【Objective】 This study was aimed to clone the coding (CDS) region of mediator complex subunit 28 (MED28) gene,predict the structure and function of MED28 protein,and analyze the expression of MED28 gene in various tissues of dairy cows,so as to provide a foundation for further exploration of the function of MED28 gene.【Method】 The CDS region of MED28 gene was amplified using PCR and cloned with cDNA template derived from myoblast RNA reverse transcription.Similarity alignment and phylogenetic tree construction of MED28 protein were performed using MegAlign and Mega 11.0 softwares,respectively.Additionally,various online platforms,including ProtParam,ProtScale,SOPMA,SWISS-MODEL,MEME and STRING,were employed to analyze the physicochemical properties and structural functions.The expression of MED28 gene in heart,liver,spleen,lung kidney,small intestine and muscle of dairy cows were detected using Real-time quantitative PCR.【Result】 The CDS region of MED28 gene was successfully amplified,the length was 537 bp which encoded 178 amino acids.Similarity alignment results showed that MED28 protein of dairy cows shared the highest similarity with Capra hircus and Ovis aries.Phylogenetic tree showed that there were the closest genetic relationship between dairy cows and Capra hircus and Ovis aries,and there was the farthest genetic relationship between dairy cows and Gallus gallus.The MED28 protein,containing 2 N-glycosylation sites and 21 phosphorylation sites,was characterized as an unstable,hydrophilic protein lacking transmembrane regions and signal peptides,predominantly functioning extracellularly.The secondary structure of MED28 protein was composed of alpha helix,beta turn,extended chain,and random coil,with alpha helix being predominant,suggesting a stable protein structure that was consistent with predicted tertiary structure.Protein-protein interaction network analysis demonstrated that MED28 protein interacted with many proteins in its family,and they mainly played a role in transcriptional pathways and thyroid signaling pathways.MEME prediction indicated that the conserved motifs of MED28 protein were the same among dairy cows,Homo sapiens,Sus scrofa,Mus musculus,Equus caballus,Oryctolagus cuniculus,Capra hircus and Ovis aries.MED28 protein domain was composed of low complexity and coiled coil sequences.Real-time quantitative PCR results showed that the expression of MED28 gene in muscle of dairy cows was significantly higher than that in other tissues (P＜0.05).【Conclusion】 The genetic relationship was the closest between dairy cows and Capra hircus and Ovis aries,and the protein similarity was high.MED28 protein exhibited a hydrophilic and structurally stable nature,featuring N-glycosylation and phosphorylation sites,while lacking a transmembrane region and signal peptide.MED28 protein functioned mainly in extracellular and was involved in transcriptional pathways and thyroid signaling pathways.The expression of MED28 gene was the highest in muscle of dairy cows.The results could provide reference for further study on the function of bovine MED28 gene.

【Objective】 This study was aimed to establish a rapid detection method for Porcine reproductive and respiratory syndrome virus (PRRSV) based on the combination of CRISPR/Cas12a system and reverse transcription recombinase aided amplification (RT-RAA) technology.【Method】 The whole genome sequences of different genotypes of PRRSV were analyzed,and recombinase aided amplification (RAA) primers,crRNA and DNA reporter substrate molecules were designed in the conserved region of the 3'-UTR terminal,and the best primer pairs of RT-RAA were screened.AsCas12a protein was expressed and purified,and the plasmid was used as the standard product.After amplification by RT-RAA,the product was added to CRISPR/Cas12a system to establish the CRISPR/ Cas12A-RT-RAA detection method for PRRSV.Nucleic acids of PRRSV,Porcine circovirus type 2 (PCV2),Porcine epidemic diarrhea virus (PEDV),Classical swine fever virus (CSFV) and Porcine pseudorabies virus (PRV) were used as templates to verify the specificity of the established method,and different concentrations of pMD18T-PRRSV were used as templates to verify the sensitivity of the established method.The plasmids of 5.62×10⁶,5.62×10³ and 5.62 copies/μL were used as templates for repeat tests,and finally the clinical effect was evaluated,and the established method was compared with reverse transcription Real-time quantitative PCR.【Result】 This study successfully expressed and purified AsCas12a protein with good shear activity,with a molecular weight of 196 ku.The detection lower limit of the CRISPR/Cas12a RT-RAA method established could reach 5.62 copies/μL.This method had good specificity and could specifically detect PRRSV,but could not detect other porcine disease viruses such as PCV2,PEDV,CSFV and PRV.And it had good repeatability.This method was applied to detect 121 pig tissue samples,the positive conformity rate with reverse transcription Real-time quantitative PCR was 93.75%,and the negative conformity rate was 99.05%,with a total conformity rate of 99.17%.【Conclusion】 This study successfully established a rapid detection method for PRRSV based on CRISPR/Cas12a-RT-RAA.The method had strong specificity,high sensitivity,good repeatability,and did not rely on complex equipment.It could be used in both on-site and grassroots laboratories.

【Objective】 The aim of this study was to evaluate the protective effect of lycopene (LCP) on organ injury induced by cisplatin (CDDP) in rats.【Method】 Thirty male Wistar rats were randomly divided into 5 groups (n＝6),which were control group (CON),CDDP and different LCP treatment groups (5 mg/kg,LLCP group;10 mg/kg,MLCP group;50 mg/kg,HLCP group).Rats in CON and CDDP groups were given 1 mL/kg corn oil per day,and rats in LLCP,MLCP and HLCP groups were given the corresponding concentration of LCP per day,and consecutive for 28 days.Except CON group,the other groups were given a single intraperitoneal injection of CDDP (7 mg/kg) on day 26.After the experiment,samples of stomach,small intestine (duodenum,jejunum and ileum),liver,kidney and blood were collected.The protective effects of different concentrations of LCP on CDDP-induced acute injury in rats were evaluated by physiological and biochemical indexes detection,HE staining,ELISA and Western blotting.【Result】 Compared with CON group,body weight,feed intake and water intake of rats in CDDP group were extremely significantly or significantly decreased (P＜0.01 or P＜0.05).Serum levels of aspartate aminotransferase (AST),urea nitrogen (BUN),creatinine (CRE) and malondialdehyde (MDA),interleukin-8 (IL-8),IL-6 and tumor necrosis factor-α (TNF-α) were significantly or extremely significantly increased (P＜0.05 or P＜0.01).The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were extremely significantly decreased (P＜0.01),and the histopathological injuries of stomach,small intestine,liver and kidney were serious.Compared with CDDP group,the body weight,feed intake and water intake of rats in MLCP group were significantly increased (P＜0.05),the levels of 6 biochemical indexes such as AST and ALT and the contents of MDA,IL-8,TNF-α and IL-6 in serum were significantly decreased (P＜0.05),and the organ histomathological damage was alleviated.Western blotting results showed that compared with CON group,the expression of TNF-α and IL-6 proteins in different tissues of rats in CDDP group were extremely significantly increased (P＜0.01),while the expression of IL-10 protein was extremely significantly decreased (P＜0.01).Compared with CDDP group,the expression of TNF-α and IL-6 proteins in all tissues of MLCP group were extremely significantly or significantly decreased (P＜0.01 or P＜0.05),the expression of IL-10 protein was extremely significantly or significantly increased (P＜0.01 or P＜0.05).【Conclusion】 LCP could improve the stomach,small intestine,liver and kidney injury in CDDP-induced rats by inhibiting oxidative stress and inflammation.Under this experiment conditions,10 mg/kg LCP had the best effect.

【Objective】 This experiment was to investigate the effect of cyclocarioside H on the damage of renal podiocytes (MPC-5) induced by high glucose by inhibiting the NLRP3/ASC/Caspase1 pathway.【Method】 MPC-5 cells were incubated with different concentrations (0,3.25,7.5,15,30,60 and 120 μmol/L) of cyclocarioside H,and the cell viability was detected by CCK-8 method to determine the safe concentration.MPC-5 cells were divided into blank control (CON),D(+) -anhydrous glucose isoosmotic control (MA),high glucose (HG) and cyclocarioside H with different concentrations (3.25,7.5 and 15 μmol/L) groups (CY3.25,CY7.5 and CY15).HG and CY3.25,CY7.5 and CY15 groups were added with 30 μmol/L D(+) -anhydrous glucose,MA group was added with 5.5 mmol/L D(+) -anhydrous glucose＋24.5 mmol/L mannitol,CON group was added with DMEM medium without FBS.After incubating the cells for 24 h,the culture medium of CY3.25,CY7.5 and CY15 groups was changed to different concentrations of cyclocarioside H,and the cells were incubated again for 24 h.The cells were collected,and CCK-8 and lactate hydrogenase (LDH) assay were used to determine cell viability and damage degree,respectively.The apoptosis rate was detected by fluorescent staining with propyl iodide (PI).The co-localization of NOD-like receptor heat protein domain associated protein 3 (NLRP3) and apoptosis-associated speck-like proteins (ASC) in MPC-5 cells was observed by immunofluorescence.The contents of Pro-IL-1β and IL-1β in the supernatant were detected by ELISA.Western blotting was used to detect the expression of NLRP3,ASC,Caspase1/Pro-Caspase1,Desmin,Nephrin,MFF and Collagen Ⅳ proteins.【Result】 Compared with 0 μmol/L cyclocarioside H group,the cell viability of 7.5 and 15 μmol/L cyclocarioside H groups was significantly increased (P＜0.05),and that of 30,60 and 120 μmol/L cyclocarioside H groups was significantly decreased (P＜0.05).Therefore,3.25-15 μmol/L was subsequently selected as the safe concentration of cyclocarioside H.The cells were treated with the above safe concentration of cyclocarioside H,and the results showed that compared with HG group,cell viability in CY3.25,CY7.5 and CY15 groups was significantly increased (P＜0.05),while LDH activity in cell supernatant was significantly decreased (P＜0.05),and in a dose-dependent manner.The apoptosis rate of CY7.5 and CY15 groups was significantly decreased (P＜0.05).The results of immunofluorescence assay showed that compared with CON group,the fluorescence of NLRP3 and ASC in HG group was enhanced and the red and green fluorescence labels overlapped.Compared with HG group,the fluorescence of NLRP3 and ASC in CY3.25 and CY7.5 groups was weakened and there was no overlap of red and green fluorescence labels,while there was no positive fluorescence of NLRP3 and ASC in CY15 group.ELISA results showed that compared with HG group,the ratio of IL-1β and Pro-IL-1β in CY15 group was significantly decreased (P＜0.05).Western blotting results showed that compared with HG group,the expressions of NLRP3,ASC,Desmin,MFF, Collagen Ⅳ and Caspase1/Pro-Caspase1 in CY15 group were significantly decreased (P＜0.05),while the expression of Nephrin protein was significantly increased (P＜0.05).【Conclusion】 Cyclocarioside H could improve the damage of MPC-5 cells induced by high glucose,mainly by altering the expression of related proteins in the NLRP3/ASC/Caspase1 pathway,thereby exerting a protective effect on cells.

