conservation prioritization analysis,the Z-score normalization of gene and allelic diversity revealed the following priority order for conservation efforts,from highest to lowest:Liaoning cashmere goat,Guizhou Black goat,Tibetan goat,Xiangdong Black goat,Inner Mongolia cashmere goat,Hainan Black goat and Luoping Yellow goat. 【Conclusion】 This study demonstrated that indigenous Chinese goat breeds could be classified into 3 distinct branches,with the Liaoning cashmere goat,Guizhou Black goat and Tibetan goat identified as priority breeds for conservation.These findings provide a scientific basis for the management of genetic resources in indigenous Chinese goats and offer valuable insights for optimizing conservation policies.

Somatic cell nuclear transfer (SCNT) in pigs involves transferring the nucleus of somatic cell from pigs into enucleated oocytes,activating the reconstructed embryo to develop into a new individual through asexual reproduction,it plays a significant role in producing cloned pigs and advancing livestock breeding and biomedical research.However,the complexity of selecting and culturing donor cells and recipient oocytes in SCNT of pigs,coupled with low cloning efficiency,results in the low transfer efficiency (less than 5%).The author systematically reviews the strategies to enhance the efficiency of SCNT in pigs:①Precise enucleation techniques,such as laser-assisted,polarized light microscopy to improve enucleation rate.②Optimization of donor cells and oocytes,including low-passage fetal fibroblasts,cell cycle synchronization,dynamic hormone combination culture,etc.③Innovation in fusion and activation methods,including the optimization of electrofusion parameters,zinc ion regulation,etc.④Epigenetic rescue,such as overexpression of reprogramming factor MBD3 and inhibition of H3K9 me3 modification.In the future,it is necessary to further overcome the bottleneck of developmental efficiency through multi-technology collaboration,such as metabolic regulation combined with epigenetic modification,and promote the industrial application of cloning technology of pigs in livestock production and xenotransplantation.

【Objective】 The goal of this study was to develop a broad-spectrum nanobody (Nb) specifically targeting the polymerase acidic protein (PA) of Influenza A virus (IAV),offering novel biomaterials for both basic and applied research of IAV. 【Method】 The eukaryotic expression plasmid of IAV PA was constructed,and purified proteins were obtained using Flag tag antigen-specific antibody affinity chromatography.It was mixed with Freund’s adjuvant to immunize alpacas.The titer of antibody against IAV PA in alpaca serum collected at 14 days post the fifth immunization was determined by indirect ELISA.Peripheral blood lymphocytes (PBL) were isolated from the peripheral blood of alpacas,and total RNA was extracted and reverse transcribed into cDNA.Following nested PCR amplification,the gene coding for the variable domain of heavy chain of heavy-chain antibody (VHH) was inserted into a pComb phage vector to construct a phage display library.The capacity of the library was calculated and its diversity was analyzed.Positive Nb clones binding to PA protein were selected through enrichment and panning.The specificity of Nbs was evaluated by Western blotting and immunofluorescence assay (IFA). 【Result】 After sequencing verification,the pcDNA3.1-Flag-PA eukaryotic expression recombinant plasmid with correct sequence was obtained.The PA protein was expressed and purified to be used as an immunogen for immunizing alpacas.After five immunizations,the antibody titer against PA protein in alpaca serum reached 1∶64 000. VHH gene was successfully amplified and a nanobody phage display library with a recombination rate of 100%,sequence diversity of 90%,and a library capacity of 6.5×10⁷ CFU/mL was constructed.After three rounds of enrichment and panning,six PA protein nanobodies with different functional region sequences were obtained,namely Nb13,Nb14,Nb16,Nb20,Nb42 and Nb72,respectively.Among them,the nanobody Nb16 could specifically recognize PA proteins of different subtypes of IAV in a broad-spectrum. 【Conclusion】 In this study,a phage display library of IAV PA protein nanobodies was successfully constructed,and a broad-spectrum nanobody specifically recognizing IAV PA protein was screened and obtained.

【Objective】 This study aimed to investigate the effects of ZBED6 gene knockout on the gene expression profile in lungs of Bama Xiang pigs and further elucidate the impact of ZBED6 deficiency on pulmonary function in this breed. 【Method】 Lung tissues were collected from 8-month-old female wild-type (WT) and ZBED6-knockout (ZBED6-KO) Bama Xiang pigs,and lung weight was measured.Total RNA was extracted using TRIzol reagent,followed by quality assessment,library preparation (NEBNext^® Ultra^TM),and sequencing (Illumina HiSeq 2500).Raw data were quality-controlled using Fastp v 0.23.4,and differentially expressed genes (DEGs) were analyzed using TopHat and Cufflink,with the threshold set at |log₂FoldChange|≥1 and P≤0.05.Functional enrichment analysis of DEGs was performed using the g:Profiler online database,and RNA-Seq results were validated by Real-time quantitative PCR. 【Result】 Compared with WT Bama Xiang pigs,the lung weight of ZBED6-KO Bama Xiang pigs showed no significant changes (P＞0.05).RNA sequencing (RNA-Seq) analysis identified 480 DEGs between WT and ZBED6-KO lungs of Bama Xiang pigs,including 125 upregulated and 355 downregulated genes.Upregulated genes were primarily enriched in two immune-related pathways,while downregulated genes were significantly associated with metabolic pathways.Real-time quantitative PCR validation confirmed that the expression trends of selected DEGs were consistent with the RNA-Seq results,confirming the reliability of the sequencing data. 【Conclusion】 ZBED6 gene knockout significantly altered the gene expression profile in lungs of Bama Xiang pigs,with immune-related genes generally upregulated and metabolic-related genes downregulated.This study provided theoretical basis for understanding the role of ZBED6 gene in pulmonary function,further filling the research gap regarding the regulatory mechanisms of the transcription factor ZBED6 in mammalian lungs.

