【Objective】 The aim of this study was to clone the CDS sequence of B-cell translocation gene 2(BTG2)gene in buffalo,analyze the biological characteristics of its encoded protein,construct the overexpression vector and transfect buffalo granulosa cells,and explore the expression patterns of BTG2 gene in different tissues of buffalo,laying theoretical basis for the subsequent in-depth research on the biological function of BTG2 gene in buffalo. 【Method】 The heart,liver,spleen,lung,kidney,small intestine,rumen,uterine horn,mammary gland and ovarian tissues of buffalo were collected.Total RNA was extracted and cDNA was obtained by reverse transcription.The CDS region of BTG2 gene was cloned using buffalo ovary cDNA as template and sequenced.The sequence of BTG2 gene of buffalo and different species were compared and phylogenetic tree was constructed.The physicochemical properties,signal peptides,transmembrane structure,etc.of BTG2 protein were analyzed and predicted by online software.The OE-BTG2 overexpression vector was constructed to transfect buffalo granulosa cells,and the expression of BTG2 gene was detected after transfection.The expression of BTG2 gene in different tissues of buffalo was detected by Real-time quantitative PCR. 【Result】 The CDS region of BTG2 gene in buffalo was 453 bp in length,encoding 150 amino acids.The similarity between buffalo and Bos mutus,Bos taurus,Capra hircus,and Ovis aries exceeded 98%,with the highest similarity (99.8%) observed between buffalo and Bos mutus and Bos taurus.The phylogenetic tree outcomes demonstrated that buffalo had the closest genetic relationship with Bos mutus and Bos taurus,and had the farthest genetic relationship with Mus musculus.BTG2 protein was a hydrophilic protein,which had a btg1 domain at positions 1-108 of the amino acid sequence.A total of 15 phosphorylation sites and 1 O-glycoylation site were observed.It had no signal peptide or transmembrane structure,mainly distributed in the nucleus.BTG2 protein mainly interacted with CNOT7,CNOT8,CNOT11,CNOT9,CNOT1,CNOT10,ATF3,ANKRD49,HES6 and PRMT1 proteins.The secondary structure of BTG2 protein in buffalo was dominated by alpha helix,extended chain and random coils,the prediction result of the tertiary structure was consistent with it.The OE-BTG2 overexpression vector was successfully constructed and transfected into buffalo granulosa cells.Compared with control group (pcDNA3.1(+)),the expression of BTG2 gene in overexpression group (OE-BTG2) was extremely significantly increased (P＜0.01).Real-time quantitative PCR results revealed that BTG2 gene was expressed in heart,liver,spleen,lung,kidney,small intestine,rumen,uterine horn,mammary gland and ovarian tissues of buffalo,with a higher expression in liver than that in other tissues (P＜0.05). 【Conclusion】 BTG2 gene exhibited high conservation among different species,and BTG2 gene was widely expressed in different tissues of buffalo,with the highest expression in liver.The results provided a theoretical basis for further study on the function of BTG2 gene in buffalo.

Spermatogenesis is a critical process in mammalian reproduction,in which spermatogonial stem cells (SSCs) play an indispensable role.As the central cells in spermatogenesis,Sertoli cells (SCs) not only provide physical support and a stable microenvironment for SSCs but also intricately regulate their proliferation,differentiation,apoptosis,and immune privilege by secreting various cytokines and signaling molecules.This complex interplay significantly influences the self-renewal and differentiation of SSCs.This review focuses on the mechanisms by which SCs regulate SSCs development,aiming to offer new perspectives and insights for SSCs research and to advance its applications in reproductive biology and clinical practice.

【Objective】 This study aimed to explore and compare the effects of two different immune stress models on the growth performance and intestinal immune status of broiler. 【Method】 A total of 270 one-day-old male Ross 308 chicks were randomly divided into 3 groups,namely the control (Con) group,the coccidia and Clostridium perfringens (CP) infection group,and the lipopolysaccharide (LPS) challenge group,with 6 replicates in each group and 15 chicks in each replicate.On the 11th and 13th days of the experiment,broilers in LPS group were challenged by intraperitoneal injection of LPS.On the 12th day and from the 16th to 20th days of the experiment,broilers in CP group were respectively gavaged with coccidial vaccine and Clostridium perfringens to induce a necrotic enteritis model.The experiment was divided into two stages:the early stage (1-21 days of age) and the later stage (22-42 days of age).At the end of each stage,the body weight was measured and the growth performance was calculated.After being stunned and sacrificed,the organ indices were measured.Segments of the ileum were taken to analyze the intestinal tissue morphology and gene expression.Flow cytometry was used to determine the proportions of immune cells in the ileal lamina propria and cecal tonsils,and the sIgA content in the ileal mucosa was measured. 【Result】 ① From 22 to 42 days of age and from 1 to 42 days of age,the feed to gain ratios of broilers in CP and LPS groups were both significantly higher than those in control group (P＜0.05).② At 21 days of age,the liver index of broilers in CP group was significantly higher than that in Con group,and the spleen index was significantly higher than that in the other groups (P＜0.05).③ At 21 days of age,compared with Con group,the ileal crypt depth of broilers in CP and LPS groups were increased significantly,and the ratio of villus height to crypt depth were decreased significantly (P<0.05).Meanwhile,the ratio of villus to crypt in LPS group was significantly lower than that of CP group (P＜0.05).④ At 21 days of age,the number of CD4⁺ T cells in the ileal lamina propria of broilers in LPS group was decreased significantly,while the number of Treg cells in CP group was increased significantly (P＜0.05).The proportions of mononuclear macrophages in both models were decreased significantly (P＜0.05).In addition,the proportion of Treg cells in the cecal tonsils of LPS group was increased significantly,while the proportion of dendritic cells decreased significantly (P＜0.05).⑤ Compared with Con group,the sIgA content in the ileal mucosa of broilers in LPS group at 21 days of age was increased significantly (P＜0.05).⑥ Compared with Con group,the expression levels of MUC-2,ZO-1,Claudin-1,and Occludin genes in the ileum of broilers in LPS group at 21 days of age decreased significantly.Among them,the expression level of MUC-2 gene in LPS group was significantly lower than that in CP group (P＜0.05). 【Conclusion】 The LPS challenge suppressed intestinal innate immune cell maturation while stimulating sIgA secretion,whereas the coccidia and CP infection induced splanchnic hypertrophy and increased intestinal Treg cells.These models differentially impaired broiler growth performance and intestinal immunity through distinct mechanisms,establishing a theoretical framework for stress mitigation.

【Objective】 This study aimed to investigate the effects of dietary supplementation with vitamin D₃ (VD₃) and 25-hydroxyvitamin D₃ (25-OH-D₃) on the tibial quality and intestinal microbiota composition of breeding ducks in late laying period. 【Method】 A total of 60 laying Pekin ducks (64 weeks of age) were randomly allotted to 3 treatments with 10 replicate pens of 2 duck each.The ducks were fed a basal diet with no vitamin D supplementation or supplemented with VD₃ or 25-OH-D₃ at 1 000 IU/kg of feed for 15 weeks.During the experiment,the average body weight and average daily feed intake were measured.After the experiment,the slaughter performance (pectoral muscle rate and cecal rate),plasma biochemical indicators and tibial quality of breeding ducks were determined.Additionally,intestinal morphology of the duodenum,jejunum,and ileum was evaluated,and cecal contents were collected for 16S rRNA sequencing to analyze microbial diversity. 【Result】 Compared with control group,①Dietary supplementation of VD₃ or 25-OH-D₃ had no significant effects on body weight,average daily feed intake,pectoral muscle rate,and cecal rate (P＞0.05).②Dietary supplementation of VD₃ or 25-OH-D₃ significantly increased plasma albumin content and the ratio of albumin to globulin (P＜0.05).③Dietary supplementation of 25-OH-D₃ significantly increased the tibial compressive strength (P＜0.05),dietary supplementation of VD₃ had no significant effect on the tibial compressive strength (P＞0.05).④Supplemented with either VD₃ or 25-OH-D₃ significantly increased the villus height of duodenum and jejunum and the ratio of villus height to crypt depth of duodenum (P＜0.05),reduced the muscle thickness of duodenum (P＜0.05).Compared with VD₃ group,dietary supplementation of 25-OH-D₃ significantly increased villus height and the ratio of villus height to crypt depth of duodenum (P＜0.05).⑤Dietary supplementation with VD₃ or 25-OH-D₃ significantly increased the relative abundance of norank_f_norank_o_Clostridia_vadinBB60_gorup and Rikenellaceae_RC9_gut_group (P＜0.05), significantly reduced the relative abundance of Roseburia,Erysielatoclostrium,Collinsella and Clostridium_sensu_stricto_1 (P＜0.05).The relative abundance of Desulfovibrio and Clostridium_sensu_stricto_1 in 25-OH-D₃ group was significantly higher than that in VD₃ group (P＜0.05). 【Conclusion】 Both VD₃ and 25-OH-D₃ could improve the tibial quality and intestinal morphology of breeding ducks in late laying period.Moreover,the effect of 25-OH-D₃ in improving tibial compressive strength,duodenal villus height and the abundance of flora related to energy metabolism in cecum was superior to that of VD₃.

【Objective】 The aim of this experiment was to study the effects of feeding Snowy Mountain chicken breeder hens with different energy and protein nutrient levels of rations on their offspring’s average daily gain (ADG),feed-to-gain ratio (F/G) and body size traits. 【Method】 Breeding hens were randomly divided into 9 groups according to a 3×3 two-factor experimental design,with 3 energy levels (11.09,11.51,11.92 MJ/kg) and 3 protein levels (14.50%,15.50%,16.50%).Breeding eggs were collected and incubated at 35 weeks of age, 2 700 1-day-old chicks were selected after hatching,and were grouped according to the breeders,1 350 males and 1 350 females,housed separately.The experimental period was 91 d.Daily weight gain and feed consumption were counted during the experimental period,and body size traits were measured at the end of the experiment. 【Result】 ① Dietary energy level had a significant effect on ADG and F/G of the male chickens at 50-70 days of age (P＜0.05),and with the increase of energy level,ADG was decreased while F/G was increased (P＜0.05).Dietary protein level had a significant effect on ADG of the male chickens at 1-14 days of age (P＜0.05),it was significantly higher in high protein group than that in medium protein group.Energy and protein levels had an extremely significant interaction on the newborn weight (P＜0.01),and significant interaction on ADG at 50-70 days of age (P＜0.05) of male chickens.② Dietary energy level had significant effects on F/G of female chickens at 71-91 days of age (P＜0.05),it was significantly higher in high energy group than that in medium energy group.Dietary protein level had significant effects on F/G at 71-91 days of age,ADG and body weights at 1-91 days of age,and body weights at 91 days of age of female chickens (P＜0.05),the high protein group exhibited a significantly higher F/G compared to the low protein group during the 71-91 days of age,while the medium protein group demonstrated markedly lower body weight at 91 days of age than both the low and high protein groups.③ Dietary energy and protein levels exhibited no significant effects on body size traits of male chickens (P>0.05),however,pectoral depth in male chickens demonstrated a tendency to increase with elevated dietary energy levels (P＝0.085).④ Dietary energy levels significantly affected breast width in female chickens (P＜0.05),the medium energy group exhibited significantly higher values than the low and high energy groups (P＜0.05),tibial circumference demonstrated a quadratic tendency with increasing dietary energy levels (P＝0.053).Dietary protein levels exerted significant effects on tibial circumference,pectoral depth (P＜0.05).Tibial circumference in the low protein group was significantly greater than that in the medium and high protein groups (P＜0.05).Both low and high protein groups exhibited significantly greater pectoral depth compared to the medium protein group,with maximum keel length observed at a protein level of 16.50%. 【Conclusion】 Based on a comprehensive evaluation of offspring growth performance parameters,the recommended dietary energy and protein levels for Snow Mountain breeding hens during the laying period were 11.51 MJ/kg and 16.50%,respectively.