【Objective】 This study was conducted to investigate the effect of chebuli extract (CE) on the intestinal morphology,intestinal and liver inflammatory responses of lipopolysaccharide (LPS)-stimulated broilers,so as to provide a theoretical basis for the development of functional feed additives.【Method】 This experiment adopted a two-factor experimental design.40 healthy 1-day-old Ross 308 broilers were selected and randomly divided into control and CE treatment groups.Each group had 5 replicates with 4 broilers in each replicate.The broilers in control group were fed basal diet,and broilers in CE treatment group was supplemented with 1 000 mg/kg CE on the basis of basal diet.The experimental period was 29 days.On the 29th day,2 broilers in each replicate were intraperitoneally injected with 1 mg/kg BW LPS,and the other 2 broilers were intraperitoneally injected with normal saline.Slaughter sampling was performed 3 h after LPS injection.The liver,spleen,thymus and bursa of Fabricius of broilers were weighed to calculate organ coefficients.Intestinal tissue morphology,intestinal barrier-related genes,and intestinal and liver inflammation-related genes of broilers were measured.【Result】 ① Compared with broilers without LPS stimulation,LPS stimulation significantly reduced villus height,crypt depth,villus height/crypt depth and villus area of duodenum,and jejunum villus height and ileum villus area in broilers (P＜0.05);Adding CE significantly reduced the jejunum crypt depth,and significantly increased villus height,villus height/crypt depth and villus area of jejunum and ileum (P＜0.05).Compared with LPS-stimulated broilers,adding CE significantly increased villus height of duodenum,jejunum and ileum,villus height/crypt depth of duodenum and ileum,and villus area of jejunum and ileum (P＜0.05).② Compared with broilers without LPS stimulation,adding CE significantly increased the expression of Mucin-2,zonula occludin 1 (ZO-1) and Occludin genes in broilers (P＜0.05).Compared with LPS-stimulated broilers,adding CE significantly increased the expression of Claudin-1, Mucin-2 and ZO-1 genes in broilers (P＜0.05).③ Compared with broilers without LPS stimulation,LPS stimulation significantly increased the expression of matrix metalloproteinase 9 (MMP9),interleukin-1β (IL-1β),IL-8 and tumor necrosis factor-α (TNF-α) genes in liver of broilers (P＜0.05);Adding CE significantly reduced the expression of MMP9,IL-1β and IL-8 genes in liver of broilers (P＜0.05).Compared with LPS-stimulated broilers,adding CE significantly decreased the expression of MMP9 and interferon-γ (IFN-γ) genes in liver of broilers (P＜0.05).【Conclusion】 CE improved the intestinal morphology and structure of LPS-stimulated broilers,enhanced the intestinal barrier function,and mitigated the inflammatory response in liver of LPS-stimulated broilers.The results provided a theoretical basis for the application of CE as a natural feed additive.

【Objective】 The aim of this study was to explore the effects of adding different concentrations of carvacrol to the diet on the growth performance,serum biochemical indexes and intestinal microbiota of mink during the breeding period,so as to find out the optimal concentration of carvacrol which was beneficial to mink in various aspects.【Method】 Forty Short-haired black mink were randomly divided into four groups of 10 minks each,with one replication for each mink.Minks in control group were fed with basal diet,while minks in groups A,B and C were supplemented with carvacrol at concentrations of 100,200 and 300 mg/kg in addition to the basic diet,respectively.The pre-test period was 7 d and the main test period was 42 d.The growth performance,serum biochemical indexes and intestinal microbiota were tested at the end of the feeding period.【Result】 The results showed that adding carvacrol had no effect on the growth performance (except for final body weight) of minks (P＞0.05).300 mg/kg carvacrol could significantly increase the activity of catalase (CAT) and crypt depth in minks (P＜0.05).100 mg/kg carvacrol could significantly increase the content of immunoglobulin A (IgA) in minks (P＜0.05).200 and 300 mg/kg carvacrol could extremely significantly reduce the content of malondialdehyde (MDA) (P＜0.01),and significantly increase the jejunal villus heigth in minks (P＜0.05).High throughput sequencing results showed that a total of 9 phyla dominated by Firmicutes were detected in the bacterial community with abundance greater than 1%,and 43 genera dominated by Lactococcus and Streptococcus.【Conclusion】 The addition of 300 mg/kg carvacrol to mink feed could improve jejunal intestinal development and intestinal microbiota structure,and enhance the health status of minks.

【Objective】 The aim of this trial was to investigate the effects of heat stress on fecal flora structure and metabolites in goats.【Method】 Six adult castrated rams of Dazu Montenegrin sheep with similar age and body weight of 28.4 kg±3.2 kg were selected,and a single factor self-control experimental design was used.They were subjected to three stages of treatment:Severe heat stress (H group,temperature and humidity index (THI)=91),moderate heat stress (M group,THI=84),and no heat stress (L group,THI=71).Each treatment stage was 15 days,and the transition period was 7 days.Rectal feces were collected at 14:00 on the last day of each stage for microbial 16S rRNA sequencing and metabolomics analysis.【Result】 There was no significant difference in the diversity of goat fecal flora among the three groups (P＞0.05).Compared with group L,in H and M groups,the relative abundance of Bacteroidetes was significantly decreased (P＜0.05),and the relative abundance of Proteobacteria was significantly increased (P＜0.05). The relative abundances of Christensenaceae R-7 group,Clostridium sensu stricto 1,and Family XⅢ AD3011 group were significantly increased (P＜0.05),whereas the relative abundances of Bacteroides and unclassified_f_Lachnospiraceae were significantly decreased (P＜0.05). The contents of atrazine,cholic acid,taurocholate,oleic acid,hyperforin,and taurocholic acid in feces were significantly increased (P＜0.05),whereas the contents of Pro-Ala,Arg-Thr and homoveratric acid were significantly decreased (P＜0.05).Differential metabolites in feces under heat stress conditions were significantly enriched in the pathways of taurine and hypotaurine metabolism,ABC transporters,glutathione metabolism and gap junction (P＜0.05).【Conclusion】 Heat stress led to changes in the structure of goat fecal flora,which was not conducive to goat intestinal health. It also upregulated the biosynthetic metabolic pathways of arginine and primary bile acids,which helped goats adapt to high humidity and heat environments. It also led to an increase in the content of toxic substances in goat metabolites,which might cause damage to the intestinal barrier and nervous system.

【Objective】 The objective of this experiment was to investigate the effect of different protein levels on the reproductive performance,digestive metabolism,and plasma biochemical indicators of Tarim pigeons during breeding period under "2＋2" production mode,in which a pair of breeding pigeons feeds 2 squabs simultaneously.【Method】 60 pairs of healthy Tarim breeding pigeons aged 12-18 months,with similar reproductive performance,were randomly divided into 5 groups,with 12 pairs in each group,and each pair bred 2 squabs.The pigeons in groups Ⅰ,Ⅱ,Ⅲ,Ⅳ and Ⅴ were fed with different crude protein (CP) levels (12%,13%,14%,15% and 16%).The experiment had a 3-day pre-experimental period and a 28-day experimental period.At the end of the breeding period,the performance,digestion and metabolism and plasma biochemical indicators of pigeons were measured.【Result】 ① The dietary protein levels significantly affected the average daily feed intake (ADFI) of pigeons during the breeding period,and ADFI of pigeons in group Ⅱ was significantly higher than that in groups Ⅳ and Ⅴ (P＜0.05).The weight loss of pigeons was gradually increased during breeding period.② With the protein levels increased,the apparent digestibility of CP in pigeons was gradually increased.The dietary protein levels significantly affected the apparent digestibility of CP in pigeons during breeding period,and the apparent digestibility of CP in group Ⅰ was significantly lower than that in groups Ⅳ and Ⅴ (P＜0.05).③ The dietary protein levels significantly affected the levels of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in plasma of pigeons during breeding period,the HDL level of pigeons in group Ⅲ was significantly lower than that in other groups,and the LDL level of pigeons in group Ⅲ was significantly higher than that in other groups (P＜0.05).【Conclusion】 Under the conditions of this experiment,13% CP was recommended to be applied as the protein dietary requirement of Tarim pigeons during breeding period in "2＋2" pattern.

【Objective】 The aim of this study was to explore the effects of dietary supplementation with guanidinoacetic acid on the growth performance,serum biochemical indices,antioxidant capacity and immune function of finishing pigs,and provide reference for the application of guanidinoacetic acid in pig breeding industry.【Method】 Thirty-six healthy Duroc×Landrace×Yorkshire three-way crossbred sows with similar body weights were randomly divided into 3 groups with 6 replicates per group and 2 sows per replicate.Finishing pigs in control group were fed basal diet,and finishing pigs in experiment group were supplemented with 0.1% guanidinoacetic acid (GAA group) and 0.1% guanidinoacetic acid + 0.04% betaine (GAA+BT group) in basal diet,respectively.The experiment lasted for 50 d.At the end of the experiment,finishing pigs were weighed on empty stomachs in replicates and calculated for growth performance,one pig was selected from each replicate for vena cava blood sampling to determine serum biochemical indices,antioxidant capacity and immune function.【Result】 ① Dietary supplementation with guanidinoacetic acid had no significant effect on growth performance of finishing pigs (P＞0.05).② Compared with control group,the contents of total protein,globulin,interleukin-2,interleukin-10,interferon-γ and immunoglobulin A in serum of finishing pigs in GAA and GAA+BT groups were significantly increased,and the immunoglobulin G content in serum of finishing pigs in GAA group was significantly increased (P＜0.05).The levels of creatinine,uric acid and lactate dehydrogenase in serum of finishing pigs in GAA and GAA+BT groups were significantly decreased,and the malondialdehyde content in serum of finishing pigs in GAA+BT group was significantly decreased (P＜0.05).③ Compared with control and GAA groups,the total antioxidant capacity in serum of finishing pigs in GAA+BT group was significantly increased (P＜0.05).④ Compared with control and GAA+BT groups,the tumor necrosis factor-α content in serum of finishing pigs in GAA group was significantly increased (P＜0.05).【Conclusion】 Dietary supplementation with guanidinoacetic acid did not affect the growth performance of finishing pigs,however,it could improve the metabolism level,enhance the antioxidant capacity and immune function of finishing pigs,and it was effective both alone and in combination with the addition of betaine.

【Objective】 As an organic calcium source with small molecular weight,calcium formate has a good application potential in improving the production of laying hens.The purpose of this study was to reveal the effect of dietary calcium formate supplementation on the production performance,blood indexes and egg quality of elderly laying hens,and the appropriate level of supplementation.【Method】 A total of 360 62-week-old healthy Hy-Line Brown laying hens were randomly divided into 4 treatment groups,with 6 replicates and 15 chickens per replicate.The hens in control group were fed a basal diet,and the hens in three experimental groups were fed 0.5%,1.0% and 2.0% calcium formate to partially replace the basal diet of stone powder,respectively,and the calcium level of each group was same.The formal feeding period was 12 weeks.The egg production was recorded every day,and the feed weekly.At weeks 2,4,6 and 8,egg quality was determined.At the end of the feeding,one layer per replicate was selected for blood samples collection,and blood routine and serum biochemical indexes were measured,and organ index and tibial mass were determined after slaughter.【Result】 Compared with control group,calcium formate supplementation had no effects on the performance of laying hens and the egg quality at the second and fourth weeks (P＞0.05),but increased the eggshell ratio,eggshell thickness and eggshell strength at the eighth and twelfth weeks (P＜0.05),and the 0.5% addition level had the best effect.Calcium formate supplementation reduced the content of mononuclear leukocytes,basophils and neutrophils in the blood of laying hens (P＜0.05),and increased the content of hemoglobin and hematocrit (P＜0.05).Calcium formate supplementation decreased the serum total protein,globulin,albumin and creatinine contents (P＜0.05),and increased the total bilirubin content (P＜0.05).The pancreas,spleen,leg muscles and shinbone index of laying hens were not affected by calcium formate supplementation (P＞0.05),but the work required for shinbone fracture increased (0.05＜P＜0.1).【Conclusion】 Dietary addition of calcium formate to partially replace stone powder did not affect the production performance of elderly laying hens,while could improve the health of the body and the quality of eggshells,and the recommended addition level was 0.5%.