【Objective】 This study was conducted to investigate the effect of heat shock protein GroEL on the growth of Brucella in vitro and its cellular localization in macrophages. 【Method】 In this study,with Brucella abortus strain A19 as the research object,homologous recombination and SacB reverse screening technology were used to construct GroEL gene deletion strain (A19ΔGroEL) and overexpression strain (A19-HA/GroEL) of Brucella abortus strain A19.The in vitro growth curves of GroEL gene deletion and overexpression strains were plotted to detect the effect of GroEL on the in vitro growth of Brucella.Indirect immunofluorescence assay was used to detect the localization of GroEL in RAW264.7 macrophages.And the localization of GroEL in Brucella was observed by immunoelectron microscopy. 【Result】 PCR results showed that a specific band with a size of 1 520 bp was amplified,indicating that the GroEL gene deletion strain A19ΔGroEL was successfully constructed.Western blotting results showed that the target band with size of 60.2 ku was obtained,indicating that overexpression strain A19-HA/GroEL was successfully constructed.The results of the bacterial growth curve showed that there was no significant difference in the in vitro growth trends among Brucella abortus A19,A19ΔGroEL and A19-HA/GroEL (P＞0.05).The results of indirect immunofluorescence assay showed that the GroEL was mainly localized in the cytoplasm of RAW264.7 macrophages.The results of immunoelectron microscopy observation showed that GroEL was mainly located in the outer and inner membranes of Brucella. 【Conclusion】 The results showed that the GroEL gene deletion and overexpression strains of Brucella abortus were successfully constructed in this study,which initially revealed that the deletion of GroEL gene did not affect the growth of Brucella in vitro,and GroEL was mainly localized in the cytoplasm of RAW264.7 macrophages and the outer and inner membranes of Brucella,which provided the theoretical basis and data support for the in-depth investigation of the biological function of GroEL.

【Objective】 In December 2024,a flock of 28-day-old broilers in a commercial White-feathered broiler farm in Pingdu city of Shandong province,showed symptoms of swollen hock joints and paralysis,and deaths occurred successively.To determine the cause of the disease in this flock,this study conducted necropsy and laboratory diagnosis on the diseased and dead chickens submitted for examination. 【Method】 Liver,spleen tissues,fibrinous exudate from the serous layer of diseased chicken organs,synovial secretion,and pharyngeal swab were collected for laboratory diagnosis.Real-time PCR/RT-PCR was used to detect nucleic acids of pathogens that cause similar clinical signs including Riemerella anatipestifer (RA),Avian reovirus (ARV) and Mycoplasma synoviae (MS),to identify the etiology of the outbreak.The bacterial characteristics and drug sensitivity of infected chickens were determined through bacterial isolation and culture of joint cavity secretions,Gram staining microscopy observation,serotype identification,16S rRNA gene sequence analysis,and drug sensitivity testing.Tissue homogenates were inoculated into LMH cells for virus amplification.The major structural-protein genes were amplified by PCR and sequenced to define the molecular epidemiology of the viruses infecting the chickens. 【Result】 Real-time quantitative PCR/RT-PCR detection showed that RA and ARV had Ct values＜35,indicating positivity.MS had no Ct value,indicating negativity.The bacteriological test results showed that the isolated strain appeared as semi transparent circular protrusions with neat edges and smooth surface colonies on the blood plate and TSA plate,the bacteria were single and paired Gram negative short rods,suspected to be RA.The serotype identification results showed that the isolated strain from chicken was RA serotype 10.16S rRNA gene amplification sequencing was analyzed by BLAST to be located in the same branch as the RA reference strain that from duck,with a similarity greater than 99%,the isolate was identified as RA.The drug sensitivity test results showed that the strain was sensitive to ceftriaxone,cefotaxime and polymyxin B,but resistant to neomycin,gentamicin,kanamycin,ampicillin,amoxicillin and ciprofloxacin.PCR detection of cell culture showed that the ARV σC gene nucleic acid was positive.Further analysis of the gene fragment revealed that the strain belonged to the GC1-sub genotype，not in the same branch as the classical vaccine strains S1133 and 1733. 【Conclusion】 This study identified the mixed infection of RA and ARV as the cause of the disease in the chicken farm through comprehensive diagnostic methods.The RA isolate exhibited multidrug resistance,whereas the ARV isolate belonged to the GC1-sub cluster.These findings provided a scientific basis for the targeted prevention and control of broiler diseases in Qingdao area.

【Objective】 This experiment aimed to study the prevention effect of small-cell partition pigsty on Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus type 2 (PCV2) immune and immune-exit. 【Method】 Conventional closed pigsty was set up for immune-exit group (CCP-N) and PCV2 immune group (CCP-PCV2).Small-cell partition pigsty was set up for immune-exit group (SPP-N),PCV2 immune group (SPP-PCV2),and PRRSV immune group (SPP-PRRSV).Each group consisted of 10 replicates,with 1 pig per replicate,and raised for 200 days from weaning to slaughter.Pigs in immune-exit groups in both housing systems received no vaccination. PCV2 immune and PRRSV immune groups were administered one dose of the respective vaccine on the 20th and 30th day after enrollment,respectively.Blood samples were collected on days 0,60,140 and 200 for PRRSV N protein and PCV2 serum antibody detection.At the end of the experiment,the PRRSV GP5 protein antibody levels in serum and PRRSV nucleic acid negative rate in tissues were tested,and lung lesion conditions were observed and statistically analyzed. 【Result】 During the experimental period,the antibody levels of PRRSV N protein in SPP-N group were consistently low,and significantly lower than the other groups (P＜0.05).The positive rate of PRRSV in SPP-N group was 0,and consistently significantly lower than the other groups.At the end of the experiment,the antibody levels of PRRSV GP5 protein in SPP-N group were extremely significantly lower than the other groups (P＜0.01).The single negativity rate of PRRSV N protein antibody in serum and the double negativity rate of PRRSV N protein and GP5 protein antibodies in SPP-N group were both 100%,and were higher than the other groups.The lung tissue lesion scores in SPP-N group were the lowest and extremely significantly lower than the other groups (P＜0.01).The negative rate of PRRSV nucleic acid in tissues and the triple negative rate of PRRSV N protein antibody,GP5 protein antibody and tissue PRRSV nucleic acid in pig serum were 100%,which was higher than the other groups.During the experimental period,the antibody level and positive rate of PCV2 in all groups peaked at day 60 and remained stable until the end of the experiment. 【Conclusion】 In swine farms with mixed infection of PRRSV and PCV2,the effect of small-cell partition pigsty for PRRSV and PCV2 immune-exit were significant effectiveness.Small-cell partition pigsty could effectively block PRRSV transmission,markedly improve lung health in pigs,and achieve long-term PRRSV negative farming.And it was an effective equipment for carrying out PRRSV and PCV2 immune pigsty breeding.