【Objective】 This experiment was conducted to investigate the effects of mixed addition of different levels of fermented cottonseed meal,fermented rapeseed meal and fermented palm kernel meal on growing development and meat quality traits of medium-growing Yellow-feathered chickens aged 1 to 84 days, and to provide reference for the application of mixed fermentation meal in chickens. 【Method】 A total of 480 1-day-old female ephedra chickens were randomly divided into four treatment groups according to the principle of consistent body weight,the control group (without the addition of fermented feed,CON) and the groups with low (F1),medium (F2),and high (F3) levels of fermented feed.There were 6 replicates in each group and 20 chickens in each replicate.The experiment was divided into three stages,1 to 30 days of age,31 to 59 days of age and 60 to 84 days of age.The experiment period was 84 days.The growth performance was calculated at the end of each stage.Two chickens in each replicate were selected with body weight closest to the average,then whole blood was collected and plasma was separated for biochmical indices analysis.The immune organs were dissected and weighed to calculat immune organ indexes.Carcasses were segmented to measure carcass traits,and breast muscles were dissected to evaluate meat quality traits. 【Result】 Compared with CON group,①The feed to gain ratio of chickens at 1 to 30 days and 60 to 84 days of age in F3 group were significantly increased (P＜0.05),meanwhile,European efficiency index at 1 to 30 days of age,body weight at 59 and 84 days of age,average daily feed intake at 31 to 59 days of age,average daily gain and European efficiency index at 60 to 84 days of age,and average daily gain and average daily feed intake at 1 to 84 days of age in F3 group were significantly decreased (P＜0.05).Average daily gain and European efficiency index at 31 to 59 days of age and European efficiency index at 1 to 84 days of age in F2 and F3 groups were significantly decreased (P<0.05),feed to gain ratio of chickens at 31 to 59 days of age and 1 to 84 days of age in F2 and F3 groups were significantly increased (P＜0.05).② The plasma immunoglobulin A (IgA) and interleukin-10 (IL-10) contents of chickens at 30 days of age in F3 group were significantly decreased (P＜0.05),meanwhile,the plasma triglyceride (TG) content of chickens in F1,F2 and F3 groups at 59 days of age was significantly decreased (P＜0.05).③ The thymus index of chickens at 30 days of age in F2 group was significantly increased (P＜0.05).The carcasses brightness values of abdominal at 59 days of age and shoulder at 84 days of age in F3 group were significantly increased (P＜0.05).The breast muscle rate at 59 days of age,the evisceration rate and breast muscle rate,and abdominal fat rate at 84 days of age in F2 and F3 groups were significantly increased (P＜0.05).The a^*_{24 h} of breast muscle in F2 and F3 groups was significantly decreased (P＜0.05),and the b^*_{24 h} of breast muscle in F3 group was significantly decreased (P＜0.05). 【Conclusion】 Supplementation of low levels of fermented cottonseed meal,fermented rapeseed meal and fermented palm kernel meal at the same time in the diet of medium-growing Yellow-feathered chickens aged 1 to 84 days could replace the soybean meal without affecting growth performance,carcass traits and meat quality traits.The recommended addition levels of total fermented mixed meal in the three stages were not more than 6%,8% and 10%, respectively.

【Objective】 The purpose of this experiment was to investigate the effects of peanut vine,corn,and urea compound extruded feed on the growth performance,rumen fermentation parameters and microbial composition of fattening Hu sheep,and provide a reference for the application of peanut straw compound extruded feed in fattening Hu sheep. 【Method】 A total of 90 Hu sheep with similar weight and good body condition during the fattening period were randomly divided into 3 groups,with 6 replicates in each group and 5 sheep in each replicate.In the diets of the three groups of Hu sheep,the compound expanded feed was used to replace the concentrate in equal amounts,with replacement levels of 0 (control group),10% (group Ⅰ),and 20% (group Ⅱ),respectively.The adaptation period was 15 days,and the main test period was 60 days.At the beginning and end of this experiment,sheep were weighed,feed intake and residual material samples were recorded every day,and blood and rumen juice were collected at the end of this experiment for the determination of growth performance,serum biochemistry,rumen fermentation parameters and microflora. 【Result】 Compared with control group,①The average daily feed intake,average daily weight gain,and final body weight of Hu sheep in groups Ⅰ and Ⅱ were extremely significantly or significantly increased (P＜0.01 or P＜0.05),and the feed-to-gain ratio was significantly decreased (P＜0.05).②The serum urea nitrogen level of Hu sheep in group Ⅱ was significantly increased (P＜0.05).③The concentrations of acetic acid,propionic acid,total volatile fatty acids,and ammonia nitrogen in rumen fluid of Hu sheep in groups Ⅰ and Ⅱ were extremely significantly increased (P＜0.01),and pH was extremely significantly decreased (P＜0.01).The acetic to propionic acid ratio in rumen fluid of Hu sheep in group Ⅱ was significantly decreased (P＜0.05).④The Ace index of rumen microbiota of Hu sheep in group Ⅱ was significantly increased (P＜0.05).Principal coordinate analysis (PCoA) results indicated there was a clear separation of the rumen microbiota among the different groups.⑤At the phylum level,the relative abundance of Bacteroidetes in rumen fluid of Hu sheep in group Ⅱ was significantly increased (P＜0.05),while the relative abundance of Firmicutes was significantly decreased (P＜0.05).⑥At the genus level,the relative abundance of Ruminococcus in rumen fluid of Hu sheep in group Ⅰ was significantly increased (P＜0.05)，and the relative abundance of Prevotella in group Ⅱ was significantly increased (P＜0.05). 【Conclusion】 The composite extruded feed produced by mixing peanut vine,corn and urea as ingredients to replace concentrates could improve the growth performance,promotes rumen fermentation and increases rumen microbial diversity in fattening Hu sheep.

【Objective】 This study was aimed to provide a scientific basis for the germplasm characteristics of Xupu geese and their hybrid utilization by comparing the liver tissue,intestinal morphology and intestinal flora in different breeds of goslings. 【Method】 A total of 60 healthy goslings each of Xupu geese,Landes geese and hybrid geese (Xupu geese ♂ × Landes geese ♀) without feeding after hatching were selected,half male and half female,with 6 replicates in each breed and 10 per replicate.After slaughter,the basic indicators were measured,liver and intestinal samples were taken to determine the tissue morphology,and intestinal floras were determined. 【Result】 The intestinal lengths of Landes geese and hybrid geese were significantly higher than those of Xupu geese (P＜0.05).The numbers of fat droplets and vacuoles in liver sections of Xupu geese were lower than those of Landes geese and hybrid geese,the crypt depths of duodenum and ileum were significantly higher than those in Landes geese and hybrid geese (P＜0.05),and the villus height/crypt depth ratios of duodenum and ileum were significantly lower than those in Landes geese and hybrid geese (P＜0.05).The ACE index and Chao index of hybrid geese were significantly higher than those of Xupu geese and Landes geese (P＜0.05),and the cecal intestinal flora was richer than that of the other two breeds at the phylum and genus levels. 【Conclusion】 The breed factor had significant effects on liver tissue,intestinal morphology and intestinal flora of goslings,among which the hybrid goslings showed significant hybridization advantages.The results provided a reference basis for germplasm characteristics,intestinal flora regulation and subsequent production of goose fatty liver in Xupu geese.

【Objective】 This study was conducted to explore the effects of dietary bile acids or compound essential oils supplementation in the diets on the performance,egg quality and lipid metabolism of hens during the extended laying period,specifically from 81 to 89 weeks of age,in order to improve lipid metabolism of hens during the extended laying period through nutritional regulation. 【Method】 A total of 180 healthy 80-week-old Jinghong laying hens were randomly divided into 3 groups with 5 replicates of 12 chickens each.Hens in control group were provided basal diets,in the bile acids group were supplemented with 250 mg/kg bile acids in the basal diet,and in the compound essential oils group were supplemented with 90 mg/kg compound essential oils in the basal diet.All hens were fed basal diets for a 1-week adaptation followed by an 8-week experimental period.The egg production was recorded every day,and the feed accounts were settled weekly.At the end of weeks 0,4 and 8,egg quality was determined.At the end of week 8,blood samples were collected from 2 laying hens in each replicate to determine the serum lipid metabolism related indexes.Then,the laying hens were slaughtered to measure the liver lipid metabolism related index,the activity of enzymes related to lipid metabolism in the ileum and the gene expression. 【Result】 Compared with the control group,①Neither supplementation of bile acids or compound essential oils had effect on the laying performance (P>0.05),at the end of week 8,egg yolk color score in the bile acids group were higher than those in the control group (P＜0.05).② Both supplementation of bile acids and compound essential oils decreased the contents of serum triglycerides,cholesterol and low-density lipoprotein cholesterol,and the triglyceride content in the liver (P＜0.05),and increased the high-density lipoprotein cholesterol content in the liver of laying hens (P＜0.05).Adding bile acids also reduced the content of low-density lipoprotein cholesterol in the liver of laying hens (P＜0.05).Both supplementation of bile acids and compound essential oils increased the ileal lipase activity and expression levels of small peptide transfer carrier (PepT-1) (P＜0.05). Expression of atty acid binding protein (FABP-1) was also increased by bile acids supplementation (P＜0.05). 【Conclusion】 Dietary supplementation of bile acids and compound essential oils could regulate intestinal lipid absorption and improve lipid metabolism of hens during the extended laying period.

Mycotoxins (MCTs) are the toxic secondary metabolites produced by fungi such as Aspergillus and Fusarium in moldy grains and feeds,contaminate over 50% of cereal feeds in the intensive farming.The toxins can enter the bodies of livestock through the food chain,causing damage to multiple organs including the liver,kidney,and immune system.Notably,as a key metabolic and detoxifying organ,the liver is the most significantly affected.Representative MCTs such as aflatoxin B₁ (AFB₁),zearalenone (ZEA),and deoxynivalenol (DON) can induce hepatocyte necrosis,steatosis,inflammatory infiltration and other pathological changes,seriously endangering livestock health and reducing farming profitability.Resveratrol (Res),a natural polyphenolic compound derived from peanuts,grapes and other plants,has great potential in mitigating MCTs toxicity due to its remarkable antioxidant,anti-inflammatory and anti-apoptotic properties in recent years.The protective effects are achieved through multi-pathway synergistic mechanisms.For instance,in terms of antioxidant activity,Res activates antioxidant enzymes (SOD,CAT,etc.),and neutralize toxin-induced free radicals to alleviate oxidative stress mainly through the Nrf2/Keap1 signaling pathway.In terms of anti-inflammatory effects,Res suppresses the NF-κB pathway and upregulates SIRT1 expression,thereby reducing pro-inflammatory cytokines (TNF-α and IL-6) and mitigating hepatic inflammation.Additionally,Res exerts anti-apoptotic effect by regulating the Bcl-2/Bax value,inhibiting the Caspase cascades,and promoting mitochondrial autophagy to prevent hepatocyte apoptosis and protect the liver.The author systematically summarizes the protective effects and the molecular mechanisms of Res against MCTs-induced liver damage in livestock,aiming to provide a theoretical basis for its application in alleviating MCTs-induced liver damage in livestock,and offer scientific support for it potential as a novel feed additive.

【Objective】 This study aimed to investigate the effects of Turpiniae folium extract (TFE) on growth performance,immune function,antioxidant capacity,and intestinal health in broilers stimulated with lipopolysaccharides (LPS). 【Method】 A total of 360 one-day-old female broiler chickens were randomly divided into three groups:Control,LPS,and TFE+LPS groups,with 6 replicates of 20 birds each.The trial lasted for 26 days.The chickens in control and LPS groups were fed a basal diet,while in TFE+LPS group were received the same basal diet supplemented with 500 mg/kg TFE.The broilers in LPS and TFE+LPS groups were intraperitoneally injected with 1 mg/kg BW LPS solution at 21,23,and 25 days of age,while the broilers in control group were received an equivalent volume of saline.On day 26 of the trial,growth performance was measured,and two broilers per replicate with body weights closest to the replicate average were selected for blood sampling and slaughter to collect organ tissues and cecal contents.Immune and antioxidant parameters in the serum and liver,jejunum tissue morphology,and cecal microbiota composition were analyzed. 【Result】 Compared to control group,the final body weight,average daily gain (ADG),and average daily feed intake (ADFI) of broilers in LPS group were significantly reduced (P＜0.05).The final body weight,ADG,and ADFI of broilers in TFE+LPS group were significantly higher than those in LPS group (P＜0.05),and they had no significant difference compared with control group (P＞0.05).Compared with control group,LPS stimulation significantly increased the levels of serum soluble CD8 ligand (sCD8),interleukin-6 (IL-6),tumor necrosis factor-α (TNF-α),and malondialdehyde (MDA) in broilers (P＜0.05),while also significantly elevating the hepatic interleukin-1β (IL-1β) content (P＜0.05).Additionally,LPS stimulation significantly reduced serum total antioxidant capacity (T-AOC) and hepatic glutathione peroxidase (GSH-Px) activity compared to control group (P＜0.05).The broilers in TFE+LPS group had significantly lower serum IL-6,TNF-α,MDA,and hepatic IL-1β levels than LPS group (P＜0.05),with no significant difference compared to control group (P＞0.05).LPS stimulation significantly increased the jejunal crypt depth (CD),villus height-to-crypt depth ratio (VH/CD),and the relative abundance of Bacteroidetes and Desulfobacterota in broilers (P＜0.05).The jejunal CD of broilers in TFE+LPS group was significantly higher than that in control group，but significantly lower than that in LPS group (P＜0.05).The Chao1 index,observed species,and relative abundance of Desulfobacterota in the cecum of broilers in TFE+LPS group were significantly higher than those in LPS group (P＜0.05),while the relative abundance of Parabacteroides was significantly lower than that in LPS group (P＜0.05),and there was no significant difference compared to control group (P＞0.05). 【Conclusion】 TFE significantly alleviated LPS-induced declines in immune function and antioxidant capacity, intestinal morphological damage and cecal microbiota disruption in broilers,thereby effectively attenuating the negative effects of LPS on broiler growth performance.