【Objective】 This experiment was conducted to study the effects of high and low nutrient level diets on growth performance,nutrient digestion and antioxidant property of Anhei crossbred cattle.【Method】 A comparative experimental design was used and twelve healthy Angus (♂)×Black Simmental (♀) cross cattle with similar body weight (206.08 kg±21.87 kg) and age (about 6 months,half male and half female) were randomly divided into two groups with 6 cattle in each group and fed low and high nutrient level diet respectively.The pre-trial and normal trial periods were 12 and 62 days,respectively.The comprehensive net energy (NE_mf) of the high nutrient level formula diet in the early (1-31 d) and late (32-62 d) stages were 5.77 and 6.00 MJ/kg,and the crude protein (CP) was 12.47% and 10.44%.The NE_mf of the low nutrient level formula diet in the early and late stages were 5.42 and 5.57 MJ/kg,and the CP were 9.66% and 9.05%.Body weight and body size were measured on days 1 and 62.The digestion experiment was done in the last 8 days,and the digestibility of organic matter (OM),CP,ether extract (EE),neutral detergent fiber (NDF) and acid detergent fiber (ADF) were measured.【Result】 In whole stage,the average daily gain,average daily feed intake and the abdominal circumference of the cattle in the high nutrient level group were significantly higher than those in the low nutrient level group by 39.09%,22.20% and 6.17% (P＜0.05).The F/G of the high nutrient level group had a lower tendency than that of the low nutrient level group (P=0.087).The digestibility of OM,CP,EE,NDF and ADF of Anhei crossbred cattle was not affected significantly by dietary nutrient levels (P＞0.05).The serum total antioxidant capacity of the cattle in the high nutrient level group was significantly lower than that in the low nutrient level group (P＜0.05),glutathione peroxidase activity and malondialdehyde content were not affected significantly by the treatments (P＞0.05).【Conclusion】 In conclusion,compared with the low nutrient level diet,the high nutrient level diet could better promote the growth and development of Anhei crossbred cattle.

【Objective】 Digestive dysfunction caused by high-concentration feeding in fattening lambs has been paid more attention in recent years.The objective of present study was to investigate the effects of two probiotics (Bacillus licheniformis and Clostridium butyricum) added alone or in combination in diet on growth performance,nutrient apparent digestibility and serum biochemical indexes of fattening lambs.【Method】 One hundred and twenty-five 3-4 months fattening lambs with body weight at 30.89 kg±0.38 kg were randomly allocated into five groups including control group,antibiotic group (20 mg/kg monensin) and three probiotic groups (Bacillus licheniformis 4×10¹⁰ CFU/kg,Clostridium butyricum 2×10⁸ CFU/kg,and Bacillus licheniformis 4×10¹⁰CFU/kg+Clostridium butyricum 2×10⁸ CFU/kg).The experiment lasted for 60 days including the early stage (1-30 d) and late stage (31-60 d).Growth performance and nutrient apparent digestibility in lambs were measured for two experimental stages.Serum biochemical parameters in lambs were measured on 1 st,30th and 60th day of the experiment.【Result】 There was no difference in average daily feed intake among all groups (P＞0.05).However,throughout the entire experimental period,compared with the control group the combination of two probiotics significantly increased average daily gain (P＜0.05), and feed/gain ratio (F/G) of three probiotic groups were all decreased. Moreover,in the late period,the F/G in the combined group was significantly lower than that in other two groups (P＜0.05).Additionally,the combination group exhibited a significant increase in apparent digestibility of dry matter during both early and late periods (P＜0.05).In the early period,all three probiotic groups showed a significant improvement in acid detergent fiber digestibility compared with control group (P＜0.05).Furthermore,serum glucose concentrations in three treatment groups increased by 16.97% to 28.18% during the late period when compared with control group (P＜0.05).【Conclusion】 The dietary supplementation of Bacillus licheniformis and Clostridium butyricum alone or in combination could improve growth and digestive performance of lambs,and the effect of combined supplement was optimal.

【Objective】 This experiment was aimed to investigate the impact of incorporating varying proportions of dried potatoes into the diets of growing and fattening pigs on their growth performance,carcass traits,meat quality,and routine blood indexes.The primary objective was to determine the optimal ratio of potatoes substitution for corn in pig rations and assess its effects on pig performance,thereby providing a scientific foundation for the utilization of potatoes as pig feed.【Method】 A total of 192 healthy three-way crossbred (Duroc×Landrace×Large White) growing and fattening pigs (half male and half female) with similar weights (33.0 kg±3.2 kg) were randomly divided into four groups, 6 replicates per group and 8 pigs per replicate. The pigs in control group received the basal diet,while the pigs in experimental groups were fed diets in which 10％,20％ and 30％ of the total corn content in the basal diet was replaced with an equivalent amount of dried potatoes.This feeding regimen was maintained for 120 days,during which the growth performance,carcass traits,meat quality traits,and blood indexes of each group were assessed.【Result】 The inclusion of 10％,20％ and 30％ dried potatoes in the ration did not cause significant effects on most of the indices related to fattening pig growth performance,carcass traits,and routine blood (P＞0.05) compared to control group.However,the eye muscle area of fattening pigs in 30％ potatoes addition group exhibited a significant increase (P＜0.05).Moreover,a significant reduction in backfat thickness was observed with the addition of 20% and 30% dried potatoes (P＜0.05).Regarding meat quality,the incorporation of 20％ and 30％ dried potatoes led to a significant increase in intramuscular fat content of longissimus dorsi muscle (P＜0.05) and meat color brightness (L^*_{45 min}) (P＜0.05),and a significant decrease in muscle shear strength (P＜0.05).There was a tendency for the drip loss rate to decrease with increasing potatoes content (0.05≤P＜0.1).The addition of 10% dried potato significantly increased the hematocrit and distribution width of red blood cells in blood of fattening pigs (P＜0.05).【Conclusion】 The addition of 10% dried potatoes significantly increased some of the red blood cell indices associated with anemia.However,the inclusion of 20％ and 30％ dried potatoes notably reduced backfat thickness in growing and fattening pigs while increasing intramuscular fat content and decreasing muscle shear force.It was recommended to incorporate dried potatoes at a level of 20％ to 30％ in the feed for growing and fattening pigs.

【Objective】 The aim of this experiment was to investigate the effects of supplemental feeding of different doses of branched-chain amino acids (BCAAs) to Yili mares during lactation on the growth performance and fecal flora of lactating foals,in order to provide a reference basis for safeguarding the growth and health of foals during lactation.【Method】 Twenty lactating mares and their foals (lactating foals between 2 and 3 months of age,with an initial weight of 97.60 kg±13.24 kg,lactating mares weighing 392.90 kg±12.18 kg) were selected and randomly divided into four groups of five mares each,the control group and the experimental groups Ⅰ,Ⅱ,and Ⅲ,and were kept under the same feeding management conditions for 67 d (7 d pre-feeding period,60 d experimental period).During the test period,mares in control and test groups were supplemented with 2 kg of concentrate per day,and mares in test groups Ⅰ,Ⅱ,and Ⅲ were supplemented with 38,76 and 114 g/d of BCAAs,respectively.Body measurements and body weights were taken on the day before experiment and 60th day of the experiment,and feces were collected by rectal fecal sampling on the 60th day,and fecal flora diversity of foals was analyzed.【Result】 On the 60th day of the trial,the breast circumference and body weight of foals in the test group Ⅲ were significantly higher than those in control group (P＜0.05),and the body weight of foals in the test group Ⅱ was significantly higher than those in control group (P＜0.05).The number of fecal flora OTUs was 4 553,4 483,4 607 and 3 839 in test groups Ⅰ,Ⅱ,Ⅲ,and control groups,respectively.There was no significant difference in the Alpha diversity index as well as the number of species observed among the groups (P＞0.05).The relative abundance of Spirochaetota in test group Ⅱ was significantly higher than that in control group and test group Ⅰ(P＜0.05),and the relative abundance of Lachnospiraceae was significantly higher than that of control and test group Ⅲ (P＜0.05).The relative abundance of Rumenococcaceae was significantly higher in both test groups Ⅰ and Ⅱ than control group (P＜0.05),and the relative abundance of Rikenellaceae in test group Ⅲ was significantly higher than that in control group (P＜0.05).At the genus level,the relative abundance of Treponema was the highest and significantly higher in test group Ⅱ than that in test group Ⅰ (P＜0.05),and the relative abundance of Ruminococcus was significantly higher than in the other three groups (P＜0.05).The relative abundance of Prevotellaceae_UCG_004 in test group Ⅲ was highly significantly higher than that of control group and test group Ⅰ (P＜0.01).The differential items exhibited by LEfSe analysis and PICRUSt2 function prediction were all related to intestinal fermentation metabolism,short-chain fatty acid production,and amino acid metabolism.【Conclusion】 Supplemental feeding of different doses of BCAAs to Yili mares could promote the growth of breast circumference and body weight of lactating foals,increase the abundance of beneficial intestinal flora,and decrease the abundance of harmful flora,and the best results were obtained by supplementing mares with 76 g/d of BCAAs.

Biologically active substances such as short-chain fatty acids (SCFAs) and bile acids produced by the metabolism of many microorganisms are present in animals.Among them,SCFAs are mainly produced by the fermentation of dietary fiber bacteria,which can be used as both an energy source for intestinal mucosal cells and a signaling substance.SCFAs can not only regulate the intestinal flora and enhance the intestinal barrier function,but also participate in the regulation of the nervous system through various pathways.Therefore,SCFAs are considered to be important factors affecting host health.In the context of a total antibiotic ban,finding suitable antibiotic alternatives has become a hot research topic.Exogenous addition of appropriate amounts of SCFAs can improve the enrichment of beneficial bacteria in the animal gut,increase the expression of tight junction proteins,and enhance the immune function of the body.In addition,SCFAs have some therapeutic potential in the study of central nervous system diseases,such as autism and depression.Most of SCFAs produced by gut microbial metabolism are acetic acid,propionic acid and butyric acid.Currently,the studies on SCFAs are dominated by butyric acid,while the studies on the combined effects of mixtures of SCFAs are relatively few,and the application areas are yet to be expanded.The authors summarized the potential effects of SCFAs on animal intestinal health and gut-brain signaling as well as the possible regulatory mechanisms,which provides a theoretical basis for the development of SCFAs as an exogenous additive or drug to be promoted in the modulation of animal health and disease treatment and other applications.

In recent years,dietary fiber (DF) as a non-grain feed resource has become a research hotspot in the development of low-protein feed and it is an important nutritional component of feed for pigs.Numerous studies have confirmed that DF plays multiple roles in regulating pig intestinal health and alleviating intestinal disorders,with its mechanism being highly complex.DF regulates the intestinal microbiota by selecting microbial communities and maintaining an anaerobic environment in the intestine.Simultaneously,it directly stimulates immune cells,upregulates the expression of tight junction proteins,and exerts immune-regulatory effects.Additionally,DF increases the number of intestinal crypts and the viscosity of chyme,and its metabolites provide the primary energy sources for intestinal epithelial cells and symbiotic microorganisms,promote the development and renewal cycle of intestinal crypt cells and epithelial cells,and thus improve the intestinal tissue morphology.The author systematically sorts out and summarizes the relevant research in recent years,and summarizes the definition and classification of DF and its research results in preventing pig intestinal disorders.Starting with the optimizing microbial community structure,improving the mucosal barrier and intestinal morphology,the specific function and potential mechanism of DF in regulating the intestinal health of pigs have been analyzed,and the existing problems and future research focus have been proposed,which provides new perspectives and evidence for further investigating the impact of DF on pig intestinal health,while also laying a theoretical foundation for the development of non-grain feed resources rich in DF and developing grain-saving and sustainable green pig breeding.