Bacterial infections pose a significant challenge to the health of humans and animals globally.Pathogenic bacteria carry multiple virulence genes that play various unique roles during infection,enabling them to survive and reproduce in the hostile environment of the host.With the increasing prevalence of antibiotic resistance and the emergence of “superbugs”,bacterial infections have become increasingly difficult to treat.Therefore,it is urgent to discover new bacterial virulence genes using effective screening methods,to elucidate their pathogenic mechanisms,and provide new vaccine and drug targets.This article systematically reviews and categorizes relevant literature on bacterial virulence genes using literature search websites such as Sci-Hub,ScienceDirect,Google Scholar,Web of Science,PubMed,and China National Knowledge Infrastructure (CNKI).It focuses on screening methods for bacterial virulence genes and their applications,particularly in the gene mutation,in vivo expression techniques,omics,gene editing and transposon bioinfoemation,aiming to provide insights and a theoretical foundation for the discovery of new bacterial virulence genes.

【Objective】 The aim of this experiment was to prepare monoclonal antibody against the structural domain of 208-222 amino acid (208-222aa) in VP7 protein of group A Porcine rotavirus (PoRV),and investigate its immunogenicity,in order to provide a reference for the establishment of a detection method for group A PoRV. 【Method】 AlphaFold3 software was used to predict the structure of the VP7 trimeric proteins of the four major popular G genotypes (G3,G4,G5 and G9),and the characteristic antigenic epitopes in the 208-222aa region of the VP7 proteins were compared and fitted.The truncated VP7 protein was designed and fused with GST and MBP tags,respectively.The recombinant proteins GST-VP7 and MBP-VP7 were expressed using the Escherichia coli expression system,respectively.Purification of recombinant protein by affinity chromatography.The purified MBP-VP7 recombinant protein was used to immunize mice,and hybridoma cell lines were prepared and screened using synthetic 208-222aa peptide to obtain cell lines that specifically secreted antibodies. 【Result】 Structural analysis of the 208-222aa region showed that this structural domain had high conformational conservation in different genotypes of PoRV VP7 proteins.Through the prokaryotic expression system,highly pure soluble expressed recombinant proteins GST-VP7 and MBP-VP7 were successfully obtained.Through the screening of the 208-222aa peptide,two hybridoma cell lines capable of secreting specific monoclonal antibodies against this domain were obtained:10F11G and 5F6B.Indirect immunofluorescence assay showed that the antibodies 10F11G and 5F6B could effectively recognize natural PoRV particles. 【Conclusion】 This study successfully obtained two hybridoma cell lines that could stably secrete monoclonal antibodies specific to the 208-222aa domain of VP7 protein of PoRV.This results provided basic materials for the preparation of group A PoRV related antibodies and the development of detection methods.

【Objective】 The aim of this study was to explore the haplotype diversity (Hd),genetic differentiation and population expansion of Fasciola hepatica (F.hepatica) collected from different regions in Hunan province,and to illuminate their phylogenetic relationships. 【Method】 Total 36 F.hepatica isolated from six regions including Zhangjiajie,Huaihua,Shaoyang,Loudi,Yongzhou,and Xiangxi in Hunan province were used as samples,and the cox1 and ITS-1 genes of F.hepatica were amplified by PCR,and then sequenced.The Hd of F.hepatica from six regions in Hunan was analyzed with the ClustalX,Dnasp 5.0,and Network 4.6 software.The genetic differentiation and population expansion of F.hepatica were explored with the software Dnasp 5.0 and Arlequin version 3.0,and the phylogenetic relationships was studied using ClustalX and PhyML 3.0. 【Result】 The cox1 gene sequence of F.hepatica showed a base AT preference,with 36 base mutation sites and 22 haplotypes (A1-A22，Hd＝0.9571),the genetic differentiation coefficients (Fst) of cox1 gene were 0.013 to 0.278,its gene flow (Nm) were -1.895 to 18.981, Tajima’s D values were -0.996 to 0.564,and the Fu’s Fs values were -2.452 to 0.391.The ITS-1 gene sequence of F.hepatica showed a base GC preference,with 17 base mutation sites and 8 haplotypes (B1-B8，Hd＝0.7762).The Fst of ITS-1 gene were -0.174 to 0.564,and the Nm were -5.932 to 7.103,Tajima’s D values were -1.337 to 1.389,and the Fu’s Fs values were -1.072 to 2.139.Moreover,36 F.hepatica collected from different regions in Hunan province together clustered into a branch of the phylogenetic tree. 【Conclusion】 This study revealed that there was a certain level of haplotype diversity of F.hepatica in some areas of Hunan province,and there had been varying degrees of genetic differentiation.All of them had experienced multiple population expansion events,but they still had close genetic relationships.