【Objective】 The purpose of this experiment was to investigate the application effects of replacing corn completely with wheat in diet on the production of Yellow-feathered chickens. 【Method】 A total of 1 260 healthy 1-day-old rapidly-growing Yellow-feathered male chickens were selected and randomly divided into 7 treatment groups with consistent body weight，with 6 replicates per group and 30 chickens per replicate.The 7 groups were as follows：corn-soybean meal diet (C) group，wheat completely replacing corn (W) group，wheat low-energy (WL) group，xylanase (WL＋X) group，phytase (W＋P) group，xylanase＋phytase (WL＋X＋P) group，and 4-fold xylanase and phytase (WL＋4X＋4P) group.The experimental period was 63 days.At 57 days of age，2 birds with body weights (BW) close to the average were randomly selected from each replicate and placed into metabolic cages for a metabolic trial to determine energy utilization efficiency.At 63 days of age，broilers were weighed after fasting on a replicate basis，and average feed intake (ADFI),average daily gain (ADG) and ratio of feed to gain (F/G) were determined.Two birds with body weight close to the average were selected from each replicate for blood sampling and slaughtering to measure plasma biochemical indicators,slaughter performance,and meat quality of the breast muscle.And the tibia was separated for measurement of tibial mineral content and bone density. 【Result】 At 1-21 days of age, the ADFI of chickens in group W was significantly greater than that in group C (P＜0.05),and the ADFI of chickens in group WL was significantly greater than that in group WL＋X (P＜0.05).At 1-42 days of age,the F/G of chickens in group C was significantly less than that in group W (P＜0.05),the F/G of chickens in group W was significantly less than that in group WL (P＜0.05),and the F/G of chickens in group WL＋X was significantly less than that in group WL (P＜0.05).At 1-63 days of age,both the ADFI and F/G of chickens in group W were significantly greater than those in group C (P＜0.05),and the latter was significantly less than that in group WL (P＜0.05)；the F/G of chickens in group WL＋X was significantly less than that in group WL (P＜0.05).The breast muscle rate of chickens in group WL＋X was significantly greater than that in group W (P＜0.05),the breast muscle rate of chickens in group WL＋4X＋4P was significantly greater than that in group C (P＜0.05).The energy utilization rate of chickens in group WL＋4X＋4P was significantly greater than that in the other four groups except groups C and WL＋X＋P (P＜0.05),the energy utilization rate of chickens in group C was significantly greater than that in group W and group W＋P (P＜0.05).The drip loss of breast muscle of chickens in group W was significantly less than that in groups WL＋X and WL＋4X＋4P (P＜0.05). 【Conclusion】 The addition of xylanase complex to wheat-based diets of Yellow-feathered chickens was conducive to improving feed utilization rate，saving dietary energy，reducing broiler mortality,while supplementing phytase could save the use of inorganic phosphorus and improve carcass quality.

【Objective】 This experiment was aimed to study the effects of adding squalene (SQ) to the diet on the growth performance,carcass traits,meat quality,antioxidant capacity,and economic benefits of growing-finishing pigs,so as to provide a basis for the popularization and application of SQ in pig production. 【Method】 100 Duroc×Landrace×Yorkshire three-way crossbred pigs with an initial body weight of 32.50 kg±2.50 kg,were randomly allocated into two groups.Each group had 5 replicates,and each replicate consisted of 10 pigs with a half male and half female.Pigs in control group were fed a basal diet,while pigs in SQ group were fed the basal diet supplemented with SQ at a dose of 240 mg/kg.The experimental period lasted for 98 days.After the experiment,pigs were weighed to determine the growth performance,blood was collected to detect serum antioxidant index,after pigs were slaughtered,liver tissues were collected for antioxidant index detection.Meanwhile,carcasses were divided to determine carcass traits,and the longissimus dorsi muscles were collected to determine meat quality and antioxidant index. 【Result】 Compared with control group,the final body weight and average daily gain of pigs in SQ group were significantly increased (P＜0.05),and the feed to gain ratio (F/G) was significantly decreased (P＜0.05).The preslaughter body weight,carcass weight,and carcass straight length of pigs in SQ group were significantly increased (P＜0.05).pH at 45 min,redness value at 45 min (a^*_{45 min}),yellowness value at 45 min (b^*_{45 min}),and lightness value at 24 h (L^*_{24 h}) of longissimus dorsi muscle of pigs in SQ group were significantly increased (P＜0.05),while the 24 and 48 h drip loss,and shear force were significantly decreased (P＜0.05).The activities of glutathione peroxidase (GSH-Px),catalase (CAT),and total antioxidant capacity (T-AOC) in serum of pigs in SQ group were significantly increased (P＜0.05).The activities of superoxide dismutase (SOD) and CAT and T-AOC in liver were significantly increased (P＜0.05),and the malondialdehyde (MDA) content was significantly decreased (P＜0.05).The T-AOC and SOD activity in longissimus dorsi muscle of pigs in SQ group were significantly increased (P＜0.05),while the MDA content was significantly decreased (P＜0.05). 【Conclusion】 Supplementation of 240 mg/kg SQ in diet could improve the growth performance and meat quality,and enhance the antioxidant capacity of growing-finishing pigs.

【Objective】 This study was aimed to improve the genomic prediction accuracy for three feed efficiency-related traits in purebred Duroc boars by integrating data from Duroc boars and their crossbred offspring (Duroc×Landrace×Yorkshire pigs,DLY commercial pigs) using cross-population genomic selection. 【Method】 Phenotypic and genotypic data for average daily gain (ADG),average daily feed intake (ADFI),and feed to gain ratio (F/G) were collected from 1 138 Duroc boars and 4 346 DLY commercial pigs.Single-trait (ST-GBLUP) and multi-trait (MT-GBLUP) genomic best linear unbiased prediction models were employed,two reference population strategies were compared:①Only Duroc boars (group 1);②Combined Duroc boars and DLY commercial pigs (group 2).The effect on the prediction accuracy for these target traits in Duroc boars was evaluated. 【Result】 ADG,ADFI,and F/G showed moderate heritabilities (0.292-0.455) in both populations.Genetic correlations for the same traits between populations ranged from 0.386 (F/G) to 0.602 (ADG).Compared with group 1,the strategy in group 2 consistently improved the prediction accuracy for Duroc boars.Under the ST-GBLUP model,accuracies for ADG,ADFI,and F/G increased by 4.90%,17.18%,and 25.74%,respectively,and under the MT-GBLUP model,the respective increased by 1.69%,12.98%,and 16.32%,respectively. 【Conclusion】 Constructing a combined reference population by integrating data from purebred Duroc boars and DLY commercial pigs for cross-population genomic selection could effectively enhance the prediction accuracy for ADG,ADFI,and F/G in purebred Duroc boars.

【Objective】 This study was aimed to investigate the effects of the natural non-caloric sweetener rebaudioside A (Reb A) on the reproductive system and reproductive performance in female mice,so as to provide a reference for its further application in production. 【Method】 Twenty-four healthy female ICR mice (29.11 g±1.21 g) were divided into control and Reb A groups,with 3 replicates per group and 4 mice per replicate.Mice in control group received 200 mg/(kg·d) saline via oral gavage daily for 14 days,while mice in Reb A group received 200 mg/(kg·d) Reb A.Daily body weight,feed intake,water consumption,litter size,and offspring weight were recorded.Another 24 healthy female ICR mice (28.88 g±0.93 g) were grouped and treated similarly.The estrous cycle was monitored via vaginal smear combined with vulvar observation.The levels of estradiol,follicle-stimulating hormone (FSH),luteinizing hormone (LH),and progesterone in serum were measured by enzyme-linked immunosorbent assay (ELISA).Ovarian histomorphology,follicular types and counts were assessed using hematoxylin and eosin (HE) staining.Superovulation and in vitro fertilization were performed to evaluate ovulation count,cleavage rate,and blastocyst rate.Reactive oxygen species (ROS) and glutathione (GSH) levels in oocytes were measured by fluorescence staining.Uterine and ovarian weights were recorded,and organ coefficients were calculated. 【Result】 Compared with control group,14-day Reb A administration did not alter body weight,feed intake,or water consumption in female mice,but significantly increased litter size and offspring weight (P＜0.05).Reb A also significantly reduced the proportion of abnormal estrus (P＜0.05),however,there was no significant effect on the levels of estradiol,follicle-stimulating hormone,luteinizing hormone and progesterone in serum(P＞0.05).Additionally,Reb A could significantly increase the uterine weight and preantral follicle count in ovaries (P＜0.05),significantly elevate cleavage and blastocyst rates during in vitro fertilization (P＜0.05),significantly reduce ROS level in oocytes (P＜0.05),and extremely significantly enhance GSH level (P＜0.01). 【Conclusion】 Under the experimental conditions,oral administration of 200 mg/(kg·d) Reb A did not negatively affect growth performance in mice,but improve oocyte quality,embryonic developmental potential,and reproductive capacity.

【Objective】 Calycosin (CAL),a naturally occurring isoflavone compound derived from medicinal plants such as Astragalus membranaceus,exhibits diverse pharmacological properties,including antioxidant and anti-inflammatory activities.This study was aimed to investigate the mechanism of CAL alleviated testicular dysfunction induced by bisphenol A (BPA),a pervasive environmental endocrine-disrupting chemical. 【Method】 Thirty 6-week-old male Kunming mice were randomly assigned into three groups (n=10 per group):Control group (vehicle-treated with corn oil),BPA group (oral gavage of 50 mg/kg BPA daily),and BPA+CAL group (received 50 mg/kg BPA daily for 1 week followed by 40 mg/kg CAL daily).Body weight was monitored throughout the experiment.On day 22 post-intervention,blood samples were collected via retro-orbital puncture for ELISA quantification of the levels of testosterone (T) and estradiol (E₂) in serum.Mice were euthanized by cervical dislocation,and testicular tissues were collected for histopathological evaluation via HE staining.Protein extracts from testicular tissues were subjected to Western blotting for analyzing endoplasmic reticulum stress markers,including Bip,ATF6,p-PERK,eIF2α,and ATF4. 【Result】 ①Compared with control group,mice in BPA group exhibited a significant reduction of body weight in some experimental days (P＜0.05),while BPA+CAL-treated group showed a non-significant upward trend in body weight relative to BPA group (P＞0.05).②Testicular weight in BPA group was significantly lower than that of control group (P＜0.05),but this decline was significantly ameliorated by CAL intervention (P＜0.05).Compared with control group,there was significant structural damage in testicular tissue of mice in BPA group,but the structural damage was significantly improved after CAL treatment relative to BPA group.③ Compared with control group,the T level of serum of mice in BPA group was significantly increased (P＜0.05),while the E₂ level was significantly decreased (P＜0.05).Compared with BPA group,the T level of serum of mice in BPA+CAL group was significantly decreased (P＜0.05),while the E₂ level was significantly increased (P＜0.05).④Western blotting analysis results demonstrated that the expression of stress-related proteins (Bip,ATF6,p-PERK,eIF2α,and ATF4) in testicular tissue of mice in BPA group were significantly higher than those of control group (P＜0.05).However,CAL treatment attenuated this response,showing a downregulation trend of these proteins compared with BPA group,suggesting that CAL mitigated BPA-induced testicular injury by suppressing endoplasmic reticulum stress pathways. 【Conclusion】 CAL could mitigate BPA-induced testicular injury in mice,regulate sex hormone homeostasis,inhibit endoplasmic reticulum stress response in testicular tissue,and thereby improve reproductive function in mice.