【Objective】 The aim of this study was to investigate the effects of adding cecropin antimicrobial peptide to diet on the growth performance,serum immune indicators,antioxidant function,and rumen microbiota structure of Hu sheep.【Method】 36 5-month-old male Hu sheep with similar body weight (33.47 kg±1.24 kg) were randomly divided into 2 groups with 3 replicates per group and 6 sheep per replicate.The sheep in control group (CON) was fed with basic feed, and in antimicrobial peptide group (AMP) was supplemented with 0.5 g/kg of cecropin antimicrobial peptide in basal diet.After a 30 day feeding experiment,serum and rumen fluid samples were collected, and the growth performance,serum immunoglobulin,cytokine content,antioxidant function,and rumen fluid microbial community structure of sheep were determined.【Result】 The final weight and average daily gain (ADG) of Hu sheep in AMP group were significantly higher than those in CON group (P＜0.05),while feed-to-gain ratio (F/G) was significantly lower than that in CON group (P＜0.05).The levels of immunoglobulin G (IgG),IgM,superoxide dismutase (SOD),and total antioxidant capacity (T-AOC) in serum of Hu sheep in AMP group were significantly higher than those in CON group (P＜0.05).Cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) of Hu sheep in AMP group were significantly lower than those in CON group (P＜0.05).The addition of cecropin antimicrobial peptides to the diet did not significantly affect the rumen microbiota richness indices Sobs index,Chao1 index,and Ace index,as well as diversity indices Shannon index and Simpson index in Hu sheep (P＞0.05).Relative abundances of Proteobacteria and Christensenellaceae_R-7_group in the rumen of Hu sheep in AMP group were significantly lower than those in CON group,while relative abundances of Bacteroidales_RF16_group,norank_f__F082,and NK4A214_group were significantly higher than those in CON group (P＜0.05).【Conclusion】 Adding 0.5 g/kg of cecropin antibacterial peptide to the diet was beneficial for improving the growth performance of fattening Hu sheep,enhancing immune and antioxidant performance,reducing the secretion level of inflammatory factors,and the abundance of harmful bacteria in the rumen,regulating the structure of rumen microorganisms.

Yak is known as the "Boat of the plateau",and yak meat is one of the most important meats in the life of Tibetan plateau herders.Yak meat is high in protein,low in fat,rich in many kinds of amino acids,etc.It is a natural green food.Due to geographical constraints,yaks can only live in high altitude areas,so the production scale can’t be expanded and the yak meat yield can’t be increased.Therefore,clarifying the quality characteristics of yak meat is of great significance for the development of yak meat products and the regulation of meat quality.Yak breeds are diverse,and there are differences in the nutritional composition of yak meat in different regions.The protein,fat,mineral,and vitamin contents of yak meat vary among different breeds,and the chemical composition,pH,shear force,drip loss,and cooking loss of yak meat vary among different ages.Different muscle parts lead to differences in related indicators.From the perspective of food safety,the results of detecting heavy metal residues,microbial indicators,and veterinary drug residues in yak meat all comply with national standards,making it a safe and reliable meat choice for consumers.Based on recent research on the quality of yak meat,the author systematically reviews the quality characteristics of yak meat from the aspects of nutritional quality,edible processing quality,and safety.The results shows that yak meat has advantages such as high nutritional value,safety,and pollution-free,but also has disadvantages such as poor sensory and edible quality and taste.But overall,the quality of yak meat is good,but further regulation is still needed.

【Objective】 The experiment aimed to explore the effects of supplementing different levels of yeast culture on milk production,milk composition,and blood biochemical indicators of lactating mares,in order to provide reference for the development and use of feed additives to improve mare lactation ability.【Method】 Eighteen Yili mares with an average milk production of (2.65±0.34) kg,similar parity and delivery date,and lactation for two months were selected and randomly divided into three groups:Control group,experimental groups Ⅰ and Ⅱ.Among them,the control group was raised by pure grazing,while the experimental groups Ⅰ and Ⅱ were supplemented with 45 and 60 g/(d·horse) of yeast culture on this basis.After pre feeding for 5 days,a 60 day supplementation experiment was conducted.Before the feeding period (recorded as day 0),on the 30th and 60th days (11:00,13:00,15:00,and 17:00 each day),the milking amount of each horse was measured during each milking,and milk samples from the whole day were collected to determine milk protein,milk fat,lactose,and non-fat solids in the milk.On the 60th day of the experiment,blood samples were collected on an empty stomach before feeding,and total protein (TP),globulin (GLB),albumin (ALB),uric acid (UA),glucose (GLU),triglycerides (TG),total bilirubin (T-Bil) and other indicators were measured. 【Result】 ①On day 0,there was no significant difference in milk production among the three groups (P＞0.05).On the 30th day,the milk production of experimental group Ⅱ was significantly higher than that of control group (P＜0.05).On the 60th day,compared with control group,the milk production of experimental groups Ⅰ and Ⅱ was significantly increased by 19.33% and 27.30%,respectively (P＜0.05).② On days 0 and 30,there were no significant differences in percentage of milk protein,milk fat,lactose,and non-fat solids content among the three groups (P＞0.05).On the 60th day,compared with control group,the milk fat of experimental groups Ⅰ and Ⅱ was significantly increased by 30.89% and 35.77%,respectively (P＜0.05),and the non-fat solids of experimental group Ⅱ was significantly increased by 1.90% (P＜0.05).③ Compared with control group,there was no significant difference in the levels of TP,ALB,GLB,UA,GLU,TG,and T-Bil in plasma of experimental groups Ⅰ and Ⅱ (P＞0.05).【Conclusion】 Supplementing lactating mares with yeast culture could increase milk production and improve the quality of milk components.The effect was better when supplemented with 60 g/(d·horse) yeast culture,but it had no significant effect on biochemical indicators in the blood.

【Objective】 This study was aimed to investigate the regulatory mechanism of long non-coding RNA (lncRNA) in reproduction of Tibetan and Chuanxiang Black pigs,and screen the related lncRNA.【Method】 The pregnant uterine body samples from Tibetan and Chuangxiang Black pigs with different litter sizes were collected,a specific porcine uterine body mRNA library was established,and sequencing was performed on the Illumina PE150 HiSeq platform.After quality control,alignment and splicing,the differentially expressed lncRNA was screened and its target gene was predicted.GO function and KEGG pathway enrichment analysis were performed on the target genes.In addition,7 differentially expressed lncRNAs were randomly selected for Real-time quantitative PCR verification.【Result】 There were 32 co-differentially expressed lncRNAs in uterine body of Tibetan and Chuanxiang Black pigs,and a total of 168 target genes were obtained.GO function analysis showed that the target genes of co-differentially expressed lncRNAs in samples with different reproductive ability were mainly enriched in items such as molecular metabolism,transcriptional activity regulation,cell proliferation regulation,uterine body modification, trace element metabolism,etc.KEGG pathway analysis showed that the target genes of co-differentially expressed lncRNAs were mainly enriched in glycolysis,oocyte division,cell life regulation,Ras signaling pathway,RIG-Ⅰ signaling pathway,FoxO signaling pathway,HIF-1 signaling pathway,regulation of homeostasis process,etc.Real-time quantitative PCR results showed that the expression of 7 differentially expressed lncRNAs were consistent with RNA-Seq results.【Conclusion】 This study found 32 lncRNAs in uterine body of Tibetan and Chuanxiang Black pigs that might be involve in the pregnancy process of sows by regulating potential target genes,so as to provide a reference basis for further research on the regulatory mechanism of lncRNA in sow reproduction.These lncRNAs could be used as new reproductive trait biomarkers for the breeding process of pigs.

【Objective】 In order to improve fitting accuracy on egg production curve in chicken,the machine learning method was explored to model the weekly egg production rate for 2 indigenous breeds,and compared with the nonlinear regression method.【Method】 Data were collected from local chickens populations recorded from 22 to 50 weeks.The hens were housed in a single cage in an enclosed house,kept in artificial light for 16 hours during the laying period.The chickens were divided into two groups,each with 150 chickens.Group Ⅰ was a synthetic line of Meat-type Yellow chickens,and group Ⅱ was a local dual-purpose breed.The non-linear regression method in IBM SPSS Statistics 21.0 software was used to fit the egg production curve,including the Logistic,McNally,Yang,and Grossman models.The machine learning model was constructed with MATLAB R2014a,using a multilayer perceptron.The neural network was trained with the quasi-Newton method for 300 iterations.The egg production curve was fitted with least squares support vector machine,and the regularization and kernel function parameters were optimized with Bayesian inference.【Result】 According to the evaluation criteria of MSE,R²,and AIC,the Grossman model had the best degree of fitting among the four non-linear regression models,and the McNally model performed the worst.The peak egg production rate predicted by the McNally model deviated from the real value,the peak egg production rates obtained from the Logistic, Yang, and Grossman models were consistent with the observed value.There were differences between the parameter estimates of the curves fitted for the two groups.The persistence of the lay for group Ⅱ was better than that of group Ⅰ.Based on the MSE,R² and graphical evaluation,the neural network fitted better than the traditional nonlinear regression,and the support vector machine was slightly better than the neural network.The optimized parameters for the neural network were 2 hidden layers,containing 5 neurons in each layer.For support vector machine,the regularization parameter of group Ⅰ was 30.97,and the kernel parameter was 0.0701;The regularization and kernel parameter of group Ⅱ was 566.53 and 0.1754,respectively.【Conclusion】 Machine learning method could be used to fit the egg production curve in these populations.Compared to those classic univariate regression,machine learning methods could harbor more variates and provide more accurate predictions.

【Objective】 The purpose of this study was to investigate candidate genes associated with body size in the genomes of Chinese indigenous pigs using whole-genome selection signal detection methods,and analyze the selection patterns during their evolution and domestication of Chinese indigenous pigs.【Method】 Based on whole-genome resequencing data of 310 indigenous pigs,two methodologies were employed:Cross population extended haplotype homozygosity (XP-EHH) and fixation index statistic (F_ST),a comprehensive genome-wide selection signal scan was conducted.The intersection of the top 0.1% sites identified by both methods was designated as candidate loci,and their upstream and downstream 200 kb regions were designated as potential selection regions.Through enrichment analysis,the biological functions of selected signals were further explored.【Result】 A total of 633 potential selection loci were detected by XP-EHH and F_ST methods,633 potential regions were obtained,and 133 genes were annotated.The quantitative trait locus (QTL) enrichment analysis showed that the selection signals in different body types of Chinese indigenous pigs were related to meat and carcass traits.The results of chromatin state enrichment analysis showed that the tissues with differences in different body types of Chinese indigenous pigs were concentrated in viscera,digestive system and cerebellum.GO function and KEGG pathway enrichment analysis showed that 16 genes were significantly enriched in 6 functional items (P＜0.05,count＞3),mainly concentrated in growth and metabolism-related pathways.Among them,ACSM5,ACSM4,FXR1 and FOXA2 were candidate genes,which were mainly enriched in fatty acid synthesis and metabolism,muscle growth and development and other signaling pathways.【Conclusion】 In this study,two detection methods were used to analyze Chinese indigenous pigs with different body sizes,and a series of genetic differentiation genes were screened.The candidate genes involved in the regulation of growth and development in pigs,such as ACSM5,ACSM4,FXR1 and FOXA2,were excavated.