【Objective】 The purpose of this experiment was to construct a recombinant LigiLactobacillus saerimneri (L.sae) expressing an immunogen fused with chicken DEC-205 binding peptide,and investigate its effects on peripheral blood monocyte-derived dendritic cells (PB-MoDCs) in chickens. 【Method】 The lactic acid bacteria expression plasmid p612 containing the HCE constitutive promoter,T7 g10 enhancer,ssUSP signal peptide,and the cell wall anchoring protein LPXTDG was constructed.The Flag tag and the chicken DEC-205 short peptide (DL (binding peptide)/PS (control peptide)) column fusion was inserted into the chicken Infectious bursal disease virus (IBDV) VP2 gene sequence,and the recombinant plasmids p612-VP2-PS and p612-VP2-DL were constructed.After the recombinant plasmid was identified by double enzyme digestion,they were electroporated into the LigiLactobacillus saerimneri M11 to construct recombinant Lactobacillus plasmids p612-VP2-PS/M11 and p612-VP2-DL/M11.The expression and distribution of proteins were identified by Western blotting and immunoelectron microscopy.The phagocytic effect of immature PB-MoDC cells on the recombinant Lactobacillus was observed through fluorescence microscopy.After the recombinant bacteria acted on PB-MoDC cells for 4 h,the transcriptional levels of cell surface markers (MHC-Ⅱ,CD40,CD80,CD83 and DEC-205) and inflammatory factors (IFN-γ,IL-1β,IL-12,IL-6,TNF-α and CXCLi1) were detected using Real-time quantitative PCR. 【Result】 The experiment successfully obtained LigiLactobacillus saerimneri M11 carrying the empty vector p612,the recombinant plasmid p612-VP2-DL (expressing VP2 and binding peptide) and p612-VP2-PS (expressing VP2 and control peptide).Western blotting results showed that the target protein could be detected in the bacterial precipitates of both recombinant Lactobacillus p612-VP2-PS/M11 and p612-VP2-DL/M11.Immunoelectron microscopy observations revealed that the recombinant protein was mainly distributed in the cytoplasm and the inner wall，and the recombinant bacteria could be phagocytosed by immature PB-MoDC cells.Compared with p612-VP2-PS/M11 group,the transcriptional levels of MHC-Ⅱ,CD80, CD40,CD83,IL-1β,IL-12,IFN-γ and CXCLi1 genes in PB-MoDC cells of p612-VP2-DL/M11 group were significantly or extremely significantly upregulated (P＜0.05 or P＜0.01).There were no significant difference in the transcriptional levels of DEC-205,IL-6 and TNF-α genes in PB-MoDC cells between the two groups (P＞0.05). 【Conclusion】 This study successfully constructed a recombinant chicken intestinal Lactobacillus that expresses immunogenic fusion peptides of chicken DEC-205 on its surface.This strain could effectively stimulate the maturation of PB-MoDC cells.This experimental results provided a theoretical basis for developing a new targeted delivery strategy for livestock and poultry vaccines.

【Objective】 To provide experimental basis for its pharmacological research and clinical medication,the regulatory effect of Shenjiang Zhili mixture (SJZL) on mouse immune function in vivo was investigated. 【Method】 Fourty-eight BALB/c mice were randomly divided into low- (5 g/kg),middle- (10 g/kg) and high-dose group (20 g/kg) of SJZL and blank control group,with 12 mice in each group.Mice in each drug group were given intragastric administration once a day.And mice in blank control group were regularly administered with equivalent saline every day for 6 days.After 3 and 6 days of administration,6 mice were randomly selected from each group.Spleen index and thymus index were calculated after weighing.The splenic cell suspension was prepared and the lymphocyte proliferation rate was determined by the CCK-8 kit.Mouse peritoneal macrophage suspension was prepared,and the phagocytic ability of mouse peritoneal macrophages was detected by neutral red method.Blood was collected from the mouse eyeball,and the levels of interleukin-2 (IL-2),IL-6,tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in serum were detected by ELISA Kit. 【Result】 The clinical index including mental state,feed intake,water intake,stool and urine of mice in each group were normal.Compared with blank control group,there were no significant differences in weight,spleen index and thymus index in each treatment group (P＞0.05),the proliferation rate of T cells in SJZL high-dose group were extremely significantly increased (P＜0.01),the proliferation rate of B cells in SJZL high-dose group was significantly or extremely significantly increased (P＜0.05 or P＜0.01),the phagocytosis rate and metabolism of mouse peritoneal macrophages in high-dose group were significantly or extremely significantly increased (P＜0.05 or P＜0.01).After 6 days of administration,the serum levels of IL-2,IL-6 and IFN-γ in high-dose group were significantly or extremely significantly higher than those in blank control group (P＜0.05 or P＜0.01).In addition,the proliferation rate of T cells and B cells,the phagocytosis and metabolism of peritoneal macrophages,and the levels of IL-2,IL-6 and IFN-γ were increased with increasing days of treatment. 【Conclusion】 Under the conditions of this experiment,10 and 20 g/kg of SJZL could promote the proliferation of T cells and B cells,enhance the phagocytosis and metabolism of macrophages,and increase the secretion of related cytokines in mouse serum,thereby enhancing immunity in mice.

【Objective】 This study aimed to investigate the antioxidant activity of Orobanche coerulescens extract and the protective effects of its total glycosides and polysaccharides on cisplatin-induced oxidative damage in the rat reproductive system. 【Method】 The water extraction process of Orobanche coerulescens was optimized using response surface methodology. In vitro and in vivo antioxidant assays were performed to evaluate the antioxidant activity of the extract.Furthermore,total glycosides and polysaccharides isolated from the water extract were assessed for their protective effects against cisplatin-induced reproductive system injury in rats. 【Result】 Considering production feasibility and extraction cost,the optimal extraction conditions were determined to be:Extraction time of 90 min,extraction temperature of 100 ℃,and a material-liquid ratio of 1∶30.The in vitro antioxidant results demonstrated that Orobanche coerulescens water extract exhibited significant scavenging activity against ABTS,DPPH,and hydroxyl radicals,with antioxidant activity increasing in a dose-dependent manner.In vivo experiments showed that rats in the low-dose polysaccharide group had significantly lower serum reactive oxygen species (ROS),malondialdehyde (MDA),and protein carbonyl (PCO) contents compared to model control group (P＜0.05).Serum superoxide dismutase (SOD) activity was significantly higher in low-,medium-,and high-dose total glycoside groups and low-dose polysaccharide group,while serum reduced glutathione (GSH) contents were significantly increased in low- and medium-dose total glycoside groups and medium-dose polysaccharide group (P＜0.05).Both total glycosides and polysaccharides at all doses significantly inhibited cisplatin-induced increases in serum follicle-stimulating hormone (FSH) in female rats,with the most pronounced effects observed in low- and medium-dose total glycoside groups and low-dose polysaccharide group (P＜0.05).Serum estradiol (E₂) levels were increased in all total glycoside and polysaccharide groups,and testosterone (T) levels were significantly elevated in low-dose total glycoside group (P＜0.05).Low-dose total glycosides and polysaccharides significantly reduced testicular index,prevented a decrease in ovarian index (P＜0.05),preserved ovarian follicle morphology,and inhibited the reduction of granulosa cells and follicular fluid.High doses of total glycosides and polysaccharides significantly decreased epididymal index (P＜0.05),improved spermatogonia and sperm counts,and preserved the morphology of testicular supporting cells. 【Conclusion】 Orobanche coerulescens extract exerted significant protective effects against cisplatin-induced reproductive system damage in rats through its potent antioxidant activity and hormonal regulation.This study provided experimental evidence and theoretical support for the clinical application of Orobanche coerulescens extract.