【Objective】 This study was aimed to precisely locate and screen candidate genes that affected the number of thoracic vertebrae in Beijing Black pigs,providing crucial information for in-depth analysis of the gene regulatory mechanism of the number of thoracic vertebrae in Beijing Black pigs. 【Method】 In this study,645 Beijing Black pigs were selected.A genome-wide association study (GWAS) was carried out by increasing the density of single nucleotide polymorphism (SNP) of gene in the quantitative trait loci (QTL) on Sus scrofa chromosome 7(SSC7).Combined with linkage disequilibrium (LD) analysis,the candidate genes that affected the number of thoracic vertebrae in Beijing Black pigs were screened.Meanwhile,transcriptome differentially expressed gene analysis was performed on five 17-day-old embryos from the bi-directional selection lines of Beijing Black pigs with different numbers of thoracic vertebrae.Software packages such as DOSE,org.Ss.eg.db,topGO,and clusterProfiler were used to conduct GO function and KEGG pathway enrichment analysis on the differentially expressed genes.Key genes affecting the number of thoracic vertebrae were identified through multi-omics joint analysis. 【Result】 LD analysis was performed on the most significant loci of the SSC7 GWAS results.Finally,the region related to the number of thoracic vertebrae in Beijing Black pigs was located at SSC7:97 619 505-97 779 063 bp,it contained five genes,namely VRTN,SYNDIG1L,NPC2,ISCA2,and LTBP2.GO function enrichment analysis results showed that there were 401,31,and 50 significantly enriched terms in the three major categories of biological process,cellular component,and molecular function,respectively.Among them,the biological processes related to thoracic vertebrae development included terms such as the positive regulation of Wnt signaling pathway and bone mineralization,and the molecular functions covered terms such as calcium ion binding.In the KEGG pathway enrichment analysis,the differentially expressed genes were significantly enriched in 41 pathways.Among them,MAPK and the PI3K-Akt signaling pathways were closely related to thoracic vertebrae development.By integrating the analysis results,the above-mentioned GO terms and KEGG signaling pathways collectively contained 82 candidate genes,such as AKT1,CSF1,HOXA11,and LTBP2. 【Conclusion】 LTBP2 gene was jointly screened through multi-omics analysis and could be regarded as an important candidate gene affecting the number of thoracic vertebrae in Beijing Black pigs.In addition,for the first time,transcriptome expression difference analysis was performed on embryos with different phenotypes of the number of thoracic vertebrae to screen candidate genes,providing new insights for exploring the mechanism affecting the number of thoracic vertebrae in pigs.

【Objective】 The purpose of this study was to analyze the population genetic structure and genetic diversity of the conserved population of Youxian partridge duck,so as to better protect and utilize this genetic resource. 【Method】 In this study,one male duck was selected from each of the 30 families in the Youxian partridge duck conservation farm for wing vein blood collection and DNA extraction.Genetic information was obtained through whole genome resequencing (WGS).Based on the resequencing data of the 30 male ducks,indicators such as observed heterozygosity (Ho),expected heterozygosity (He),population inbreeding coefficient (Fis),polymorphic information content (PIC),gene diversity index (Nei),effective number of alleles (Ne) and Shannon’s diversity index (SHI) were adopted to evaluate the population genetic diversity.G matrix,principal component analysis (PCA),population structure analysis and runs of homozygosity (ROH) analysis were used to reveal the genetic structure of the conserved population. 【Result】 18 853 217 single nucleotide polymorphisms (SNPs) loci of the sampled population of Youxian partridge duck were obtained after detection and screening.The G matrix analysis revealed that most individuals exhibited distant genetic relatedness,with only a minority displaying closer kinship relationships.PCA divided the samples into three subgroups,and most of the samples were relatively clustered.Among the subgroups,the fixation index (Fst) between subgroup 1 and subgroup 3 was 0.051,showing a moderate degree of differentiation.The Fst between the other subgroups were all less than 0.050.In the analysis of population genetic structure,when K=2,the population was optimally divided into two groups.The He of the Youxian partridge duck population was slightly greater than Ho (0.299 vs.0.288).The PIC,Nei and Ne were 0.258,0.324 and 1.524,respectively.A total of 493 ROH fragments were detected in the population.The total length of ROH was 333.80 Mb,and the average value of F_ROH was 0.011. 【Conclusion】 The ducks in the conservation farm of Youxian partridge duck had moderate genetic diversity.The genetic structure of the population was relatively stable.There was a certain degree of inbreeding relationship but at a relatively low level.For the subsequent conservation work,it was necessary to strengthen the introduction of external bloodlines,rationally plan mating schemes,improve internal management to maintain a low inbreeding level.

【Objective】 This study was aimed to investigate the effects of forsythiaside A (FA) on estrogen synthase in cumulus cells of healthy adult female yak (5-8 years old) and explore its mechanism,so as to provide a scientific basis for FA application in yak reproductive management. 【Method】 Yak cumulus cells during the logarithmic growth phase were used for cytotoxicity assays with concentration gradients (0,5,10,20,30,and 40 μmol/L) and time gradients (12,24,36,and 48 h) to determine optimal FA treatment conditions.Real-time quantitative PCR and Western blotting were performed to analyze the expression of hypoxia-inducible factor 1α (HIF-1α),estrogen synthetase (CYP11A1,CYP17A1,and CYP19A1) genes and proteins.HIF-1α inhibitor LW6 was used to block HIF-1α synthesis,with cells divided into control group,FA group,and FA+LW6 group,after 12 h of in vitro culture，the expression of HIF-1α,CYP11A1,CYP17A1,and CYP19A1 genes and proteins in cumulus cells and estradiol concentration in supernatants of yaks were detected. 【Result】 Cytotoxicity assays showed that compared with 0 μmol/L FA group,5 μmol/L FA incubation for 12 h significantly increased cell viability and upregulated the expression of HIF-1α,CYP11A1,CYP17A1,and CYP19A1 genes and proteins in yak cumulus cells (P＜0.05).LW6 inhibition experiment revealed that compared with control group,FA treatment significantly increased the relative expression of HIF-1α,CYP11A1,CYP17A1,and CYP19A1 genes and proteins (except for CYP19A1) in yak cumulus cells (P＜0.05),the fluorescence intensity of HIF-1α was significantly enhanced,and the estradiol concentration was significantly increased (P＜0.05).FA+LW6 treatment significantly decreased the expression of all four genes and proteins in yak cumulus cells (P＜0.05),with significantly decreased estradiol concentration (P＜0.05). 【Conclusion】 FA could regulate the synthesis of estrogen synthase through the expression of HIF-1α gene,thereby promote estrogen secretion in yak cumulus cells.The results provided experimental evidence for FA as a natural reproductive regulator to improve yak breeding performance.

【Objective】 This study was aimed to investigate the population genetic structure and relatedness of breeding rams in the core group of Turpan Black sheep,evaluate their genetic diversity,and provide scientific data support for formulating rational breeding strategies and conservation plans. 【Method】 Single nucleotide polymorphism (SNPs) of 128 Turpan Black sheep rams were detected using simplified genome sequencing technology.Genetic diversity parameters and runs of homozygosity (ROH) were calculated using PLINK 1.90 and R 4.4.1 software.Population structure was analyzed through phylogenetic tree construction with Mega 10.0 software. 【Result】 The average observed heterozygosity (Ho),expected heterozygosity (He) and minor allele frequency (MAF) were 0.16,0.18 and 0.23,respectively,with a polymorphic information content (PIC) of 0.82.The 128 rams were clustered into 5 subpopulations based on principal component analysis (PCA) and phylogenetic analysis.The genetic distance (D-value) ranged from 0.1626 to 0.2749,with an average of 0.2635.The genetic distance and phylogenetic G matrix were consistent,indicating that the majority of individuals within the Turpan Black sheep population had distant phylogenetic relationships.A total of 2 239 ROHs were detected,of which 61.37% were 0-5 Mb in length.The average inbreeding coefficient (F_ROH) was 0.04,suggesting low inbreeding levels. 【Conclusion】 The breeding core group of Turpan Black sheep demonstrated high genetic diversity,with distant kinship among most individuals and low inbreeding levels within the population,indicating a robust foundation for genetic resource conservation and utilization.The results provided a scientific reference for developing breeding programs and sustainable utilization strategies for the Turpan Black sheep core population.

【Objective】 Growth hormone 1 (GH1) is a synthetic metabolic hormone belonging to the growth hormone/prolactin protein family,which plays an extremely important role in animal growth and development.This study was aimed to screen the single nucleotide polymorphism (SNP) of GH1 gene and conduct association analysis with growth traits in Guanling cattle,in order to provide scientific basis for molecular breeding of Guanling cattle. 【Method】 Using Primer Premier 3.0 software,three pairs of primers were designed based on the bovine GH1 gene sequence retrieved from the NCBI database (accession No.:NC_037346.1) to amplify GH1 gene in Guanling cattle.SNP were detected via bidirectional direct sequencing and sequence alignment.HaploView software was employed to perform linkage disequilibrium (LD) analysis on the identified SNP and identify associated haplotypes,thereby elucidating the genetic interactions and structural relationships among these loci.The correlation between GH1 gene and growth traits in Guanling cattle was evaluated using SPSS 21.0 software,and the potential function of GH1 protein was predicted. 【Result】 Three SNPs were detected in exon 5 of GH1 gene in Guanling cattle:g.44119526 C＞G,g.44119662 C＞T,and g.44119676 C＞A.g.44119526 C＞G and g.44119676 C＞A showed moderate polymorphism,while g.44119662 C＞T exhibited low polymorphism.g.44119662 C＞T and g.44119676 C＞A were in Hardy-Weinberg equilibrium (P＞0.05),while g.44119526 C＞G was extremely significantly deviated from Hardy-Weinberg equilibrium (P＜0.01).Three SNPs formed four haplotypes (H1,H2,H3,and H4) and five diplotypes (H1H1,H1H2,H1H3,H1H4,and H2H3).The association analysis results revealed that the back waist length of CC genotype of g.44119676 C＞A in GH1 gene of Guanling cattle was significantly higher than that of AA genotype (P＜0.05),and the back waist length of H1H1(CCCCCC) diplotype was significantly higher than that of H2H3(GCCCAA) diplotype (P＜0.05).The protein function prediction results showed that GH1 gene encoded 217 amino acids and was a stable protein.The secondary and tertiary structures were mainly composed of alpha helix and random coil. 【Conclusion】 The mutation of g.44119676 C＞A on exon 5 of GH1 gene in Guanling cattle had a significant impact on the back waist length.The diplotype H1H1 could be used as a genetic marker to improve the growth trait of Guanling cattle.

Birds play a crucial role in both natural ecosystems and human societies,and the technology for identifying bird sex is vital for ecological research and the sustainable development of animal husbandry.Traditional sex identification methods,such as anal swelling,are generally limited by the experience and proficiency of the operators,and by low efficiency.In recent years,molecular sex identification technology,including PCR,molecular markers technology,and isothermal amplification technology have advanced rapidly. These applications have improved the accuracy and simplicity of sex identification in birds.Especially the introduction of isothermal amplification technology has accelerated the development of the sex identification technology and become a new tool in the sex identification of birds.Compared with PCR and molecular markers techniques,isothermal amplification has shown significant advantages in bird sex identification with its high efficiency,specificity and simple operation.Isothermal amplification technology can complete sample amplification and reach the detection level in 30 to 60 minutes at a single temperature.The authors reviewed the conventional methods of sex identification,using loop-mediated isothermal amplification (LAMP),recombinase polymerase amplification (RPA)/recombinase-aided polymerase amplification (PAA),and cross-priming amplification (CPA) in sex of birds as examples,illustrating the progress of the different isothermal amplification techniques in the sex identification of birds,and providing reference for future related research.

【Objective】 The purpose of this study was to compare and analyze the similarities and differences in the structure and function of heat shock protein beta-1 (HSPB1)between Wuyi Black pig and Duroc pig,and provide a reference for studying animal anti-stress mechanism. 【Method】 The HSPB1 gene of Wuyi Black pig and Duroc pig was cloned and sequenced,and bioinformatics methods were used to compare similarities.A phylogenetic tree was constructed.And a comparative analysis was conducted on the differences in the characteristics,structure,and function of HSPB1 protein between the two pig breeds,as well as the expression of each tissue in the two breeds. 【Result】 The HSPB1 gene was successfully cloned from both Wuyi Black pig and Duroc pig.The coding sequence (CDS) of each gene was 624 bp in length,encoding a protein composed of 207 amino acids.The nucleotide and amino acid sequence similarities between the two breeds were 99.0% and 98.6%,respectively，with variations in nucleotide sites 123^G→T and 233^T→C,as well as differences in amino acids 41^E→D and 78^L→P.The nucleotide sequence evolutionary tree showed that both were closely related to Desmodus rotundus;The evolutionary tree of amino acid sequences showed that both were closely related to Ovis aries.Both had the same gene promoters and transcription factor binding sites,and their HSPB1 proteins displayed consistent features in terms of signal peptides,transmembrane regions,conserved motifs,physicochemical properties,and hydrophilicity/hydrophobicity profiles.Further comparative analysis identified one novel phosphorylation site uniquely recognized by glycogen synthase kinase-3 (GSK-3) and four differential sites potentially involved in interactions with chemical effectors.However,the mitochondrial proportion of HSPB1 protein in Wuyi Black pig was lower than that in Duroc pig.Moreover,seven distinct regions of secondary and tertiary structural divergence were observed between the two proteins.Although both HSPB1 proteins exhibited strong interactions with phosphorylation-related kinases,the Wuyi Black pig had an additional functional annotation related to cytoskeletal organization. HSPB1 gene was broadly expressed across various tissues in both pig breeds,with no significant differences between breeds.In both breeds,the highest expression was observed in the longissimus dorsi muscle. 【Conclusion】 HSPB1 genes of Wuyi Black pig and Duroc pig were successfully amplified.Compared with Duroc pig, HSPB1 in Wuyi Black pig showed higher cytoplasmic content and differences in phosphorylation and chemical factor binding sites.However,the expression was found to be similar across corresponding tissues in both breeds,with the highest expression in longissimus dorsi muscle.The results provided a theoretical foundation for further studying the specific role of porcine HSPB1 in anti-stress response.