【Objective】 This study was aimed to explore the kinship and inbreeding coefficient of Yunshang Black goats main mating rams,construct an efficient joint breeding system for meat goats,and improve the production performance and sustainable development of Yunshang Black goats.【Method】 100 main mating rams from 5 core breeding farms (XD,ZY,ML,SB and TJ) of Yunshang Black goats were sequenced by genotyping-by-sequencing (GBS) technology,and softwares such as BWA,SAMTOOLS,PLINK v 1.90,Gmatrix v 2 and Mega X were used for the identification of high-quality single nucleotide polymorphism (SNP) after quality control,principal component analysis (PCA),the construction of identical by state (IBS) and G matrix,phylogenetic tree analysis,calculation of kinship coefficient and estimation of inbreeding coefficient,respectively.【Result】 215 926 high-quality SNPs were obtained after GBS and quality control,and 8 692 long runs of homozygosity (ROH) were detected ranging from 10.59 to 591.23 Mb in length with an average of 245.74 Mb.The average IBS genetic distance in Yunshang Black goats main mating rams population was 0.118±0.011,and the results of the kinship G-matrix were consistent with the results of IBS distance matrix.100 Yunshang Black goats main mating rams were divided into 3 branches and 28 families according to the phylogenetic tree and kinship analysis.There was only one ram in family 2,11,13,14,16,17,18,20,23,24 and 25.There were 18,10,8,5 and 8 families in XD,ZY,ML,SB and TJ nuclear breeding farms,respectively.The average inbreeding coefficient of population genome based on ROH (F_ROH) was 0.099641,the F_ROH of 29 rams was less than 0.0625,that of 39 rams was between 0.0625-0.125,and that of 32 rams was more than 0.125,which indicated that there was a large inbreeding accumulation.【Conclusion】 In summary,100 Yunshang Black goats main mating rams were divided into 28 families,among which 11 families had only one ram.Breeding should be strengthened to prevent the loss of their bloodlines.In the formulation of breeding plan,attention should be paid to the individuals with F_ROH more than 0.125 to prevent inbreeding depression,and reasonable exchange of breeding rams should be carried out according to different breeding objectives.

【Objective】 The aim of this study was to improve and optimize the simple cryopreservation technique for glycerol straws in chicken semen.【Method】 A single factor design experiment was conducted to compare the motility,viability,and abnormality rate of frozen semen using 6% cryopreservation (3,6 and 9 months) and freshly prepared glycerol protective solution.The one-step freezing rate was optimized using liquid nitrogen surface fumigation method.The motility,viability and abnormality rate of sperm,fertilization rate and hatching rate of breeding eggs after freezing and glycerol removal were tested in frozen semen prepared by the optimized one-step freezing and the two-step freezing developed.Using a one-step freezing method to freeze semen from different breeds of roosters,and applying different insemination cycles to hens of different ages,the effect of insemination was evaluated through insemination experiments.【Result】 ① There was no significant differences in motility,viability,or abnormality rate of semen preserved in the newly-prepared glycerol cryoprotectant or the preserved glycerol cryoprotectant for 3,6 and 9 months (P>0.05). ② The optimal freezing procedure of the one-step freezing was that the straw was set 3 cm above the liquid nitrogen surface for 3 min to achieve sperm motility above 60% in all replicates,and above 65% in 65% replicates.③The motility and viability of sperm were 62.84% and 76.00% in one-step freezing,which were extremely significantly higher than those in two-step freezing (58.17% and 68.62%) (P＜0.01).The sperm abnormality rate was no significantly difference between the two groups (P＞0.05).There was no significant difference in the average sperm motility,average fertilization rate,and average hatching rate of breeding eggs between the one-step and two-step frozen semen centrifugation before and after glycerol removal (P＞0.05).The highest daily fertilization rates were 68.34% and 80.00%,respectively.④ There was no significant difference in the fertilization rate and hatching rate of the eggs prepared by one-step freezing method between Heifeng chicken and Xiangdong chicken after frozen sperm insemination (P＞0.05).The fertilization rate of eggs after insemination to 30 week old hens was extremely significantly higher than that of 68 week old hens (P＜0.01).The fertilization rate of eggs in the 3 round 4 insemination mode was significantly extremely higher than that in the 1 round 2 insemination mode (P＜0.01).【Conclusion】 It could achieve a better effect of insemination by using -20 ℃ frozen glycerol as cryoprotectant and one-step cryopreservation technology.Attention should be paid to the time of semen refrigeration,the age of the hen,and the number of insemination rounds during the insemination for a stable and better effect.

【Objective】 The aim of this study was to obtain immortalized porcine alveolar macrophages (iPAMs) with cluster of differentiation 163 (CD163) gene knockout,and investigate the role of CD163 protein in the invasion process of Porcine respiratory and reproductive syndrome virus (PRRSV) at the cellular level.【Method】 The sgRNA plasmid (pX330-sgCD163) targeting the exon 7 of CD163 gene was transfected into iPAMs,followed by monoclonal cell selection and genotype identification using a limited dilution method after 48 h of transfection.CD163 gene knockout iPAMs were screened by Western blotting and off-target analysis.The PRRSV infection characteristics of CD163 gene knockout iPAMs were analyzed by Real-time quantitative PCR,Western blotting,indirect immunofluorescence assay (IFA) and 50% tissue culture infective dose (TCID₅₀).【Result】 Sequencing results showed that one of the 100 monoclonal cells was CD163 gene knockout iPAMs,with an efficiency of 1%.Western blotting results showed that there was no expression of CD163 protein in CD163 gene knockout iPAMs,and no off-target effects were detected at the predicted off-target sites.Real-time quantitative PCR results showed that compared with wild-type iPAMs group,the viral copy number of CD163 gene knockout iPAMs group was extremely significantly decreased (P＜0.01).The results of Western blotting and IFA showed that there was no expression of PRRSV-N protein in CD163 gene knockout iPAMs group after 24 h of post-infection.TCID₅₀ assays also revealed that there was no cytopathic lesions in CD163 gene knockout iPAMs group.【Conclusion】 This study successfully constructed CD163 gene knockout iPAMs using CRISPR/Cas9 editing system,which completely resisted PRRSV infection.The establishment of this cell line provided novel research materials for investigating the interaction mechanism between PRRSV and CD163 protein in future studies.

【Objective】 In order to determine the characteristics of fecal microbiota of diarrheal calves infected with Bovine norovirus (BNoV),this study was conducted to compare the diversity and species composition of fecal microbiota between healthy calves and diarrheal calves with BNoV infection.【Method】 Firstly,PCR and RT-PCR were conducted to detect diarrhea-associated pathogens in the feces of healthy and diarrheal calves.Then 16S rRNA amplicon sequencing technology was performed to compare the diversity,composition and function of microbiota in the feces of healthy calves with negative pathogen detection (group H) and those of diarrheal calves with positive BNoV detection only (group BNoV).【Result】 Compared with group H,the OTUs and Chao1 index of fecal microbiota were significantly increased (P＜0.05),the Shannon and Simpson indexes were decreased extremely significantly (P＜0.01) in group BNoV. Compared with group H,the abundance of Proteobacteria,Escherich-Shigella and Butyricococcus was extremely significantly increased (P＜0.01) in group BNoV.The abundance of Actinomycetes,Collinsella, Megasphaera, Olsenella, Bifidobacterium and Faecalibacterium was extremely significantly or significantly reduced (P＜0.01 or P＜0.05) in BNoV group.Bifidobacterium, Collinsella, Megasphaera, Olsenella and Faecalibacterium were significantly enriched in group H,while Butyricococcus,Escherichia-Shigella,Bacteroides fragilis and Clostridium were significantly enriched in group BNoV.Compared with group H,cellular processes and signaling,lipid metabolism,metabolism,xenolism biodegradation and metabolism,metabolism of terpenoids and polyketides of fecal microbiota were significantly or extremely significantly up-regulated (P＜0.05 or P＜0.01) in group BNoV,while membrane transport,replication and repair,translation,nucleotide metabolism,genetic information processing were significantly or extremely significantly down-regulated (P＜0.05 or P＜0.01).【Conclusion】 The richness,diversity,species composition and function of fecal microbiota in calves with diarrhea associated with BNoV infection were significantly different from those of healthy calves,suggesting that the intestinal microbiota of BNoV infected calves was disturbed.

【Objective】 The aim of this experiment was to construct a triple epitope vaccine against Porcine epidemic diarrhoea virus (PEDV),Porcine transmissible gastroenteritis virus (TGEV) and Porcine delta coronavirus (PDCoV) simultaneously to prevent swine-associated diseases caused by the above pathogens.【Method】 In this study,immunoinformatics online softwares were used to predict and analyze the B and T cell epitopes of the S protein of three types of Swine enteric coronavirus (SECoVs).A new epitope peptide named PPT was constructed.PPT was subjected to bioinformatics analysis using online softwares such as ExPASy,VaxiJen,TMHMM,SOPMA,SOLpro and AlphaFold2.The immune response was simulated using the C-IMMSIM online software.PPT was connected to the COE sequence of PEDV,the SAD sequence of TGEV,and the CTD sequence of PDCoV through T2A and cloned into the eukaryotic expression vector pEGFP-N1.After PCR and double enzyme digestion identification,the obtained recombinant plasmid was transfected into HEK293A cells.DAPI staining,CCK8,RT-PCR and Western blotting tests were performed to verify the expression of the recombinant plasmid in vitro.【Result】 The constructed epitope protein PPT consisted of 17 epitope peptides.According to the bioinformatics analysis of software,the protein was a non-transmembrane protein with stable structure,antigenicity,solubility,high hydrophilicity,and no allergenicity.The results of C-IMMSIM showed that PPT could increase the number of DC cells in the organism,induce B and T cells immune responses,and stimulate the levels of immunoglobulins IgG,IgM and cytokines interferon-γ (IFN-γ) and interleukin-2 (IL-2).PCR and double enzyme digestion identification of the recombinant plasmid showed specific target bands at 3 359,2 375,1 064,944,764 and 467 bp,corresponding to the expected sizes.After the transfection of the recombinant plasmid into HEK293A cells,green fluorescence was observed.RT-PCR amplification yielded bands matching the size of the target gene at 3 359,2 375,1 064,944,764 and 467 bp,respectively.CCK-8 assay results indicated that the recombinant plasmid had no significant cytotoxic effect on cells.Western blotting analysis showed specific bands at 31.7,16.1,37.9 and 27.5 ku,corresponding to the molecular weights of the target genes,confirming the successful expression of the recombinant plasmid in HEK293A cells.【Conclusion】 Based on the PPT epitope protein designed by computer software analysis,the triple vaccine was successfully constructed and expressed in vitro,which provided a basis for evaluating the immunological effect of the triple epitope vaccine PEDV-TGEV-PDCoV.

【Objective】 This study was aimed to clone and analyze the chemokine-binding protein (CBP) gene of the oral and visceral strains of Ovine contagious pustular dermatitis virus (ORFV),and provide basic data for the subsequent study of the immune escape mechanism of CBP in ORFV.【Method】 Specific primers were designed using the DNA extracted from oral scabs and small intestine of sick lambs as templates,PCR amplification, cloning and sequencing were performed,and the genetic and evolutionary relationship of CBP gene and the physicochemical properties and structural characteristics of the expressed protein were analyzed.Recombinant plasmids PGEX-4T-1-CBP-K and PGEX-4T-1-CBP-N were constructed using the prokaryotic expression system pGEX-4T-1 and transformed into Escherichia coli Rosetta (DE3) competent cells,and the protein expression was detected using SDS-PAGE.The antigenicity of the protein was analyzed by Western blotting.【Result】 The total length of CBP gene in ORFV oral infection strain was 873 bp,and the total length of CBP gene in ORFV visceral infection strain was 846 bp,and the latter had 24 bp base loss at 217 bp.The phylogenetic tree showed that oral and visceral infection strains did not belong to the same evolutionary branch,and the genetic distance was far away.The CBP gene of the oral infection strain strain was closely related to the Jilin isolates in China.The CBP gene of the visceral infection strain was closely related to that of the Malaysian strain.The CBP gene of the oral infection strain encoded 291 amino acids,while the CBP gene of the visceral infection strain encodes 282 amino acids.The protein secondary structure was mainly composed of alpha-helix and random coil,the tertiary structure had 5 differences,mainly alpha-helix and beta sheet.The prokaryotic expression plasmids pGEX-4T-1-CBP-K and pGEX-4T-1-CBP-N were successfully constructed.CBP fusion proteins were expressed mainly in the form of inclusion bodies with a molecular mass of about 60 ku in Escherichia coli Rosetta (DE3) competent cells.【Conclusion】 In this study a 24 bp deletion in the visceral infection strain compared to the oral infection strain. The prokaryotic expression plasmids pGEX-4T-1-CBP-K and pGEX-4T-1-CBP-N were successfully constructed,which could express CBP fusion protein in Escherichia coli,the results of this study could provide reference for revealing the role of CBP in the interaction between ORFV and host.It could also provide a basis for finding new prevention and treatment methods of ovine contagious pustular dermatitis.