【Objective】 This experiment was conducted to investigate the antimicrobial resistance and virulence of clinically isolated Escherichia coli (E.coli),provided insights for the prevention and treatment of avian colibacillosis. 【Method】 In this study,a chicken-derived clinical isolate was purified and identified by morphological observation and molecular biology methods.Its antimicrobial susceptibility was determined by drug sensitivity test,and the whole-genome was sequenced using the Illumina NovaSeq platform. 【Result】 The isolated strain formed pink,round and smooth colonies on MacConkey agar,and microscopic examination revealed Gram-negative bacilli.16S rDNA sequencing showed over 98% similarity with E.coli (accession No.:NR_024570.1) in GenBank database.PCR amplification using mcr-1 gene-specific primers revealed that the isolated E.coli carried the polymyxin resistance gene mcr-1.Antimicrobial susceptibility test demonstrated that the isolate was resistant to twelve classes of antimicrobials,including penicillins,cephalosporins,aminoglycosides and polypeptides,etc.,exhibiting multidrug resistance.The complete genome size was 5 057 884 bp.Functional annotation analysis based on KEGG,COG,GO and CAZy databases demonstrated that the strain possessed complete amino acid transport systems,cell proliferation and differentiation mechanisms,and metabolic functions.Comparative analysis with CARD and VFDB databases identified virulence genes encoding effector secretion systems,flagella and brain endothelial invasins.Moreover,the strain carried multiple antimicrobial resistance genes against aminoglycosides,sulfonamides,quinolones,peptides,tetracyclines and β-lactams,contributing to its high virulence and multidrug resistance. 【Conclusion】 In this study,a chicken-derived Escherichia coli isolate carrying the mcr-1 gene was obtained,exhibiting multidrug resistance and high virulence.Its genomic structure and environmental adaptability collectively contributed to its strong pathogenicity.The findings provided valuable data for the prevention and treatment of avian colibacillosis.

【Objective】 Lactic acid bacteria (LAB) are a group of beneficial microorganisms for humans,animals and plants,which are widely applied in food industry,pharmaceutical health,and agricultural fields.Due to the significant differences in living environments between wild animals and livestock/poultry/humans,their intestinal microbiota exhibited distinct diversity.This study aimed to investigate the presence,biological characteristics,and antibacterial activity of LAB in fecal samples of different wild animals,with the goal of identifying candidate strains for microbial preparations. 【Method】 A total of 570 fecal samples were collected from various animals in a wildlife park in Jiangsu province.The samples were first subjected to primary screening using MRS selective medium.Isolates were preliminarily identified as LAB through colony morphology observation,Gram staining microscopy,and biochemical tests.Non-hemolytic isolates were further screened,and their species/genus were confirmed by 16S rRNA gene sequence alignment.A series of in vitro tests were conducted,including acid tolerance,bile salt tolerance,antibacterial activity,drug sensitivity,auto-aggregation ability,hydrophobicity,and animal safety evaluation. 【Result】 Forty suspected LAB strains were isolated,among which three exhibited hemolytic activity.The remaining thirty-seven strains were identified by 16S rRNA gene analysis as belonging to eleven species,including Weissella confusa,Lactobacillus reuteri and Lactobacillus salivarius,etc.After screening through a series of in vitro tests,it was found that the isolate Lactobacillus fermentum ZX2 exhibited outstanding performance.Compared with other isolates,it had high acid tolerance,being capable of growing directly in MRS medium at pH 3.0,and could tolerate 0.3% bile salt.It showed a certain degree of in vitro antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa,but no antibacterial activity against Clostridium perfringens type D-G07 and super drug-resistant Escherichia coli DJ2.Its auto-aggregation rate could reach 16% within 5 h,and its hydrophobicity could exceed 95%.Additionally,it showed certain sensitivity to chloramphenicol,tetracycline,penicillin,ampicillin,cefazolin and ofloxacin,and was non-pathogenic to mice. 【Conclusion】 In this study,thirty-seven strains of lactic acid bacteria were isolated from the feces of wild animals.Among them,Lactobacillus fermentum ZX2 exhibited excellent probiotic properties and intestinal colonization ability,could be used as a potential probiotic strain for the development of microecological preparations.

【Objective】 This study aimed to identify the cause of a respiratory disease outbreak in pigs at a breeding enterprise in Jingzhou city,Hubei province,and analyze the biological characteristics of the pathogenic bacteria,thereby providing a theoretical foundation for the prevention and control of swine diseases. 【Method】 PCR was used to screen common pathogens of swine respiratory diseases in the clinical samples,such as Streptococcus suis,Pasteurella multocida,and Porcine reproductive and respiratory syndrome virus (PRRSV).Bacterial isolation was performed on nasopharyngeal swabs from diseased pigs.The isolated strain was identified for serotype and molecular typing through morphological characteristic identification,16S rRNA gene phylogenetic analysis,and multilocus sequence typing (MLST).The pathogenicity and drug resistance of the isolated strain were analyzed by virulence gene detection,artificial mouse infection test,biofilm formation ability determination,drug resistance gene detection,as well as antimicrobial and traditional Chinese medicine susceptibility tests. 【Result】 The pathogen screening results showed that the diseased pigs were positive for Streptococcus suis,while other pathogens such as Pasteurella multocida and PRRSV were negative.One dominant bacterial strain was successfully isolated from the nasopharyngeal swabs of the diseased pigs.Morphological identification indicated it was a Gram-positive Streptococcus,and 16S rRNA gene phylogenetic analysis revealed that the isolated strain clustered with Streptococcus suis,thus identifying it as Streptococcus suis and named JZXW0103.Serotype identification and MLST analysis confirmed that the isolated strain JZXW0103 was ST25 Streptococcus suis serotype 2.The isolated strain carried virulence genes including mrp,sly,orf2,gapdh and sao,exhibited β-hemolytic activity,and could cause hemorrhage and inflammation in organs such as liver,lung and brain of infected mice.The isolated strain JZXW0103 had weak biofilm-forming ability,carried 9 drug-resistant genes including bla_TEM, aadA1,aadA2,etc.,was resistant to 10 antimicrobial agents such as ampicillin,ceftazidime and oxacillin,and was sensitive to 11 antimicrobial agents including penicillin,cefotaxime,cefoperazone,etc.,as well as the traditional Chinese medicine Scorpio. 【Conclusion】 In this study,one strain of Streptococcus suis serotype 2 belonging to ST25 was successfully isolated,which had certain pathogenicity and multiple drug resistance,providing a reference for the prevention and control of Streptococcus suis infection.