【Objective】 This study aimed to design and develop the multi-epitope nanoparticle vaccine against gB and gD proteins of Feline herpesvirus type 1 (FHV-1), and provide a theoretical basis for antigen screening and optimization of FHV-1 multi-epitope nanoparticle vaccines. 【Method】 In this study, a variety of bioinformatics tools were used to analyze the physicochemical properties, transmembrane structure, N-glycosylation site, secondary structure, B cell epitope, immunogenicity and conservation of FHV-1 gB and gD proteins. The recombinant plasmid pET20b-FBDF was constructed and transformed into Escherichia coli BL21 (DE3) competent cells to induce expression. The recombinant protein FBDF were purified by Ni column affinity chromatography and dialyed concentration treatment, and then identified by SDS-PAGE, the protein content was determined by BCA. The morphology and particle size of self-assembled nanoparticles were observed by transmission electron microscope (TEM) and dynamic light scattering (DLS). The prepared immunogens were immunized subcutaneously in BALB/c mice to evaluate their immunogenicity. 【Result】 Multiple amino acid domains in FHV-1 gB and gD proteins exhibited excellent hydrophilicity, antigenicity, structural flexibility, and surface accessibility characteristics. FHV-1 gB and gD proteins had 11 and 5 potential glycosylation sites, respectively. The secondary structures of FHV-1 gB and gD proteins were primarily composed of alpha helix, beta sheet, beta turns, and random coil, with random coil constituting the predominant structural component. And FHV-1 gB and gD proteins produced 17 and 6 B-cell linear epitopes, respectively. Three dominant antigenic epitopes with high antigenicity and conservation were screend， which were gB₁: 197－208, gB₂: 314－325 and gD: 221－232, respectively. The epitope was connected with Ferritin by flexible linker (GGGGS). After codon optimization, the recombinant plasmid pET20b-FBDF was constructed. SDS-PAGE results showed that the recombinant protein FBDF was successfully expressed at 30 ℃, 0.5 mmol/L IPTG for 16 h. The results of TEM and DLS showed that the purified FBDF protein could be self-assembled into nanoparticles with a diameter of 16.55 nm. The results of mouse immunization showed that serum antibody titers peaked after 14 days of triple immunization, and the antibody titers in FBDF+Al(OH)₃, FBDF+CpG and FRCPV groups were 1∶680 000, 1∶15 600 and 1∶3 500, respectively, which were significantly higher than those in PBS and Ferritin groups (P＜0.05). Vaccination with FBDF could induce mice to produce higher levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), and neutralizing antibodies, with neutralizing antibody levels up to 1∶2⁵. 【Conclusion】 The multi-epitope nanoparticle vaccine FBDF constructed in this study had good immunogenicity and could induce humoral and cellular immune responses in BALB/c mice， which laid a foundation for the research of FHV-1 multi-epitope vaccine.

【Objective】 The aim of this study was to investigate the biological characteristics of Riemerella anatipestifer (RA),which was isolated from diseased ducks,and evaluate the protective effect of 3 commercially available vaccines for infectious serositis in ducks against the newly isolated strain through animal challenge tests,in order to provide a reference for the prevention and control of infectious serositis in ducks. 【Method】 The liver and brain tissues of diseased ducklings were collected to isolate pathogenic bacteria.The isolated strains were identified by morphological observation,16S rRNA gene PCR identification and sequencing,phylogenetic tree construction,and serotype typing.The growth curves of the isolated strains were determined by spectrophotometry and plate counting.The drug sensitivity was analyzed by disk diffusion method.The median lethal dose (LD₅₀) and tissue bacterial load were determined through animal experiments.Finally,the immune protective effects of 3 commercial inactivated vaccines on the isolated strains were evaluated through challenge tests. 【Result】 One strain of RA was isolated in this experiment,and it was serotype 2.The results of the drug sensitivity test showed that the isolate was resistant to 10 drugs such as ampicillin,ceftazidime and kanamycin,and was sensitive to amoxicillin,cefotaxime,florfenicol and streptomycin.The determination results of the growth curve showed that after 6-10 h of culture,the isolate entered the logarithmic growth phase,and the viable bacteria count could reach 10⁹ CFU/mL after 8 h of culture.The LD₅₀ for ducklings was 7.65×10^4.7 CFU,and the bacterial load in heart of ducklings was the highest after infection.2 weeks after immunizing 7-day-old duckings with 3 commercial inactivated vaccines,the isolate was used for challenge.The results showed that the multivalent RA inactivated vaccines of types 1+2+7 had the best protective effect,with a protection rate of up to 70%,while the protection rates of the other two vaccines were less than 50%. 【Conclusion】 In this study,one strain of multi-drug resistant and highly pathogenic RA serotype 2 was isolated，the immune protective effect of commercially available RA vaccines on the isolated was limited.This result provided a theoretical basis for the current prevention and control of infectious serositis in ducks.

【Objective】 The experiment was to explore the mechanism of virulence reversion of Duck hepatitis A virus genotype 3 (DHAV-3) attenuated strain.The biological characteristics of the DHAV-3 attenuated strain and its revertant strain were compared through ducklings experiments and genomic sequence analysis. 【Method】 One-day-old Pekin ducklings were inoculated with DHAV-3 attenuated strain Y140,derived by 140 passages of isolate YDF in chicken embryos,and its revertant strain Y140/4R,derived by 4 passages in duckings,and monitored for clinical signs,gross lesions,morbidity and mortality.Viral RNA levels in liver,kidney and serum of ducklings were measured by Real-time quantitative PCR.Sequencing and analysis were conducted on the genomic sequences of the Y140 strain and the first to fourth passage viruses of Y140 to explore the molecular mechanisms related to virulence reactivation. 【Result】 Ducklings inoculated with Y140 strain showed no clinical symptoms or pathological changes.Ducklings inoculated with Y140/4R strain presented the characteristic clinical symptoms and gross lesions of duck viral hepatitis,and the mortality was 40%.Compared with Y140 strain,the viral RNA levels in liver and kidney of surviving ducks 3 days after infection with Y140/4R strain were significantly increased (P＜0.05),and the viral RNA levels in liver and kidney of dead ducks 2-3 days after infection were extremely significantly increased (P＜0.01).2-4 days after infection,the viremia level produced by Y140/4R strain was significantly higher than that of Y140 strain (P＜0.05). The genomic sequencing results showed that starting from the second generation,mutations I149V,V236I and A25G occurred in 2C protein,3D protein and 3'-UTR,respectively.Starting from the third generation,the E202D mutation occurred in VP1 protein. 【Conclusion】 Passage of duckings could drive mutations in the genome of DHAV-3 attenuated strain，leading to the emergence of virulence variants and significantly enhancing the replication ability of virus in target organs and the ability to form viremia.

【Objective】 This study was conducted to investigate the reasons why Listeria monocytogenes Lm873 exhibited attenuated virulence despite harboring virulence island 4 (LIPI-4),and assess the potential public health risk of strain Lm873 triggering human listeriosis outbreaks based on genomics. 【Method】 Taking the Listeria monocytogenes attenuated strain Lm873 as the research object,the differences in growth curve between it and the hypervirulent strain Lm928 under different temperatures,pH,NaCl concentration conditions,the differences in motility under different temperatures and the oxidative stress resistance under different concentrations of oxidants were compared.The whole genome of strain Lm873 was sequenced,and its genome characteristics,virulence genes,genome evolution,and functional annotation were analyzed. 【Result】 No significant differences in growth profiles were observed between the two strains at different temperatures and pH of 3.0,7.0 and 9.0 (P＞0.05).However,a significant reduction in the growth rate of strain Lm873 was detected at 12 h in a medium with pH 5.0 compared with strain Lm928 (P＜0.05).The growth patterns in 4.5% and 7.0% NaCl solutions were comparable for both strains (P＞0.05),yet strain Lm873 exhibited significantly lower growth in 0.5% NaCl solution at 8 h and in 10.0% NaCl solution at 12 h compared with Lm928 (P＜0.05).In the motility test,the average diameter of the motility halo of strain Lm873 was significantly lower than that of strain Lm928 when cultured at 28 and 37 ℃ (P＜0.05).Strain Lm928 grew on the medium containing CdCl₂,but Lm873 did not.There was no significant difference in the growth ability of strains Lm873 and Lm928 on the medium containing 0.25,0.5 and 1 mmol/L CuCl₂.On low concentration H₂O₂ medium,strain Lm928 grew,while Lm873 did not,whereas neither strain Lm928 nor Lm873 grew on high concentration H₂O₂ medium.Genomic analysis revealed that the genome of strain Lm873 was approximately 2 972 618 bp in length with GC content of approximately 37.88%,exhibiting distinct genetic features.Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Lm873 shared the highest similarity with HPB2262,suggesting close evolutionary relationships among them.Furthermore single nucleotide polymorphism (SNP) analysis positioned it closely alongsided Chinese clinical isolate SHL012 and the U.S.-based listeriosis-causing strain FSLJ1-194,demonstrating substantial genetic similarity.Comparative genomic analysis revealed that strain Lm873 lacked genes associated with cadmium ion transport and cation-transporting ATPases compared to Lm928.KEGG pathway analysis further demonstrated that Lm873 harbored fewer annotated genes involved in flagellar assembly.Additionally,Lm873 carried multiple antibiotic resistance genes conferring multidrug resistance phenotypes. 【Conclusion】 The presence of LIPI-4 in Lm873,yet its reduced virulence,was attributed to its diminished transmission capacity,infectivity,and environmental resilience.Phylogenetic analysis suggested that Lm873 could possess the potential to trigger outbreaks of human listeriosis.

Intestinal flora regulates the biological process of organisms and affects the health and diseases of hosts.microRNAs (miRNAs),as important regulators,are related to health and disease.It is found that there is a bidirectional regulatory relationship between intestinal flora and miRNA.Therefore,more and more researches focus on revealing the influence of intestinal flora-miRNA interaction on many biological processes such as health and diseases.In this paper,firstly,the main mechanism of the interaction between intestinal flora and miRNA is combed and summarized.miRNA can enter bacteria and specifically affect the growth of intestinal microorganisms,as well as their proliferation activity,steady state,colonization,structure,composition,abundance,adaptability and gene expression.Conversely,intestinal flora can also regulate miRNA,mainly by affecting the expression of host miRNA,metabolites,secondary microbial metabolites,probiotics and lipopolysaccharide.Then,based on the theory that "the lung and the large intestine are exterior to interior" and the intestinal flora connects the lung and the large intestine,it is discussed that the interaction of intestinal flora-miRNA has the potential to mediate the operation of the "lung-intestine axis".Then,by summarizing the effects of intestinal flora and miRNA on inflammation,it is concluded that the intestinal flora-miRNA axis can mediate many diseases and inflammatory pathways.Finally,combined with the interaction between traditional Chinese medicine and intestinal flora,the regulation of traditional Chinese medicine on miRNA and the effect of intestinal flora on exogenous miRNA,it is found that the intestinal flora-miRNA axis has great potential to mediate the prevention and treatment of diseases by traditional Chinese medicine.To sum up,the in-depth study of the interaction mechanism of intestinal flora-miRNA axis provides a reliable basis for the discussion of the pathogenesis,prevention and treatment mechanism of diseases and the mining of their targets.