【Objective】 Salmonella is a common zoonotic pathogen that could cause a variety of foodborne diseases,and Salmonella Typhimurium is one of the important serotypes of Salmonella,which had important public health research significance.The purpose of this study was to study the effect of transporter-related gene oppCDF on the biological characteristics of Salmonella Typhimurium,and to provide a theoretical basis for the preparation of a new attenuated vaccine against Salmonella.【Method】 In this study,the relationship between oppCDF gene and the biological characteristics of Salmonella Typhimurium was analyzed by comparing the biochemical characteristics,genetic stability,drug resistance,adhesion,invasiveness,half lethal dose (LD₅₀) of Salmonella Typhimurium wild-type strain and the successfully constructed oppCDF gene deletion strains,as well as the bacterial load capacity of mouse liver and spleen.【Result】 The results showed that the biochemical characteristics of Salmonella Typhimurium with oppC,oppD and oppF genes deletion did not change significantly and could be inherited stably,indicating that the phenotype of oppCDF gene did not change.Compared with wild-type strain STM LT2,gene deletion strain STM LT2ΔoppC became susceptible to erythromycin,and the sensitivity of chloramphenicol decreased.STM LT2ΔoppD was less sensitive to cephalosporin,STM LT2ΔoppF was more sensitive to compound sulfamethoxazole,and all three gene deletion strains were insensitive to ampicillin.The LD₅₀ of the three gene deletion strains showed that,compared with wild-type strain STM LT2,the virulence of STM LT2ΔoppC decreased,but the virulence of STM LT2ΔoppD and STM LT2ΔoppF increased by 1.6 and 8.6 times,respectively.Compared with wild-type strain STM LT2,the adhesion of STM LT2ΔoppC was significantly reduced (P＜0.05),the invasiveness was reduced,and the colonization capacity was extremely significantly reduced (P＜0.01). The adhesion and invasiveness of STM LT2ΔoppD were decreased,but the colonization was extremely significantly increased (P＜0.01). And the adhesion of STM LT2ΔoppF was significantly decreased (P＜0.05),but the invasiveness and the colonization was extremely significantly increased (P＜0.01).【Conclusion】 oppC gene deletion could reduce the pathogenicity of Salmonella Typhimurium,but oppD and oppF genes deletion could enhance the pathogenicity of Salmonella Typhimurium,and the above results provided a theoretical basis for the study of the pathogenicity of Salmonella.

Porcine reproductive and respiratory syndrome (PRRS) is a global disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV),which causes severe reproductive disorders in sows and respiratory distress in piglets.The severity of the disease caused by PRRSV infection is related to the viral load and the cytokines produced in the body.PRRSV activates inflammatory signaling pathways through structural and non-structural proteins encoded by itself,resulting in the release of a large number of cytokines and chemokines to induce inflammatory responses,but excessive inflammatory responses,especially cytokine storms,may lead to serious tissue damage and aggravate the disease.Therefore,inhibiting the inflammation caused by PRRSV may be the main strategy to effectively prevent and control PRRS.At present,the commonly used anti-inflammatory drugs include recombinant interferon,non-steroidal anti-inflammatory drugs (NSAID) and steroids,etc.Traditional Chinese medicine preparations have also been confirmed to have a certain therapeutic effect on PRRS.However,the conditions,degree and mechanism of inflammation induced by PRRSV infection and the role of anti-inflammatory drugs are not yet clear.The author intends to review the inflammatory response induced by PRRSV and its mechanism of action,as well as the development and application of related anti-inflammatory drugs,in order to provide new ideas for the prevention and control of PRRS.

【Objective】 The aim of this study was to isolate a phages that specifically lysed multidrug-resistant Escherichia coli and clarify the biological properties and genomic characteristics of the isolated phages furtherly.【Method】 In this study,Escherichia coli was isolated from the lung tissue of dead goats and its drug resistance was analyzed by drug sensitivity test.Using Escherichia coli ER2738 as host bacteria,the phage was initially propagated from farm sewage,and the host spectrum of the phage was determined by drop method.The phage of lytic Escherichia coli was isolated and purified by double agar plate method,and its morphology,optimal multiplicity of infection (MOI),growth curve,pH and temperature tolerance were further studied.Finally,the whole genome sequence was determined by high-throughput sequencing platform,and the characteristics of the genome and genetic evolution were analyzed.【Result】 A multidrug-resistant Escherichia coli strain named YNEC001 was isolated from the lung of dead goats.A phage (BP32) that could specifically lyse YNEC001 strain was isolated.Electron microscopic observation showed that phage BP32 belonged to Myoviridae,with the optimal MOI of 0.1,the incubation period was about 30 min,and the lysis period lasted about 60 min,and the average amount of lysis was about 660 PFU/cell.Phage BP32 could maintain stable activity at 30-50 ℃ and pH 4.0-10.0.The genome length of phage BP32 was 166 346 bp,the GC content was low (35.42%),no drug resistance genes and virulence factors were found,and the genome contained 261 open reading frame (ORF),of which 125 were known functional genes and contained 8 tRNA.At the genome-wide level,the nucleotide similarity between phage BP32 and enterobacter phage Escherichia BF15 (MW822007) was the highest (98.25%). Based on the phylogenetic analysis of the large subunit of the terminal enzyme,BP32 was most closely related to Escherichia vB_EcoM_SP1 (MT682709),but more distant to Escherichia BF15 (MW822007).【Conclusion】 In this study,a strain of multidrug-resistant Escherichia coli YNEC001 was isolated,and phage BP32,which could specifically lyse the YNEC001 strain,was isolated.This phage had strong lytic ability and good stability,and had the potential to be developed as a therapeutic agent for multidrug-resistant Escherichia coli.

【Objective】 The spectrum-effect correlation analysis of the anti-inflammatory and bacteriostatic properties of Lianpu Shuangqing powder was constructed based on experimental design,and its pharmacological substance basis and mechanism of action were revealed through the integration of network pharmacology and in vitro and in vivo experiments.【Method】 The L₉(3⁴) orthogonal experiment was devised incorporating the proportion of Coptidis Rhizoma and Taraxaci herba,the extracted acid concentration and the ethanol concentration,resulting in nine pairs of extracts of Lianpu Shuangqing powder.HPLC was utilized to establish fingerprints for each extract.An in vitro pharmacodynamic analysis of anti-inflammatory and bacteriostatic effects was conducted utilising lipopolysaccharide (LPS)-induced inflammatory cytokines of mouse macrophages and bacteriostasis zone as pharmacodynamic evaluation indexes.The spectrum-effect relationship was established using the grey relational degree analysis and partial least squares regression analysis methods,which screened out the pharmacological substances responsible for its anti-inflammatory effects and bacteriostatic effects against Staphylococcus aureus.Pharmacodynamic constituents and targets were further identified through network pharmacology analysis.Lastly, in vivo experiment was used to verify the spectral and network pharmacology results.【Result】 HPLC fingerprints identified nine common peaks and four components (chicoric acid,coptisine,epiberberine and palmatine).The results of orthogonal test showed that the components in Coptidis Rhizoma played a dominant role in the antibacterial effect against Staphylococcus aureus,while the role of Taraxaci herba in the anti-inflammatory efficacy was more obvious.Spectrum-effect analysis screened that epiberberine and coptisine were part of the pharmacological substance basis for the anti-inflammatory and antibacterial effects of the powder.Network pharmacology research revealed that epiberberine,palmatine,coptisine,luteolin and caffeic acid were the five components with the highest weights in the network.Interleukin-6 (IL6) and tumor necrosis factor (TNF) were the two main anti-inflammatory and antibacterial targets.The docking results showed that the binding energies of five compounds to both target protein receptors were all below －5 kJ/mol,indicating a good binding affinity.In the mouse model of acute enteritis,histological sections revealed that the area of inflammatory cell infiltration in the colon of mice was significantly reduced after treatment with Lianpu Shuangqing powder.ELISA experiments showed a significant difference in the expression of inflammatory factors in colon of mice between the drug group and the model group (P＜0.05).【Conclusion】 An integrated approach involving spectrum-effect correlation analysis based on experimental design and network pharmacology had effectively delineated that the primary pharmacological substances responsible for the anti-inflammatory effects and bacteriostatic effects against Staphylococcus aureus in Lianpu Shuangqing powder were coptisine and epiberberine,with IL6 and TNF being the major target molecules.This study offered a significant exploration into the pharmacological substance basis and target actions of compound traditional Chinese medicines.

【Objective】 In order to rationally prevent and control Salmonella infection,an experiment was conducted to study the prevalence of Salmonella in livestock and poultry slaughtering and marketing in Gansu province and neighboring areas.【Method】 Bacterial isolation and identification were carried out through bacterial culture,biochemical experiments,and molecular biology methods.The serotype of Salmonella was determined using slide agglutination and multilocus sequence typing (MLST).The quantitative method of crystal violet staining was used to evaluate the biofilm formation ability of the isolated strains.The sensitivity of isolated bacteria to commonly used antibiotics was tested through drug sensitivity tests.The sterilization effect of four commercially available disinfectants on Salmonella was evaluated,and their minimum inhibitory concentration (MIC) was determined. 【Result】 The results of bacterial isolation and culture showed that the suspected Salmonella isolates showed black colonies with metallic luster,tan or gray on BS plates.In XLD plate,the colonies showed glossy and black center,or all black colonies,and the microscopic examination showed that they were Gram-negative short bacilli.The biochemical identification results showed that the isolates reacted positively with hydrogen sulfide,glucose,ornithine,and lysine decarboxylase,and reacted negatively with urease,potassium cyanide,lactose and peptone water.PCR amplification of the Salmonella-specific gene invA yielded a target band of approximately 284 bp in size,confirming that a total of 95 Salmonella strains were isolated.All the isolates belonged to three serotypes of Salmonella enterica: Salmonella Typhimurium, Salmonella Derby and Salmonella Rissen.Multilocus sequence typing identified 3 species,ST34,ST469 and ST40.All the isolates could form biofilm,and drug sensitivity test showed that all strains were completely resistant to minocycline and vancomycin,and antibiotics such as amikacin,ceftriaxone,kanamycin,cefazolin,cefadroxil,and gentamicin were more susceptible to Salmonella isolates,and 75.78% of the strains were multidrug-resistant.The MIC of the four disinfectants against the isolates varied from 0.125 to 0.25 mg/mL for 0.2% formaldehyde solution,0.0625 to 0.125 mg/mL for 0.2% benzalkonium bromide,0.3125 to 0.625 mg/mL for 0.5% iodine solution,1.25 to 2.5 mg/mL for 2% sodium hydroxide.【Conclusion】 Salmonella Typhimurium,Salmonella Derby and Salmonella Rissen were the main serotypes of Salmonella in current slaughterhouses,and they possessd the ability to form biofilms and exhibited multidrug resistance,posing a significant threat to public health.This study provided a scientific basis for strengthening hygiene management in slaughterhouses and agricultural markets in Gansu province and surrounding areas.