【Objective】 Based on network pharmacology and molecular docking techniques,this study explored the therapeutic targets and pathway regulatory mechanisms of pectin and its metabolite short-chain fatty acids (SCFAs) in regulating goat endometriosis. 【Method】 Potential targets of pectin and SCFAs were predicted using the traditional Chinese medicine systems pharmacology database (TCMSP),PharmMapper,SwissTargetPrediction,and SEA databases.Disease-related targets for goat endometriosis were obtained from the GeneCard,DisGeNET,TTD,DrugBank and OMIM databases.The intersection targets of pectin,SCFAs and goat endometriosis were screened using the Venny 2.1 tool,and a protein-protein interaction (PPI) network was constructed to identify core targets.GO functional and KEGG pathway enrichment analysis was performed using DAVID database.Molecular docking simulations between pectin,SCFAs and core targets were conducted using Autodock Tools 1.5.7,to validate their binding affinity. 【Result】 A total of 501 pectin targets,490 SCFA targets,and 3 641 disease targets were screened,with 26 intersection targets identified.PPI network analysis revealed that androgen receptor (AR),cyclin-dependent kinase 1 (CDK1),signal transducer and activator of transcription 3 (STAT3),epidermal growth factor receptor (EGFR),heat shock protein 90 alpha family class A member 1 (HSP90AA1),peroxisome proliferator-activated receptor gamma (PPARγ),fibroblast growth factor 2 (FGF2),and peroxisome proliferator-activated receptor alpha (PPARα) were the core targets of the drug-disease interaction.GO functional analysis (P＜0.05) showed enrichment in 10 biological processes,1 cellular component,and 3 molecular functions.KEGG pathway enrichment analysis indicated that pectin and its metabolites primarily regulated goat endometriosis by influencing key pathways such as EGFR tyrosine kinase inhibitor resistance,PI3K-Akt signaling pathway,PPAR signaling pathway,and progesterone-mediated oocyte maturation.Molecular docking results demonstrated that the binding energies between pectin,SCFAs and core targets (AR and PPARγ) were all less than －20 kJ/mol. 【Conclusion】 The predicted results of this study showed that,pectin and its metabolite SCFAs alleviated goat endometriosis by targeting AR,CDK1,STAT3,EGFR,HSP90AA1,PPARγ,FGF2,and PPARα,and regulating key signaling pathways such as EGFR tyrosine kinase inhibitor resistance and PI3K-Akt,thereby inhibiting cell proliferation and promoting apoptosis.This study provided a theoretical foundation for the application of pectin as a dietary fiber in goat reproduction.

【Objective】 This study aimed to clone and perform biological analysis of mannosidase alpha class 2A member 1 (MAN2A1) gene in Sushan pigs,as well as reveal the regulation of MAN2A1 gene overexpression on the replication of Swine influenza virus (SIV). 【Method】 The complete CDS region sequence of MAN2A1 gene was amplified using porcine lung cDNA as a template and ligated into the pMD19-T vector with T4 DNA ligase.The similarities of the sequence with other species and different breeds of pigs were compared and phylogenetic trees were constructed.Bioinformatics software was used to predict the MAN2A1 protein sequence of Sushan pigs.Real-time quantitative PCR was used to detect the tissue expression profile of MAN2A1 gene in Sushan pigs,as well as the expression levels of SIV M and NP genes before and after overexpression of MAN2A1 gene.AutoDock software was used to predict molecular docking between MAN2A1 protein and SIV M and NP proteins. 【Result】 The results showed that the CDS region sequence of porcine MAN2A1 gene was successfully cloned,which was 3 435 bp in length.Compared with MAN2A1 gene sequence of pig in NCBI database(accession No.：XM_003123823.6),the CDS region of MAN2A1 gene in Sushan pigs contained 6 single nucleotide polymorphism sites (SNPs),including 2 missense mutations and 4 synonymous mutations.Phylogenetic trees of MAN2A1 genes in different species showed that MAN2A1 gene in pigs had the closest relationship with cattle and sheep,followed by primates,and the farthest relationship with poultry and zebrafish.Phylogenetic trees of MAN2A1 genes in different pig breeds showed that, MAN2A1 gene in Sushan pigs had the closest genetic relationship with Chinese Wuzhishan pig and the farthest genetic relationship with Duroc.Bioinformatics analysis results showed that MAN2A1 protein in Sushan pigs contained 1 144 amino acids,with a molecular weight of 131.42 ku and a theoretical isoelectric point of 7.85.The proportions of alpha helix,beta sheet,and random coil in secondary structure were 33.65%,15.73% and 50.61%,respectively.The GMQE score for the quality assessment of tertiary structure global model was 0.9,indicating good quality.MAN2A1 protein in Sushan pigs contained three domains (amino acids 168-498,289-503,and 649-1 140).MAN2A1 protein was mainly located in the Golgi apparatus within the cytoplasm.The results of protein interaction prediction revealed that MAN2A1 protein might interact with proteins such as MAN1A1,FUT8 and COPB2.The results of tissue expression profiling showed that the expression levels of MAN2A1 gene in liver and spleen of Sushan pigs were significantly higher than those in other tissues (P＜0.05),and the expression levels in heart and muscle were significantly lower than those in other tissues (P＜0.05).This study successfully constructed an overexpression vector for MAN2A1 gene.Compared with control group,the expression levels of MAN2A1 gene in 3D4/21 cells were significantly upregulated in overexpression group (P＜0.05).When infected with H1N1 subtype SIV,overexpression of MAN2A1 gene significantly upregulated the expression of virus M and NP genes (P＜0.05).The molecular docking prediction results showed that there might be interactions between MAN2A1 protein and H1N1 subtype SIV NP,M1 and M2 proteins (iptm+ptm＞0.9),indicating that MAN2A1 protein might directly interact with NP and M proteins to regulate the replication of H1N1 subtype SIV. 【Conclusion】 MAN2A1 gene CDS region sequence of Sushan pigs was successfully cloned in this study,and the overexpression of MAN2A1 gene promoted the replication of H1N1 subtype SIV in 3D4/21 cells.The results of this study provided a theoretical basis for further study of the function and molecular mechanism of MAN2A1 gene.