【Objective】 This study aimed to explore the key host cytokines or proteins which stringed pro-inflammatory cytokines,inflammatory responses,and apoptosis together in Bluetongue virus (BTV) infection progress from the perspectives of biological processes and signaling pathways involved in pro-inflammatory cytokines and apoptosis,and further obtain clues of host factors or proteins related to the pathogenesis of BTV. 【Method】 Total RNA of BTV-1 serotype strain (Y863) infected sheep embryonic testicular cells (OA3.Ts) was extracted for RNA sequencing (RNA-Seq).R package edgeR (v 3.14.0) was used to count differentially expressed genes.Gene set enrichment analysis of differentially expressed genes were performed to obtain their gene enrichment maps under the KEGG subset of C2:curated gene sets,biological processes (BP),cellular components (CC) and molecular function (MF) subsets of C5:GO gene sets,as well as Hallmark gene sets.Real-time quantitative RT-PCR,ELISA and Western blotting were used to validate 10 genes expression of leading-edge subsets (LS) genes in the key signaling pathways or biological process.The level of apoptosis of BTV infected OA3.Ts cells was detected by Annexin V-FITC/propidium iodide (PI) staining. 【Result】 GSEA results indicated that differentially expressed genes were significantly enriched in apoptosis pathway of KEGG subset under C2:curated gene sets,response to tumor necrosis factor (TNF),response to interleukin 1 (IL-1),cellular response to IL-1,response to interferon-γ (IFN-γ),cellular response to IFN-γ,and IL-12 production terms of BP subset under C5:GO gene sets,and inflammatory response pathway under Hallmark gene sets.The results of Real-time quantitative RT-PCR showed that the transcriptional levels of gained key pro-inflammatory cytokines and proteins based on GESA analysis were significantly up-regulated,including IL-1α,IL-6,C-X-C chemokine 8 (CXCL8),caspase 7 (CASP7),CASP8,TNF receptor superfamily member 5 (CD40),and nucleotide-binding oligomerization domain receptor family pyrin domain containing protein 3 (NLRP3),with the log₂FoldChange (FC) values of 0.34,1.03,7.42,3.98,3.61,1.00 and 1.74,respectively.ELISA and Western blotting results showed that compared with control group,the expression levels of CXCL8,CD40,CASP7 and CASP8 of infected group were extremely significantly or significantly increased (P＜0.05 or P＜0.01).It was confirmed that the percentage of apoptotic OA3.Ts cells was elevated from 1.89% of control groups up to 12.78% of infected groups using Annexin V-FITC/PI staining method. 【Conclusion】 The highly expressed CD40 and CXCL8 were the key factors in stringing pro-inflammatory cytokines,inflammatory response,and apoptosis together,as well as were important potential contributors to the clinical symptoms of hemorrhage of multi-tissue,edema,disseminated intravascular coagulation,and tissue necrosis in sheep,as well as testicular degeneration and azoospermia in male sheep.

【Objective】 The purpose of this experiment was to screen strains with potential for livestock and poultry environmental restoration from Black bear feces,providing candidate strains for the development of livestock and poultry microecological preparations. 【Method】 Fresh Black bear feces were collected,and desulfurizing bacteria were initially screened using a selective medium.After isolation and purification,strains were identified via colony morphology and 16S rDNA sequencing.Growth characteristics and antimicrobial susceptibility of the isolated strain were analyzed.The biological properties were comprehensively characterized by evaluating its capacity for H₂S removal,Cr(Ⅵ) passivation,plant growth promotion,and enzyme production. 【Result】 One strain of Atlantibacter hermanii was successfully isolated，and it was named M1633-2DR.On MHA solid medium,it formed yellow,circular colonies with smooth edges and a moist surface.Gram staining revealed Gram-negative,short bacilli.In MHB liquid medium,the strain entered the logarithmic growth phase at 1 h and the stationary phase at 16 h.The isolate was sensitive to 11 kinds of antibacterial drugs such as sulbactam and amikacin,and resistant to ampicillin.Desulfurization tests results showed that the removal rate of 160 mg/L S^2- by the isolate reached 100% within 48 h,and the removal rates of 320,480 and 560 mg/L S^2- were 62.23%,49.58% and 42.44%, respectively.The tolerance concentration of Cr(Ⅵ) of the isolate could reach 3 200 mg/L.The reduction rate of 50 mg/L Cr(Ⅵ) within 96 h was 100%,and the reduction rates of 150 and 250 mg/L Cr(Ⅵ) within 168 h were 48.23% and 37.78%,respectively.Further studies had found that this strain had the abilities of phosphorus solubilization (829.28 mg/L),nitrogen fixation (9.94 μg/mL),indoleacetic acid production (43.91 mg/L) and protease production (935.80 U/mL). 【Conclusion】 In this study,one strain of Atlantibacter hermanii was isolated and obtained.It had significant advantages in H₂S removal,Cr(Ⅵ) reduction,plant growth promotion and protease production，and could be used as a candidate strain for environmental restoration in animal husbandry and the development of agricultural microbial preparations.

mRNA vaccine,which encodes the target protein or polypeptide antigens,can be delivered to cells for translation to induce an immune response against pathogens.Given the urgent need to prevent and control swine diseases,traditional vaccines can’t fully meet the needs of epidemic prevention.With short development time,high safety and strong immune efficacy,mRNA vaccines have become one of the main research directions for the prevention and control of animal diseases.Effective mRNA vaccines can reduce the incidence and spread of swine diseases.The authors summarised the types and structures of mRNA vaccines,key technologies and research progress of mRNA vaccines for porcine epidemic diarrhea,foot-and-mouth disease,porcine reproductive and respiratory syndrome,African swine fever,porcine pseudorabies,swine influenza and other swine diseases.The application of mRNA vaccines in the prevention and control of swine diseases was further prospected to provide a reference for the development and production of mRNA vaccines for swine diseases.

【Objective】 The experiment was conducted to explore the molecular genetic evolution characteristics of H3N2 subtype Avian influenza virus (AIV) strains in Guangxi. 【Method】 Disease samples of poultry throat swabs and anal swabs from various places in Guangxi from 2020 to 2024 were collected,and the H3N2 subtype AIV was identified and detected by Real-time quantitative RT-PCR.Some positive samples were selected and inoculated into SPF chicken embryos through the allantoic cavity,and the allantoic fluid of chicken embryos was collected.After extracting nucleic acids,hemagglutinin (HA) and neuraminidase (NA) genes were amplified by PCR and sequenced,and analyses of sequence similarity,phylogenetic,amino acid variation sites and genetic evolution rate were conducted. 【Result】 A total of 43 HA and NA gene sequences were obtained,with the coding region sizes of 1 701 and 1 410 bp,respectively.The similarity comparison analysis showed that the nucleotide sequence similarities between HA and NA genes obtained in this study were 86.0%-99.9% and 89.7%-99.9%,respectively.The similarities of nucleotide sequences with avian,human,porcine and canine sources in GenBank were 78.4%-99.7% and 78.1%-99.1%,respectively.HA and NA genes of H3N2 subtype AIV in Guangxi belonged to the Eurasian lineage,which was most closely related to avian origin and most closely related to human/porcine origin.Glycosylation occured at amino acids 18-20,22-24,38-40,54-56,61-63,181-183,301-303 and 499-501 of HA protein，and occured at amino acids 61-63,69-71,86-88,143-145,146-148,200-202,234-236,313-315 and 402-404 of NA protein.Some amino acid sites had undergone variations.HA protein was cleaved at sites 340PEKQTR↓GLF348 and 340PERQTR↓GLF348.Genetic evolution rate estimation showed that the genetic evolution rates of HA and NA genes were 3.26×10^-3 and 3.67×10^-3 substitutions/(site·year),respectively,and the population size of HA gene showed different dynamic evolution characteristics in various periods. 【Conclusion】 The current epidemic strains of H3N2 subtype AIV in Guangxi were closely related to the homologous epidemic strains,some HA and NA proteins havd unique amino acid mutations,and the evolutionary trend of strains of different genera was inconsistent with the characteristics of genetic diversity,which providing basic data for prevention and control of H3N2 subtype AIV.

【Objective】 The monoclonal antibody of Infectious bursal disease virus (IBDV) in chickens was labeled with R-phycoglobin fluorescein (R-PE) to establish a direct immunofluorescence assay for the rapid detection of IBDV in tissues and cells. 【Method】 After resuscitation of monoclonal antibody cells 4C12,monoclonal antibody ascites was prepared by immunizing mice.It was purified by chromatography column,identified by indirect immunofluorescence assay and ELISA,and the antibody titer was determined.The monoclonal antibodies were labeled by coupling kit,a direct immunofluorescence assay was established and the reaction conditions were optimized，and its specificity,sensitivity and stability were detected.The established method was used to detect the bursa of Fabricius and spleen of chickens infected with IBDV. 【Result】 The titer of purified 4C12 mab ascites was 1∶10⁸.A direct immunofluorescence assay for 4C12 monoclonal antibody labeled with R-PE was established.The optimal reaction conditions were determined as follows:DT40 cells were infected with IBDV for 48 h,double fixation with 4% paraformaldehyde and anhydrous ethanol as fixants,the working concentration of the monoclonal antibody of R-PE-4C12 was 24 μg/mL,and the antibody was incubated for 90 min.Under these conditions,the fluorescence effect was the best.The established direct immunofluorescence assay had no cross-reaction with ALV,REV,AEV and EDSV,and had good specificity.This method could still detect positive signals when the virus dilution was 10³ egg infection dose for 50% (ELD₅₀),and had good sensitivity.The stability test showed that R-PE-4C12 could still produce a stable fluorescence signal after being stored at 4 ℃ for 21 d.In the established direct immunofluorescence assay,R-PE-4C12 could bind to IBDV in the infected tissue to produce specific red fluorescence.Compared with the indirect immunofluorescence assay,there was no significant difference in the fluorescence effect between the two methods. 【Conclusion】 The direct immunofluorescence assay established in this study had good specificity,sensitivity and stability，and could be used for the detection of IBDV in tissues and cells,providing an rapid and effective detection scheme for IBDV in the laboratory.

【Objective】 The purpose of this experiment was to explore the anti-Porcine reproductive and respiratory syndrome virus (PRRSV) effect of dihydroartemisinin (DHA) in vitro. 【Method】 The anti-PRRSV Marc-145 cell model was established in vitro.The cytotoxicity of DHA to Marc-145 cells was detected by MTT method to determine its maximum safe concentration.Marc-145 cells were divided into 4 groups,namely blank control group (Control),positive control group (ribavirin),model group (PRRSV) and drug group (DHA).The effects of DHA against PRRSV under the modes of blocking,inhibition and direct inactivation were detected.The mRNA and protein expression levels of PRRSV N under different modes of action and at different time points were detected by Real-time quantitative PCR and Western blotting,respectively.The apoptosis induced by PRRSV infection was detected by Annexin/PI double staining method. 【Result】 The maximum safe concentrations of DHA and the positive drug ribavirin were 3.13 and 15.63 μg/mL,respectively.Under the effects of blocking,inhibition and direct inactivation,the maximum inhibition rates of DHA on PRRSV were 66.2%,88.9% and 62.1% respectively.Under the inhibitory effect,the cells basically remained monolayer intact.Under the blocking and direct inactivation effects,the cells showed the phenomenon of roundness and agglomeration. Under the inhibitory effect,ribavirin had the highest inhibition rate of PRRSV,with an inhibition rate of 68.9%,and the cells showed the phenomenon of roundness and agglomeration.The results of Real-time quantitative PCR showed that under the effects of blocking and inhibition,compared with model group,the copy number of PRRSV N gene in cells of ribavirin and DHA groups was significantly decreased (P＜0.05).Under direct inactivation,compared with model group,the copy number of PRRSV N gene in cells of DHA group was significantly decreased (P＜0.05).The results of Western blotting showed that under different modes of action,compared with model group,the expression level of PRRSV N protein in DHA group at different time points was significantly decreased (P＜0.05).The results of apoptosis detection showed that under the blocking effect,compared with model group,at 48 hours of early apoptosis,the apoptosis rate of DHA group was significantly decreased (P＜0.05).At different time points of late apoptosis,the apoptosis rates of both ribavirin and DHA groups were significantly decreased (P＜0.05).Under the inhibitory effect,compared with model group,at 72 hours of early apoptosis and late apoptosis,the apoptosis rate of DHA group was significantly decreased (P＜0.05).Under the direct inactivation,compared with model group,at 24 and 48 hours of early apoptosis,the apoptosis rate of DHA group was significantly decreased (P＜0.05).At 24 and 72 hours of late apoptosis,the apoptosis rate of DHA group decreased significantly (P＜0.05). 【Conclusion】 DHA had a significant anti-PRRSV effect in vitro,mainly by inhibiting the expression of PRRSV N gene and the synthesis of N protein,and at the same time had an inhibitory effect on cell apoptosis.