【Objective】 Bovine coronavirus (BCoV) was the main pathogen of calf diarrhea and upper respiratory tract disease.Currently,there was a lack of sensitive animal cells suitable for in vitro culture of BCoV,which limited in-depth research on the pathogenesis of BCoV.Therefore,it was planned to isolate bovine lung epithelial cells (BLEC) from calf lung tissue to establish a BCoV in vitro infection model of this animal cell.【Method】 A two-step enzymatic digestion method of trypsin and type Ⅰ collagenase was used to initially extract BLEC from newborn calf lung tissue,and then the differential attachment method of epithelial cells with different attachment time was used to purify BLEC.BLECs were identified by detecting the expression of marker cytokeratin 18 (CK-18) in purified cells using indirect immunofluorescence assay (IFA),and the pathogenic purity of the cells was detected by RT-PCR for Bovine rotavirus (BRV),Bovine viral diarrhea virus (BVDV),Bovine parainfluenza virus type 3 (BPIV3),BPIV5,Infectious bovine rhinotracheitis virus (IBRV),BCoV and Mycoplasma.The susceptibility of BCoV to BLEC was detected by RT-PCR and IFA,and the one-step growth curve of BCoV on BLEC was drawn.【Result】 The BLEC cultured from purification were uniform and stable in shape,diamond-shaped or brick-shaped.There was no contamination by common pathogens such as BRV,BVDV,BPIV3,BPIV5,IBRV,BCoV and Mycoplasma,and the epithelial cell marker CK-18 was positively expressed.Obvious cytopathic effect (CPE) could be observed 24 h after infection of BLEC with the BCoV HLJ-325 strain derived from calf diarrhea.PCR could amplify the BCoV N gene,the IFA results showed that BCoV-infected BLEC showed specific red fluorescence.The BCoV content was detected by Real-time quantitative PCR to establish the virus growth curve. The viral load of BCoV was the highest at 20 h after infection with BLEC,which was 4.46×10⁵ copies/mL.【Conclusion】 This study successfully prepared BLEC,proved that BCoV was susceptible to infecting BLEC,and constructed a BCoV in vitro infection model of BLEC,laying the foundation for studying the pathogenic mechanism of bovine respiratory diseases caused by BCoV.

【Objective】 The purpose of this experiment was to investigate the levels of enterobactin secreted by Escherichia coli isolated from the chicken intestines and the effects of enterobactin on Campylobacter jejuni growth promotion.【Method】 The indirect competitive ELISA (ic-ELISA) method was used to detect the production of enterobactin in 30 chicken enteric Escherichia coli isolates.The λRed homologous recombination system was used to construct the entB gene deletion mutant strain of Escherichia coli,and the enterobactin production of the parental and mutant strains was determined by ic-ELISA.The growth curves of the parental and mutant strains were determined in different iron-limited media.The supernatants from the parental and mutant strains were used to inoculate Campylobacter jejuni for investigating the effects of entericin secreted by Escherichia coli on the growth of Campylobacter jejuni.【Result】 The levels of enterobactin secreted by 30 chicken intestinal Escherichia coli isolates ranged from 11 to 126 μmol/L.2 entB gene deletion mutant strains of Escherichia coli were successfully constructed using λRed homologous recombination system. The qualitative results showed that Escherichia coli parental strains produced the yellow halo in the CAS medium,while no halo was observed for the entB gene deletion strain.ic-ELISA results showed that Escherichia coli parental strains produced enterobactin in the culture supernatants with a concentration of 100-150 μmol/L,while no enterobactin was detected for the entB gene deletion strain.In the iron-rich medium,there was no significant difference in the growth of Escherichia coli parental and entB gene deletion strains (P＞0.05).The D_{600 nm} values that reached the plateau in iron-limited medium were 0.6 and 0.3,respectively.The growth promotion results showed that the colony numbers of Campylobacter jejuni in the culture supernatant of Escherichia coli parental strain reached 7.8 and 8.2 lg CFU/mL at 24 and 36 h of inoculation,respectively.By comparison, the colony numbers in the culture supernatant of the entB gene deletion strain were 7.0 and 7.3 lg CFU/mL,respectively.【Conclusion】 Escherichia coli had the ability to secrete enterobactin for promoting the growth of Campylobacter jejuni in iron-limited environments.This study laid a foundation for further investigation of enterobactin-mediated interactions between intestinal bacteria.

【Objective】 This study was aimed to investigate the protective effect and mechanism of epigallocatechin-3-gallate (EGCG) on acute kindey injury (AKI) induced by cisplatin (CIS) in rats,so as to provide experimental basis for the clinical use of CIS and EGCG.【Method】 Forty male Wistar rats were randomly divided into 5 groups.Rats in CON and CIS groups were perfused with normal saline,and rats in EGCG,CE and inhibitor groups were gavaged with 40 mg/kg EGCG for 28 consecutive days.On the 26th day,rats in CIS,CE and inhibitor groups were intraperitoneally injected with 7 mg/kg CIS,rats in CON and EGCG groups were injected with an equal volume normal saline,and rats in inhibitor group was intraperitoneally injected with 5 mg/kg LY294002 for 3 consecutive days.The kindey samples of all rats were collected on the 29th day.HE staining and transmission electron microscope were used to observe the changes of renal pathology and cellular mitochondrial function,Western blotting was used to detect the expression of autophagy proteins (LC3Ⅱ/Ⅰ,ATG5,ATG12,Beclin-1 and p62) and pathway proteins (PI3K,Akt,p-PI3K and p-Akt).Immunofluorescence was used to detect the expression of autophagy-related proteins LC3II/Ⅰ and p62 in kindey of rats.【Result】 The results of HE staining and transmission electron microscope showed that pretreatment of EGCG alleviated the necrosis and exfoliation of renal tubular epithelial cells,infiltration of inflammatory cells,swelling and degeneration of cell mitochondria and rupture of mitochondrial crest induced by CIS.Western blotting results showed that compared with CON group,the expressions of LC3Ⅱ/Ⅰ,ATG5,ATG12 and Beclin-1 protein in kidney of CIS group were extremely significantly increased (P＜0.01),while the expressions of p62,p-PI3K/PI3K and p-Akt/Akt were extremely significantly decreased (P＜0.01),but this phenomenon was reversed in EGCG pretreatment group.Immunofluorescence results showed that compared with CE group,the pathological damage in inhibitor group was aggravated,the fluorescence signal of LC3Ⅱ/Ⅰ was enhanced,and p62 was weakened.【Conclusion】 Pretreatment of EGCG could inhibit autophagy by activating PI3K/Akt signaling pathway,thus alleviating CIS-induced AKI in rats,and this protective effect could be offset by PI3K/Akt signaling pathway inhibitor LY294002.

【Objective】 The experiment was conducted to understand the resistance situation of Escherichia coli (E. coli) isolated from Hequ horses and provide theoretical guidance for rational use of antibacterial agents in veterinary medicine.【Method】 Bacterial isolation and identification were carried out using bacterial morphology,Gram staining microscopy and 16S rDNA sequence analysis.Broth micro-broth dilution method and PCR method were conducted for antibacterial sensitivity tests against antibiotics and detection of antibacterial resistance genes,integrase genes,and efflux pump genes in the isolated E. coli.Meanwhile,a modified semi-quantitative crystal violet staining method was employed to assess the biofilm-forming ability.The biofilm removal ability of the combination of 13 efflux pump inhibitors with doxycycline was determined using a checkerboard method.【Result】 The isolates were purple-black colonies with green metallic luster on eosin-mercian blue agar medium,and blue colonies on E.coli/coliform chromogenic medium,and pink short rod-shaped bacteria were seen on Gram staining microscopy.64 strains were highly similar to E.coli by 16S rDNA sequencing.Among 64 strains of E. coli isolates,relatively high resistance rates were observed against quinolones,sulfonamides,and tetracyclines,with multiple drug-resistant strains emerging.Among them,20.31% (13/64) showed resistance to five classes of antibiotics.Twenty-one resistance genes,one integrase gene and six efflux pump genes were positively detected.The detection rate of integrase gene intl1 was 89.06%.The number of E. coli strains with strong, medium, weak and no biofilm forming ability were 8 (12.50%), 34 (53.13%), 20 (31.25%), and 2 (3.12%), respectively.The combination of efflux pump inhibitors with doxycycline enhanced the biofilm removal ability.【Conclusion】 The resistance of E. coli from Hequ horses was severe and it was necessary to further strengthen the supervision of the rational use of antibacterial agents in the racing horses in Henan Mongolian Autonomous county of Qinghai province to reduce and avoid the emergence and widespread transmission of multidrug-resistant strains.

【Objective】 This study was to investigate the pathogenesis of a case of canine genitourinary tract infection.【Method】 This study aseptically collected urine from sick dog and identified the bacteria by isolation and purification,Gram staining microscopy,biochemical tests,and 16S rRNA gene amplification,and determined the biological characteristics of the purified strains by drug sensitivity test,virulence gene test,drug resistance gene test,and pathogenicity test of the bacteria.【Result】 A parthenogenetic anaerobic Gram-positive coccus was isolated.The colony morphology was like a pinpoint,smooth,translucent,rounded raised,and neatly edged.The biochemical characterization showed that the causative agent was able to ferment lactose,fructose,sucrose,maltose,glucose,sorbitol,mannitol,and arginine,but not xylose,oxidase,citrate,cottonseed sugar,ornithine decarboxylase.The 16S rRNA gene sequence showed greater than 99% similarity to the published sequence of Enterococcus faecalis (E. faecalis),the isolate was identified as E. faecalis,and named EFGZ23GY01.The results of drug sensitivity test showed that this isolate was only sensitive to vancomycin and chloramphenicol,moderately sensitive to neomycin,and resistant to 25 drugs including clindamycin.The results of 8 E.faecalis virulence genes showed that this strain of E.faecalis carried efaA, ace, asa1 and gelE virulence genes.And the results of 14 resistance genes showed that this strain of E.faecalis carried resistance genes,such as tetL, sulⅠ, tetC, bla_TEM, bla_OXA-23, aadA1 and sulⅢ.Pathogenicity test showed that 5 mice showed symptoms such as depression and disorganized coat after 12 h of injection,but did not die,while 1 mouse died after 48 h of injection,indicating that the isolate was pathogenic to mice.【Conclusion】 In the present study,a strain of E. faecalis was successfully isolated from canine urine species,which carried multiple virulence and resistance genes and was pathogenic to mice.

【Objective】 The aim of this experiment was to investigate whether infection of bovine macrophages (BoMac) with Mycoplasma bovis isolate from Shihezi could affect cell proliferation activity and apoptosis through the mitochondrial apoptosis pathway.【Method】 The growth curve of Mycoplasma bovis isolated from Shihezi was drawn by medium culture and color change unit (CCU) to determine the culture time.Using Mycoplasma bovis,which was at its highest level of vitality,to infect BoMac with different infection multiples (MOI) and infection time,Real-time quantitative PCR and Western blotting tests were used to detect the expression of core molecules Bax and Bcl-2 in the mitochondrial apoptosis pathway.The cell proliferation activity level was detected by CCK8 assay,and the cell apoptosis rate was measured by flow cytometry. 【Result】 The analysis of the growth curve showed that Mycoplasma bovis isolated from Shihezi was in the logarithmic growth phase from 12 to 54 h,and in the plateau phase from 54 to 84 h (with a titer of 10⁹ CCU/mL).Therefore,the Mycoplasma bovis isolated from Shihezi cultured for 54 h was selected to infect the cells.The results of Real-time quantitative PCR and Western blotting were consistent,indicating that with the increase of infection time and MOI,the pro-apoptotic protein Bax gene and its protein showed an upward trend,while the expression of the anti-apoptotic protein Bcl-2 gene and its protein showed a downward trend.Among them,after 24 h of infection with Mycoplasma bovis isolated from Shihezi,the expression of the Bax gene was extremely significantly increased (P＜0.01),the expression of the Bcl-2 gene and Bcl-2/Bax were significantly decreased (P＜0.05).When the MOI was 1 000,Bax protein expression was extremely significantly increased (P＜0.01),Bcl-2 protein expression was significantly decreased (P＜0.05),and Bcl-2/Bax was extremely significantly decreased (P＜0.01).The CCK8 test results showed that with the increase of infection time and MOI,the activity of BoMac was extremely significantly decreased (P＜0.01),and the proliferation inhibition rate was 35% to 45%.The results of flow cytometry showed that the apoptosis rate of BoMac after infection was 25% to 45%.【Conclusion】 The results of this experiment indicated that as the infection time and MOI increasing,the Mycoplasma bovis isolate from Shihezi could induce apoptosis of BoMac through the expression of mitochondrial apoptosis pathway markers Bax and Bcl-2,providing a theoretical basis for further elucidating the pathogenic mechanism of Mycoplasma bovis.