【Objective】 This experiment was conducted to identify the main pathogenic bacteria of a wild dead Nipponia nippon,and analyze its drug resistance,pathogenicity and biological characteristics,so as to provide reference for the prevention and treatment of related diseases. 【Method】 Sterile samples of the liver,small intestine and pleural effusion were collected from the deceased Nipponia nippon in the Langshan Nature Reserve of Hunan province for bacterial isolation and culture.The strains were identified through colony morphology observation,Gram staining,and the automatic microbial mass spectrometry system.The quantitative suspension method was used to evaluate the disinfection effects of different disinfectants on the isolated bacteria.The drug sensitivity of the isolates was detected using the microbroth dilution method.Whole-genome sequencing was used to analyze the drug resistance genes,virulence factors and multilocus sequence typing (MLST) of the isolates.The phylogenetic tree was constructed using the Parsnp software,and its pathogenicity was tested through animal experiments. 【Result】 Four bacterial strains were isolated from the small intestine of the deceased Nipponia nippon during the experiment.The isolated bacteria formed white colonies on the blood agar plate,and were translucent with a shiny surface on the LB solid medium.The results of Gram staining microscopic examination showed that they were Gram-negative short bacilli.The four strains were identified as Citrobacter freundii by automated microbial mass spectrometry system and were named ZhLB2,ZhX1,ZhX4 and ZhX5,respectively.The results of the disinfectant elimination test showed that all 10 tested disinfectants had good bactericidal effects on the isolates at the recommended concentrations,with the best results for benzyltrimethylammonium and concentrated glutaraldehyde.Drug susceptibility test showed that four isolates were completely resistant to compound sulfamethoxazole,polymyxin B,cefoxitin and meropenem (4/4),and highly resistant to ampicillin,enroxacin and florfenicol (3/4).Four isolates were sensitive to cefotaxime and gentamicin,but only the isolate ZhLB2 was sensitive to tetracycline.The results of whole genome sequencing analysis showed that the four isolates carried a total of 16 drug resistance genes,including bla_CMY and qnrB,and 44 virulence factors.MLST typing showed that isolates ZhX1 and ZhX4 belonged to ST580,and ZhLB2 and ZhX5 were of unknown typing.Phylogenetic tree results showed that isolates ZhLB2 and ZhX5 were in the same branch with Citrobacter freundii from Bangladeshi chicken source and Sri Lankan water source,respectively,ZhX1 and ZhX4 were both in the same branch with Citrobacter freundii from Chinese water source.When mice were infected with 1×10⁹ CFU/mL bacterial solution,the mortality rates of the isolates ZhX1,ZhLB2,ZhX4 and ZhX5 were 70%,40%,20% and 10%, respectively.Pathological examination revealed characteristic pulmonary hemorrhage and intestinal inflammation. 【Conclusion】 In this study,four strains of Citrobacter freundii were isolated from the small intestine of Nipponia nippon.The isolates exhibited multi-drug resistance and were pathogenic to mice.The results of this study provided an important theoretical basis for the improvement of the disease prevention and control system for rare birds,and also offered a reference for guiding rational drug use in clinical practice.

【Objective】 This study used African green monkey embryonic kidney cells (Marc-145) as a model to investigate the protective effects and mechanisms of Atractylodes macrocephala polysaccharides (AP) on Porcine reproductive and respiratory syndrome virus(PRRSV)-induced damage in Marc-145 cells. 【Method】 Marc-145 cells that had grown to 80%-90% fusion were infected with PRRSV at different dilutions (10^-1 to 10^-10),and a blank control group was set up.After continued culture,the number of wells with cytopectic effect (CPE) at each dilution was recorded daily.After the lesion was stable,the half tissue culture infection dose (TCID₅₀) of the virus was calculated by Reed-Muench method.Marc-145 cells were seeded in 96-well cell culture plates at a density of no less than 1×10⁶/mL per well.The cells were infected with 100 TICD₅₀ PRRSV for 0,12,24,48 and 96 h,respectively.Random photographs were taken under a microscope at each time point to observe the cell morphology.Marc-145 cells were divided into control group (Mock) and groups treated with different concentrations of AP (200,400,800,1 600,3 200 and 6 400 μg/mL).The optimal concentration range was determined using the cytotoxicity assay.Marc-145 cells were divided into control,model,treatment (AP-treated (200,400 and 800 μg/mL) and 1 μmol/mL ribavirin) groups.The following three administration methods were used to evaluate the effect of AP on PRRSV infection:①Pre-infection administration:Cells were incubated with AP and ribavirin for 24 h,followed by simultaneous challenged with PRRSV for another 24 h in treatment and model groups.②Co-infection administration:Cells were incubated with AP or ribavirin together with PRRSV for 24 h,while model group was simultaneously challenged with PRRSV alone for 24 h;③Post-infection administration:After simultaneous challenged with PRRSV for 24 h in treatment and model groups,cells in treatment groups were incubated with different concentrations of AP or ribavirin for 24 h,while cells in model group was incubated with 2% FBS DMEM for 24 h.In all treatment groups,cells in control group were incubated with 2% FBS DMEM for 24 h as a control.After the respective treatments,all groups were replaced with 2% FBS DMEM and further cultured for 48 h before detection.The virus challenge titer used in this experiment was 100 TCID₅₀.The 50% tissue culture infective dose (TCID₅₀) assay was used to evaluate the effects of AP on PRRSV titer at different stages of infection,while Real-time quantitative PCR was used to measure the effects on relative expression of PRRSV ORF7 gene.The effects of AP on cellular superoxide dismutase (SOD) activity,malondialdehyde (MDA) and glutathione (GSH) contents were measured using colorimetric assays,and the expression of PRRSV N protein and the Nrf2/HO-1 pathway proteins was analyzed using Western blotting. 【Result】 The titer of PRRSV was calculated to be 10^-6.6 TCID₅₀/mL based on the number of CPE wells at 24 h post-infection using the Reed-Muench method.Compared with 0 h post-PRRSV infection,cells exhibited aggregation and contraction at 24 h post-infection,indicating that 24 h was the optimal infection time for this strain.After incubating Marc-145 cells with AP for 24 h,cell viability was significantly increased in the 200-800 μg/mL AP groups compared with control group (P＜0.05).Therefore,200-800 μg/mL AP was selected as the optimal concentration range for subsequent experiments.Compared with model group,AP significantly reduced the TCID₅₀ value,relative expression of PRRSV ORF7 gene,MDA content,and expression of PRRSV N protein during the endocytosis and adsorption stages (P＜0.05).It also increased SOD activity and GSH content and activated the Nrf2/HO-1 pathway proteins (P＜0.05).However,no significant effects were observed during the intracellular replication stage (P＞0.05). 【Conclusion】 The results of this experiment indicated that,200-800 μg/mL AP protectd cells from oxidative stress damage during the endocytosis and adsorption stages of PRRSV infection by activating the Nrf2/HO-1 pathway.