【Objective】 The purpose of this experiment was to study the binding mechanism of salafloxacin (SFX) and bovine serum albumin (BSA). 【Method】 The fluorescence spectroscopy method was employed to investigate the fluorescence quenching effect of SFX on BSA,the interaction type,binding mode,and number of binding sites between SFX and BSA.Synchronous fluorescence spectroscopy was used to measure the effect of SFX on the endogenous fluorescence of BSA,the influence of SFX on the fluorescence intensity of tyrosine (Tyr) and tryptophan (Trp) in BSA was analyzed.The three-dimensional fluorescence spectroscopy was employed to analyze the three-dimensional fluorescence characteristics of SFX and BSA,and the effect of SFX on the conformation of BSA was analyzed.Ultraviolet-visible spectroscopy was employed to detect the UV spectral characteristics of BSA upon addition of varying concentrations of SFX，the effect of SFX on the conformation of BSA and the type of quenching was analyzed.The binding sites and binding forces between SFX and BSA were analyzed by molecular docking method，and the effect of metal ions on the binding of SFX and BSA was analyzed by fluorescence spectroscopy. 【Result】 Fluorescence spectroscopy detection results showed that SFX quenched the endogenous fluorescence of BSA,and the quenching type of BSA was static quenching.The binding process between SFX and BSA was exothermic and spontaneous，non radiative energy transfer occurred during the binding process.The interaction between SFX and BSA was primarily mediated by hydrogen bonding and van der Waals forces.The number of binding sites was approximately 1.The molecular binding distance was 3.182 nm.Synchronous fluorescence detection showed that SFX quenched the fluorescence of Tyr and Trp residues as well as the peptide backbone in BSA.Three-dimensional fluorescence spectroscopy demonstrated a decrease in fluorescence intensity of Trp and Tyr residues induced by SFX,and the peptide chain structure of BSA was alterated.Molecular docking showed that SFX formed hydrogen bonds with Pro498,Glu470 and Lys533 residues in BSA,hydrophobic bonds with Val497 and Lys499 residues,and van der Waals forces with Tyr496 residue.The metal ions Ca²⁺,Na⁺,K⁺ and Al³⁺exhibited a promoting effect on the binding of SFX to BSA，Fe²⁺,Mg²⁺ and Cu²⁺ exhibited inhibitory effects. 【Conclusion】 SFX-BSA binding was spontaneous,forming a stable complex,and the combination would change the conformation of BSA.This study provided fundamental data for investigating SFX toxicity,in vivo transport and metabolic mechanisms.

【Objective】 The purpose of this experiment was to investigate the alleviation effect of Lactobacillus kefiranofaciens JK-24 on dextran sodium sulfate (DSS)-induced colitis in mice. 【Method】 18 C57BL/6 mice were randomly divided into 3 groups:Blank control (Control),model (DSS) and Lactobacillus kefiranofaciens JK-24 (JK-24＋DSS) groups，and 6 mice in each group.From the first day of the experiment,mice in JK-24＋DSS group were gavaged with Lactobacillus liquidacidum JK-24 suspension at 0.1 mL/10 g (with a concentration of 10⁹ CFU/mL).Mice in Control and DSS groups were gavaged with the same amount of normal saline for 21 consecutive days.From the 15th to the 21st day,mice in DSS and JK-24＋DSS groups freely drank 3% DSS solution,while mice in Control group drank water normally.During the experiment,the weight changes of mice were recorded,the fecal conditions were observed,and the disease activity index (DAI) was calculated.After the experiment,colon and blood samples of mice were collected.The length of the colon was measured and the histopathological changes of the colon tissue were observed.The levels of oxidative stress indicators and inflammatory cytokines in serum and colon tissue were determined. 【Result】 Compared with Control group,the weight of mice in DSS group decreased significantly,and the feces did not form or even showed bloody stools,DAI score increased significantly (P＜0.05).A large number of inflammatory cells were seen in colon tissues,the intestinal mucosal structure was destroyed.The contents of inflammatory factors interleukin-12 (IL-12),tumor necrosis factor-α (TNF-α),IL-10 and IL-6 in serum and colonic tissues were significantly or extremely significantly increased (P＜0.05 or P＜0.01).The activities of superoxide dismutase (SOD) and catalase (CAT) decreased extremely significantly (P＜ 0.01),while the activity of myeloperoxidase (MPO) and the content of malondialdehyde (MDA) increased extremely significantly (P＜0.01).Compared with DSS group,the body weight of mice in JK-24＋DSS group decreased relatively slowly,and the DAI score decreased in the later stage of modeling.The length of colon increased significantly (P＜0.05),the area of inflammatory cell infiltration in colonic tissue decreased significantly,and the structure of colonic mucosal tissue was basically intact.Only in some areas did glandular absence and irregular arrangement occur.The contents of IL-6,IL-10,IL-12 and TNF-α in serum and colonic tissue were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The activities of SOD and CAT in serum were extremely significantly increased (P＜0.01),the activity of MPO was significantly decreased (P＜0.05),and the content of MDA was extremely significantly decreased (P＜0.01). 【Conclusion】 Lactobacillus kefiranofaciens JK-24 had a significant alleviating effect on DSS-induced colitis in mice.The main mechanism of action was related to its improvement of the antioxidant function and reduction of the production of inflammatory factors.

【Objective】 This study aimed to explore the inhibitory effect and mechanism of lipopeptides on Ascosphaera apis,as well as its antibacterial and bactericidal activities in vitro. 【Method】 Ascosphaera apis was identified by morphological and molecular biological methods.The inhibition zone test method was used to verify whether lipopeptides could inhibit the growth of Ascosphaera apis.The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of lipopeptides against Ascosphaera apis were determined by the multiple dilution method.The effect of lipopeptides on the spore germination of Ascosphaera apis was observed using a hemocytometer.The contents of ergosterol in the cell membrane and chitin in the cell wall of Ascosphaera apis were determined by high performance liquid chromatography to determine whether the lipopeptide Iturin had an inhibitory effect on Ascosphaera apis. 【Result】 Microscopic observation revealed that the isolated fungus exhibited septate,branched hyphae along with spherical sporangia and spores.18S rDNA sequencing results showed that the isolated strain had 100% sequence similarity with Ascosphaera apis (GenBank accession No.:GQ867785.1),confirming it was Ascosphaera apis.Among the tested lipopeptides,Iturin demonstrated the strongest inhibitory effect against Ascosphaera apis,with an inhibition zone radius of 8.53 mm,while Fengycin showed a smaller inhibition zone radius (5.84 mm),and Surfactin exhibited no inhibitory activity.The MIC of Iturin against Ascosphaera apis ranged from 6.25 to 25 μg/mL,with MIC₅₀,MIC₉₀ and MIC₉₀ values of 6.25,25 and 50 μg/mL,respectively.Compared with 0 μg/mL Iturin group,the spore germination rates of Ascosphaera apis were significantly decreased (P＜0.05) in all Iturin-treated groups (6.25,12.5,25 and 50 μg/mL).Ergosterol content analysis revealed that compared with 0 μg/mL Iturin group,the ergosterol contents in the cell membrane of Ascosphaera apis were significantly decreased (P＜0.05) in 12.5,25 and 50 μg/mL Iturin-treated groups.Similarly,chitin content measurements showed that the chitin contents in the fungal cell wall were significantly decreased (P＜0.05) in 25 and 50 μg/mL Iturin-treated groups compared with 0 μg/mL Iturin group. 【Conclusion】 These results demonstrated that Iturin effectively inhibited Ascosphaera apis by reducing spore germination,disrupting ergosterol biosynthesis in the cell membrane,and impairing chitin synthesis in the cell wall.

【Objective】 This study was conducted to systematically investigate the pharmacological mechanisms of Sihuang Zhili granules against diarrheal diseases in livestock and poultry,and provide a theoretical basis for optimizing clinical diarrhea management strategies. 【Method】 The main active ingredients of Sihuang Zhili granules were analyzed using UPLC-Q-TOF-MS technology.Active ingredients were screened and their potential targets were predicted via SwissTargetPrediction database.Disease targets related to livestock and poultry diarrhea were identified through GeneCards and OMIM databases.An active component-target network was constructed using Cytoscape 3.10.3 software.The intersection between Sihuang ZhiLi granules’ targets and diarrhea-related targets was identified and visualized using a Venn diagram.A protein-protein interaction (PPI) network was subsequently built using Cytoscape 3.10.3. GO functional annotation and KEGG pathway analysis of intersection genes was performed using DAVID database.Finally,molecular docking was executed with AutoDock Vina to validate the binding activity between key components and core targets,followed by 100 ns molecular dynamics simulations conducted with Gromacs 2022 software. 【Result】 Through UPLC-Q-TOF-MS, 88 active ingredients were identified in Sihuang Zhili granules. Subsequently, 16 key components were screened out based on literature validation and analysis using SwissTargetPrediction database. The database analysis revealed that there were 289 potential targets for the drug,4 567 targets related to livestock and poultry diarrhea,and 201 intersecting targets.In terms of biological process,GO functional annotation was mainly related to protein phosphorylation,negative regulation of apoptosis,and response to exogenous stimuli.In terms of cell component,it was mainly related to neuronal cell body,plasma membrane,cytoplasm,etc.In terms of molecular function,it was mainly related to protein binding,ATP binding,and protein-containing complex binding.KEGG enrichment analysis mainly involved phosphatidylinositol signaling system,protein processing in endoplasmic reticulum,tyrosine metabolism,PI3K-Akt signaling pathway,FoxO signaling pathway,Ras signaling pathway,VEGF signaling pathway,etc.The molecular docking results showed that the binding energy of the top 10 core targets and the 6 main active ingredients of Sihuang Zhili granules was less than －5 kJ/mol,indicating that the docking effect was good.Among them,acacetin had the lowest binding energy to heat shock protein (7D1V),which was －9.4 kJ/mol.100 ns molecular dynamics simulation further confirmed that acacetin had good binding stability and activity with 7D1V. 【Conclusion】 The prediction results showed that Sihuang Zhili granules acted on tumor necrosis factor (TNF),heat shock protein 90α family class A member 1 (HSP90AA1),rapamycin target protein (mTOR),estrogen receptor 1 (ESR1) and other core targets through key active ingredients such as norwogonin,wogonin,kaempferol,5,7,2',6'-tetrahydroxyflavone,acacetin,and oroxylin A,and other core targets,through tyrosine metabolism,PI3K-Akt signaling pathway,FoxO signaling pathway,phosphatidylinositol signaling system and other pathways played a role in the treatment of livestock and poultry diarrhea.

Paeonol (Pae),the main active component derived from the dried root bark of Paeonia suffruticosa,exhibits a wide range of biological functions,including anti-inflammatory,anti-tumor,neuroprotective,cardiovascular protective,metabolic regulatory,analgesic,and antimicrobial effects.Due to its multi-target and low-toxicity characteristics,Pae has gradually become a research hotspot in the field of livestock and poultry disease prevention and treatment.Pae can alleviate inflammatory responses by inhibiting the NF-κB and MAPK signaling pathways and reducing the expression of pro-inflammatory factors,demonstrating significant anti-inflammatory effects in diseases such as bovine mastitis,fish inflammation models,and livestock arthritis.Meanwhile,Pae also exhibits anti-tumor properties by inducing tumor cell apoptosis and inhibiting cell proliferation and migration,showing potential therapeutic effects on non-small cell lung cancer,ovarian cancer,and other tumor-related diseases.In terms of neuroprotection and cardiovascular protection,Pae mitigates symptoms of neurodegenerative diseases such as Parkinson’s disease by inhibiting neuronal apoptosis and regulating neuroinflammatory responses,while also reducing pathological damage in cardiovascular diseases by suppressing myocardial cell hypertrophy,fibrosis,and oxidative stress,improving myocardial ischemia in canine acute myocardial infarction models,and exerting anti-endothelial cell aging and thrombolytic effects.Additionally,Pae has metabolic regulatory functions,improving metabolic disorders such as diabetes and hyperlipidemia by modulating insulin levels and lipid metabolism,thereby alleviating related complications.In the field of anti-inflammatory applications in livestock and poultry,research on Pae has focused on diseases such as arthritis,pneumonia,colitis,dermatitis,neuritis,periodontitis,acute pancreatitis,and endometritis.Studies have shown that Pae can alleviate inflammatory responses through multiple mechanisms,including the inhibition of the TLR4/NF-κB signaling pathway,regulation of inflammatory cytokine expression,and improvement of gut microbiota.This review systematically summarizes the biological functions of Pae and its research progress in anti-inflammatory applications in livestock and poultry,aiming to provide a scientific basis for its clinical application in veterinary medicine and promote its development as a safe and effective natural drug.

【Objective】 The purpose of this study was to evaluate the effects of chemotherapeutic drugs on the secretion of interferon-γ (IFN-γ),antitumor function,proliferation and cytotoxicity of T cells derived from peripheral blood of dogs,so as to screen chemotherapeutic drugs with little toxicity and impact on T cell function,in order to use alone or in combination with other immunotherapeutic drugs for the clinical treatment of canine tumor disease. 【Method】 Peripheral blood was collected from healthy dogs,and canine peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation.T cells were cultured and expanded in vitro,and their proportion before and after expansion was verified by flow cytometry.103 chemotherapy drugs were applied to canine T cells at a concentration of 20 μmol/L for 48 h,and the release of IFN-γ was measured by ELISA.Based on the previous experimental data in the laboratory,drugs with anti-tumor activity and a small impact on the secretion of IFN-γ in T cells were selected.After pretreating T cells at a concentration of 20 μmol/L for 12 h,the effect of the drugs on the anti-tumor function of T cells was detected by crystal violet staining,and the toxic effects of the selected chemotherapy drugs on T cells were evaluated in detail through cytotoxicity test and LDH test. 【Result】 Canine peripheral blood T cells were successfully expanded in vitro,reaching 99.70% purity.ELISA results demonstrated that of the 103 drugs tested,14 drugs (13.59%) (including docetaxel) significantly promoted the IFN-γ secretion of T cell (P＜0.05),34 drugs (33.01%) (including brusatol) significantly inhibited the release of IFN-γ (P＜0.05),while 55 drugs (53.40%) (C8-ceramide,etc.) had no statistically significant effect(P＞0.05).Combined with previous experimental data,10 candidate chemotherapy drugs including vinblastine sulfate,C8-ceramide,2'-O-(2-methoxyethyl)-cytidine and docetaxel were identified that preserved IFN-γ production in T cells while maintaining anti-tumor efficacy against canine mammary tumors.Further functional assays confirmed that drugs such as 6-mercaptopurine,docetaxel trihydrate and 2'-O-(2-methoxyethyl)-cytidine did not significantly impair T-cell-mediated antitumor activity.Among them,2'-O-(2-methoxyethyl)-cytidine showed relatively low toxicity to canine T cells,preserving ＞85% proliferation viability while causing no significant cell death. 【Conclusion】 Compounds such as vincristine sulfate,C8-ceramide,2'-O-(2-methoxyethyl)-cytidine and docetaxel had little toxicity on canine T cells.Among them,2'-O-(2-methoxyethyl)-cytidine had low toxicity on T cells and no significant inhibitory effect on their function,and it was expected to become the preferred drug for chemotherapy or combination therapy of canine tumors.

Neutrophil extracellular traps (NETs) are complex structures released by neutrophils in response to inflammatory reactions,composed of DNA,histones,and various antimicrobial proteins,forming a web-like structure.While they defend against pathogen invasion,excessive accumulation can also lead to tissue damage and exacerbation of inflammation.In recent years,the pathological role of NETs in acute lung injury (ALI) has attracted widespread attention.The authors summarize the molecular mechanisms of NET formation in ALI,their pathological effects,and their potential as therapeutic targets.This article outlines the composition,structure,and formation process of NETs,revealing the DNA backbone in NETs and the binding pattern of histones and antimicrobial proteins,as well as the activation of peptidylarginine deiminase 4 (PAD4) in neutrophils that leads to the citrullination of histones,reducing their binding affinity to DNA and ultimately resulting in the formation and extrusion of NETs.This review analyzes the signaling pathways and regulatory mechanisms of NET formation,including the production of reactive oxygen species (ROS),chromatin decondensation,and the role of autophagy,which collectively promote the formation of NETs.Subsequently,this article discusses the pathological role of NETs in ALI,including their relationship with various stimuli,their impact on alveolar injury and inflammatory responses,and the destructive effects of NET components on alveolar epithelial and endothelial cells.Additionally,this review assesses the potential of NETs as therapeutic targets for ALI,including the possibility of NET components as diagnostic biomarkers and the detection methods for NET-related biomarkers in ALI.Lastly,the strategies of NETs in the treatment of ALI are explored,including pharmacological interventions and immunomodulatory methods,and their roles in regulating the formation and degradation of NETs,so as to provide a theoretical basis for the treatment of ALI and offer new directions for future research.

【Objective】 Liquid chromatograph-mass spectrometer (LC-MS) and network pharmacology were used to explore the active ingredients of Echinacea purpurea (EP),targets and potential mechanism of EP against Porcine epidemic diarrhea virus (PEDV),so as to provide a theoretical basis for the further development of new anti-PEDV therapeutic drugs. 【Method】 A PEDV-infected IPEC-J2 cell model was constructed,and the cell model was treated with EP extracts of different concentrations.The mRNA transcription level of PEDV N protein was detected by Real-time quantitative PCR,and the effect of EP extract on PEDV replication was analyzed.The chemical constituents in EP were qualitatively analyzed by LC-MS.TCMSP,SwissTargetPrediction,NCBI databases were used to search the active ingredients of EP and the molecular targets related to anti-PEDV effects.The intersection targets of EP and PEDV were obtained and entered into the STRING database to construct protein-protein interaction (PPI) network and screen the key targets.The "EP-active ingredient-intersection target" network was constructed,and GO function and KEGG signal pathways of intersection targets were analyzed using the DAVID database to predict the core components,key targets and potential mechanisms of the anti-PEDV effect of EP.Finally,molecular docking was used to verify the binding ability of the main core components and key targets. 【Result】 In vitro results showed that compared with control group,EP extract significantly inhibited PEDV N protein mRNA transcription levels.LC-MS detection showed that EP contained 81 chemical components such as betaine,ellagic acid and cichoriic acid,and the highest classification proportion was mainly phenols,terpenoids and flavonoids.Based on network pharmacological screening,18 medicinal active ingredients such as ellagic acid,(+)-catechin and matrine in EP might exert anti-PEDV effects on 144 targets.The important core components of EP against PEDV included kaempferol,urushetin,luteolin,etc.The key targets included tumor protein 53 (TP53),interleukin-6 (IL-6),steroid receptor coactivator (SRC),mitogen-activated protein kinase 1 (MAPK1),etc.,mainly involved in Th17 cell differentiation,NOD-like receptor signaling pathway,FoxO signaling pathway,etc.,which involved in cellular immunity,apoptosis,cell cycle and other processes.The molecular docking results showed that the binding energy of ellagic acid with TP53,IL-6,SRC and MAPK1 was all less than －29.308 kJ/mol,among which ellagic acid had the strongest affinity with MAPK1. 【Conclusion】This study demonstrated that the anti-PEDV effect of EP was based on the synergistic action of multiple components, multiple targets, and multiple pathways. EP mainly used kaempferol, urushetin, luteolin and other core components to act on TP53, IL-6, SRC, MAPK1 and other targets, and regulated signaling pathways such as Th17 cell differentiation, NOD-like receptor and FoxO to treat PEDV.

Nonalcoholic fatty liver disease (NAFLD) is relatively common in clinical practice.It is a syndrome caused by nonalcoholic factors leading to secondary metabolic dysfunction of liver.Its main characteristics are steatosis of liver cells and excessive accumulation of fatin the liver.With the continuous improvement of people’s living standards,NAFLD caused by excessive fat intake has become widely prevalent among humans and pets,and has become one of the most common chronic liver diseases globally.NAFLD is a key factor leading to the increasing incidence and mortality of liver and metabolic diseases.At present,the treatment for NAFLD mainly focuses on alleviating symptomos,and it is urgent to explore more effective treatment strategies.Flavonoids from traditional Chinese medicine，with multi-component combinations,multi-target effects and diverse mechanisms,have shown great potential in the intervention of NAFLD.The author reviewed the pathways by which flavonoids improve NAFLD,such as improving insulin resistance,relieving oxidative stress,inhibiting inflammatory responses,anti-ferroptosis,anti-apoptosis,regulating autophagy levels and regulating intestinal flora,etc.Meanwhile,the molecular targets/signaling pathways of flavonoids in improving NAFLD were summarized,so as to provide references for the basic research,drug development and clinical application of flavonoids in the intervention of NAFLD.

【Objective】 The experiment aimed to evaluate the antibacterial activity of terpinen-4-ol and provide support for its structural optimization and clinical application. 【Method】 Escherichia coli (E.coli) was selected as the model bacterium in this study.The minimum inhibitory concentration (MIC) of terpinen-4-ol against E.coli was determined by double dilution method.The antibacterial curve was measured by D_{600 nm} value.The effects of terpinen-4-ol on cell morphology of E.coli were analyzed by scanning electron microscopy.Then,the effect of terpinen-4-ol on E.coli cell membrane was evaluated by measuring the concentration of extracellular alkaline phosphatase (ALP) and β-galactosidase.Simultaneously,the leakage of nucleic acid and protein was determined using ultra micro spectrophotometer.The interaction of terpinen-4-ol with E.coli DNA was detected by nucleic acid gel electrophoresis. 【Result】 The MIC of terpinen-4-ol against E.coli was 0.013 mol/L.The antibacterial effect of terpinen-4-ol on E.coli was dose-dependent,and 1 MIC of terpinen-4-ol had good antibacterial effect within 24 h.E.coli treated with terpinen-4-ol became swelling and deformation,rough surface,and adhesion between bacterial bodies.Compared with control group,0.5 MIC of terpinen-4-ol could lead to a significant increase in the content of ALP in the supernatant of E.coli (P＜0.05) and an extremely significant increase in the content of β-galactosidase (P＜0.01),and the increase was positively correlated with the concentration of terpinen-4-ol.Compared with control group,0.5 MIC of terpinen-4-ol could also increase the contents of proteins and nucleic acids in the supernatant of E.coli,and it was positively correlated with the concentration of terpinen-4-ol and the duration of action.There was no binding between terpinen-4-ol and the E.coli DNA. 【Conclusion】 Terpinen-4-ol had a good antibacterial effect on E.coli,and exerted antibacterial effects by disrupting the permeability of cell membranes,and then leading to leakage of cellular contents and bacterial death,which laid a foundation for further research on the antibacterial mechanism of terpinen-4-ol on E.coli.

Bacterial infection refers to an acute systemic infection caused by pathogenic bacteria or conditional pathogenic bacteria that invade the blood circulation to grow and multiply and produce toxins and other metabolites.It evades autophagy by destroying or utilizing autophagic virulence proteins or related molecules,affecting various organs and tissues,and causing serious impacts on the health and production performance of livestock and poultry.Due to the characteristics of diverse serotypes,high drug resistance and long survival time in nature,which makes the prevention and control of bacteria particularly complex. At the same time,bacterial infection involves multiple pathways,and most of the research mainly focuses on bacterial metabolism,nucleic acid synthesis,toxins and virulence factors,immune regulation,inflammation and immunopathological mechanisms,etc.Meanwhile,autophagy is closely related to the mechanism.Therefore,the research on bacterial infection and autophagy had gradually become one of the hot spots in the intersection of microbiology,immunology and cell biology at present.Autophagy plays an important role in the host’s defense against bacterial infections,but bacteria may also evade the host’s immune clearance by interfering with the autophagy pathway.Therefore,an in-depth analysis of the molecular mechanism of the interaction between bacteria and autophagy and the exploration of new autophagy regulatory targets are of specific significance for the development of novel antibacterial drugs and treatment regimens.The author summarized the role of autophagy in bacterial infections and discussed how host cells evade bacterial infections in the body through the autophagy mechanism,so as to provide new ideas for targeted autophagy treatment of bacterial infection.

Twin pregnancy in equine species primarily originates from dizygotic twinning resulting from multiple ovulations,and its occurrence is influenced by factors such as breed,age,reproductive status and season.Certain breeds，for example,Thoroughbreds,Catalan donkeys and Mammoth donkeys,exhibit a higher incidence of double ovulation,and aged or barren mares also show elevated rates of twin pregnancies.Seasonal variations further modulate mare reproductive activity,thereby affecting the overall incidence of twin pregnancies.Twin gestation in equines can lead to complications including abortion,dystocia,and neonatal foal mortality,ultimately resulting in wasted reproductive resources and economic losses to the horse and donkey industries.Therefore,early diagnosis and timely management are critical for improving the survival rates of singleton pregnancies.The optimal diagnostic window for twin pregnancy is on gestational days 14 to 16,with transrectal ultrasonography being the preferred method due to its ability to accurately distinguish between embryonic vesicles and endometrial cysts.Depending on the stage of gestation,various management strategies may be applied.In early gestation (＜16 days),manual twin reduction using embryonic vesicle crushing yields the highest success rates and is effective for both unilaterally and bilaterally fixed twins.For gestational ages between 28 and 32 days,transvaginal ultrasound-guided aspiration is recommended to minimize adverse effects on the remaining embryo.Beyond 35 days,as fetal organ development advances,common techniques include oscillation,thoracic compression,cranio-cervical dislocation,or pharmacologically induced abortion.For late-stage twin pregnancies,the choice of management must be carefully balanced with the health condition of the mare,overall reproductive management goals,and the need for rigorous postoperative monitoring to reduce the risk of adverse outcomes.This review comprehensively examines the etiology,influencing factors,diagnostic methods and management strategies for equine twin pregnancy,aiming to enhance the understanding of veterinary practitioners,optimize reproductive management,mitigate pregnancy-associated risks,and ultimately improve embryo survival rates to promote the sustainable development of the horse and donkey industries.