【Objective】 This experiment was conducted to investigate the prevalence,drug resistance and pathogenicity of Clostridium perfringens in Shandong region,and explore the inhibitory effects of five plant essential oils (oregano oil,cinnamaldehyde,eucalyptus oil,glyceryl laurate,and garlic oil) against this bacterium. 【Method】 One hundred intestinal samples of broilers were collected from different large-scale commercial broiler farms in Shandong region for bacterial isolation.The isolats were identified by biochemical tests,16S rRNA sequencing and PCR amplification of toxin genes.The drug sensitivity detection and pathogenicity tests were conducted on the isolates identified as Clostridium perfringens.The minimum inhibitory concentrations (MIC) of five essential oils against Clostridium perfringens were determined by broth microdilution method.Essential oils with superior antibacterial activity were selected and combined in pairwise formulations using the checkerboard assay,after which the fractional inhibitory concentration (FIC) index of the combined essential oils against Clostridium perfringens was evaluated. 【Result】 The isolated bacteria presented as circular colonies with black center and white turbidity ring around the TSC plate.Gram-staining microscopic examination showed Gram-positive results.A total of 32 isolated bacteria were isolated from 100 samples.Biochemical identification results showed that the isolates were positive for glucose,lactose,maltose,etc.,but negative for mannitol and indole.The results of 16S rRNA gene PCR amplification and sequencing comparison showed that the similarity of the isolates with Clostridium perfringens was greater than 99%.The results of multiplex PCR identification of the toxin genes showed that only a 202 bp target band was amplified.All the isolated strains identified were Clostridium perfringens type A,with an isolation rate of 32%.Antimicrobial susceptibility testing demonstrated high resistance rates to erythromycin,norfloxacin,and ciprofloxacin.The pathogenicity test showed all five isolates from different regions could cause severe necrotizing enteritis in broilers.In vitro antibacterial assays showed that the MIC of garlic oil,cinnamaldehyde and glyceryl laurate against Clostridium perfringens were 250,125 and 125 μg/mL,respectively.The FIC of the compound combinations of garlic oil＋cinnamaldehyde,garlic oil＋glyceryl laurate,and cinnamaldehyde＋glyceryl laurate were 1,2 and 2.5, respectively. 【Conclusion】 The isolation rate of Clostridium perfringens from broilers in Shandong region was relatively high,and type A strains had an absolute advantage.The isolates had strong drug resistance and pathogenicity. Garlic oil,cinnamaldehyde,and glycerol monolaurate demonstrated superior antibacterial effect against Clostridium perfringens.In addition,the combination of garlic oil and cinnamaldehyde showed an additive effect.

【Objective】 This experiment was conducted to evaluate the effect of dopamine hydrochloride on the gastrointestinal motility of White-feathered broilers,and further determine the appropriate dosage of dopamine hydrochloride for inducing a gastrointestinal motility disorder model in broilers. 【Method】 Thirty-six 1-day-old healthy White-feathered broilers were selected for the experiment and randomly divided into the control group and the low-,medium- and high-dose groups of dopamine hydrochloride.Each group was set up with 3 biological replicates.No treatment was given to the broilers in the early stage of each group.At 14 days of age,the broilers in each dose group of dopamine hydrochloride were induced to develop a gastrointestinal motility disorder model by intraperitoneal injection of the corresponding doses of dopamine hydrochloride (3.979,7.958 and 15.916 mg/kg BW).After the experiment,the gastrointestinal tissues of the broilers were collected to measure the gastric emptying rate and small intestinal propulsion rate.The activities of digestive enzymes in the gastrointestinal tract,the contents of gastrointestinal hormones,and the morphology of the intestinal mucosa were also detected. 【Result】 Compared with control group,the gastric emptying rate and intestinal propulsion rate of broilers in each dose group of dopamine hydrochloride were significantly lower (P＜0.05),and this effect was dose-dependent.There were no significant differences in the intestinal mucosa morphology of the broilers in each group.The results of the digestive enzyme activity assay showed that in the medium- and high-dose groups of dopamine hydrochloride,the activities of pancreatic amylase,trypsin and pancrelipase in different intestinal segments of broilers were significantly lower than those in control group (P＜0.05).The measurement of gastrointestinal hormone levels revealed that,compared with control group,the levels of substance P,motilin and dopamine in duodenum and serum of broilers in each dose group of dopamine hydrochloride were significantly decreased (P＜0.05)，and the level of dopamine in gizzard was also significantly decreased (P＜0.05).The 5-hydroxytryptamine content in gizzards of broilers in the medium- and high-dose groups was significantly reduced (P＜0.05).The levels of substance P and motilin in serum and duodenum，and gastric dopamine content of the broilers in each dose group of dopamine hydrochloride decreased with the increase of the dose,and the differences among the groups were significant (P＜0.05). 【Conclusion】 Dopamine hydrochloride had a significant effect on the gastrointestinal motility of White-feathered broilers,and this effect was dose-dependent.Under the conditions of this experiment,the optimal dose for inducing the gastrointestinal motility disorder in broilers by using dopamine hydrochloride was 7.958 mg/kg BW.