【Objective】 In this study,overexpression of miRNA-424-5p was performed to clarify the mechanism of the effect of miRNA-424-5p and its target gene AKT3 on the apoptosis of cow endometrial epithelial cells,and provide experimental basis and theoretical basis for elucidating the mechanism of miRNA-424-5p in the process of cow placental separation.【Method】 The experiment was divided into three groups: Blank control group (Control), negative control group (miRNA-424-5p mimics NC) and overexpressed group (miRNA-424-5p mimics). The expression of miRNA-424-5p in cow endometrial epithelial cells after miRNA-424-5p mimics was detected by Real-time quantitative PCR using stem loop method.Western blotting and Real-time quantitative PCR were used to detect the expression changes of AKT3 and apoptosis-related factors after overexpression,and Annexin V-FITC/PI was used to detect the apoptosis of cow endometrial epithelial cells.【Result】 Compared with blank control and negative control groups,the expression of miRNA-424-5p mRNA in endometrial epithelial cells was extremely significantly increased (P＜0.01),the mRNA expression of Bax,Caspase3 and Caspase9 was extremely significantly increased (P＜0.01),and the mRNA expression of AKT3 and Bcl-2 was extremely significantly reduced (P＜0.01) after transfection with miRNA-424-5p mimics.The relative expression of Bax and Caspase3 proteins was extremely significantly increased (P＜0.01),Caspase9 protein was significantly increased (P＜0.05),and AKT3 and Bcl-2 proteins were extremely significantly reduced (P＜0.01).Apoptosis rate of cells was extremely significantly increased(P＜0.01).【Conclusion】 Overexpression of miRNA-424-5p could increase the expression of apoptotic factors Bax,Caspase3 and Caspase9,and decrease the expression of Bcl-2 in endometrial epithelial cells of dairy cows through targeted regulation of AKT3,thus leading to increased apoptosis.

【Objective】 The purpose of this study was to investigate the effects of mulberry leaf polysaccharide (MLP) on the immune function of porcine alveolar macrophages and its mechanism.【Method】 The experiment took porcine alveolar macrophages as the study object,CCK-8 method was used to detect the proliferation rate of porcine alveolar macrophages treated with different concentrations of MLP,and screen the appropriate concentration of MLP.The cells were divided into blank control group (BC group),positive control group (LPS group) and MLP groups with different concentrations.The release of nitric oxide (NO) in porcine alveolar macrophages was detected by Griess method.The phagocytosis ability of porcine alveolar macrophages was detected by neutral red phagocytosis assay.The contents of interleukin-10 (IL-10),IL-6 and tumor necrosis factor-α (TNF-α) in porcine alveolar macrophages were determined by ELISA.The mRNA expression levels of TNF-α,Toll-like receptor 4 (TLR4),IL-6,IL-10 and nuclear transcription factor κB (NF-κB) were detected by Real-time quantitative PCR.The expression levels of TLR4 and p65 proteins were detected by Western blotting.【Result】 Compared with 0 μg/mL MLP group,the proliferation rate of porcine alveolar macrophages in 20,40 and 80 μg/mL MLP groups was extremely significantly increased (P＜0.01),and showed a dose-dependent effect.Therefore,20,40 and 80 μg/mL MLP were selected for follow-up tests.Compared with BC group,the phagocytosis rate and NO release of porcine alveolar macrophages in different concentration of MLP groups were extremely significantly increased (P＜0.01).ELISA results showed that the contents of IL-6 and TNF-α in porcine alveolar macrophages were significantly or extremely significantly higher than those in BC group when MLP concentration was 80 μg/mL (P＜0.05 or P＜0.01).Real-time quantitative PCR results showed that the mRNA expressions of TNF-α,IL-6 and TLR4 genes in porcine alveolar macrophages at MLP concentrations of 40 and 80 μg/mL were extremely significantly higher than those in BC group (P＜0.01).The results of Western blotting showed that when MLP concentration was 80 μg/mL,the protein expressions of p65 and TLR4 were extremely significantly higher than those in BC group (P＜0.01).【Conclusion】 MLP could activate porcine alveolar macrophages,significantly promote the release of NO and the secretion of IL-6 and TNF-α,and had good immunomodulatory activities,and its immunomodulatory mechanism was related to the activation of TLR4 on the surface of macrophages.

【Objective】 The objective of this study was to investigate the protective effect of alcohol extract from Curcuma kwangsiensis on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice.【Method】 A total of 54 male Kunming mice were randomly divided into 6 groups: Blank,model,dexamethasone positive control,and alcohol extract from Curcuma kwangsiensis low (2 g/kg),medium (4 g/kg) and high (40 g/kg) groups,with 9 mice in each group.Administration groups were given dexamethasone (5 mg/kg) or corresponding concentration of alcohol extract from Curcuma kwangsiensis (0.2 mL/10 g) by intragastric administration,respectively.Mice in blank and model groups were given the corresponding volume of 0.5% carboxymethyl sodium vitamin (CMC-Na) by intragastric administration for 5 days.1 h after the last administration,except for blank group,mice in other groups were intraperitoneally injected with 10 mg/kg LPS,anesthetized and killed 12 h later,and lung tissue was dissected.During the experiment,the general conditions of mice before and after modeling were observed,the changes of body weight and lung index of mice were counted,and the ratio of wet weight to dry weight of lung was measured.HE staining was used to observe the lung histopathological changes and lung injury score.Real-time quantitative PCR was used to detect the mRNA expression of inflammatory factors,including tumor necrosis factor-alpha (TNF-α),interleukin-6 (IL-6),myeloid differentiation factor 88 (MyD88),Toll-like receptor 4 (TLR4) and nuclear transcription factor κB (NF-κB) in lung.【Result】 Compared with blank group,the body weight growth rate of mice in model group was extremely significantly decreased (P＜0.01),the lung index,the ratio of wet weight to dry weight of lung and total lung injury score were extremely significantly increased (P＜0.01),and the mRNA relative expression of related inflammatory factors was significantly or extremely significantly increased (P＜0.05 or P＜0.01), the ALI model was suscessfully constructed.Compared with model group,the body weight growth rate of mice in each dose group of alcohol extract from Curcuma kwangsiensis was significantly or extremely significantly increased (P＜0.05 or P＜0.01).The lung index and the ratio of wet weight to dry weight of lung in medium and high dose groups of alcohol extract from Curcuma kwangsiensis were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The total lung injury score and the mRNA expression of TNF-α and IL-6 genes in lung of mice in high dose group of alcohol extract from Curcuma kwangsiensis were significantly decreased (P＜0.05).The mRNA expression of NF-κB gene in lung of mice in medium dose group of alcohol extract from Curcuma kwangsiensis was significantly decreased (P＜0.05).【Conclusion】 Alcohol extract from Curcuma kwangsiensis had a protective effect on LPS-induced ALI in mice,and its mechanism might be related to the reduction of inflammatory damage mediated by TLR4/MyD88/NF-κB signaling pathway.

【Objective】 The purpose of this experiment was to elucidate the pharmacological efficacy and molecular mechanism of Jianpi Qushi decoction (JQD) in the treatment of non-alcoholic fatty liver disease(NAFLD) in rats.【Method】 After the rat NAFLD model was established by feeding high-fat diet,the rats were randomly divided into model,orlistat (32.4 mg/kg),JQD low (0.35 g/kg) and high (0.70 g/kg) dose groups,and control group was set up,with 6 rats in each group. Rats in orlistat and JQD different dose groups were given intragastric administration,1 mL/rat,once a day for 7 weeks,and the rats in control and model groups were given intragastric administration of equal dose of normal saline.After the experiment,the blood and liver tissues of rats were collected.Serum lipid levels of rats were determined by ELISA,including total cholesterol (TC),triglyceride (TG),low density lipoprotein (LDL-C) and high density lipoprotein (HDL-C).The liver index of rats was measured and the liver antioxidant indexes,including glutathione (GSH),superoxide dismutase (SOD),catalase (CAT) and malondialdehyde (MDA) were detected.HE staining and oil red O staining were used to observe the morphological structure of liver.The active ingredients and key targets of JQD for NAFLD were analyzed using drug and disease target database.The "drug-ingredient-target" network diagram was constructed by Cytoscape 3.7.2 software.The effects of main active ingredients of JQD and key targets were verified by molecular docking and Real-time quantitative PCR.【Result】 Compared with control group,the contents of TC,TG and LDL-C in serum,MDA content in liver and liver index in model group were significantly or extremely significantly increased (P＜0.05 or P＜0.01),HDL-C content in serum,GSH content,SOD and CAT activities in liver were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).Compared with model group,the contents of TC and TG in serum,MDA content in liver and liver index of rats in JQD low and high dose groups were significantly decreased (P＜0.05),and the activities of SOD and CAT, and GSH content in liver were significantly increased (P＜0.05).The results of HE staining and oil red O staining showed that the liver cells of control group were normal and the liver cords were neat.In the model group,the liver showed obvious adipose deformation,hepatocyte atrophy,vacuoles of different sizes,and disordered liver cord morphology.The number of liver vacuoles decreased significantly in JQD low and high dose groups,and hepatic cords were clearly visible.A total of 35 active ingredients of JQD and 1 890 disease targets of NAFLD were identified by network pharmacology.The results of network topology analysis showed that the core active ingredients of JQD in the treatment of NAFLD were kaempferol,isorhamnetin and quercetin,and the core therapeutic targets were tumor necrosis factor (TNF),peroxisome proliferating-activated receptor γ (PPARG) and vascular endothelial growth factor A (VEGFA).Molecular docking results showed that the binding energies of core active ingredients and key targets were less than ―5.0 kJ/mol,and they had good binding activity with each other.Real-time quantitative PCR results showed that compared with model group,the expressions of TNF and PPARG genes in liver of JQD low and high dose groups were extremely significantly decreased (P＜0.01).The expression of VEGFA gene in JQD high dose group was extremely significantly decreased (P＜0.01).【Conclusion】 JQD had shown good therapeutic effect on NAFLD,and the mechanism might be related to reducing blood lipid level and increasing antioxidant enzyme activity in liver.

【Objective】 The purpose of this experiment was to investigate the effects of Staphylococcus aureus and Staphylococcus epidermidis on the activity of neutrophils (PMNs) and the secretion of inflammatory factors in peripheral blood of dairy cows.【Method】 The morphology and cell viability of PMNs in peripheral blood of dairy cows cultured in vitro were identified by Giemsa and Trypan blue staining,and the optimal time of action of PMNs was determined.The peripheral blood PMNs infection model induced by Staphylococcus aureus and Staphylococcus epidermidis was established to determine the killing ability of peripheral blood PMNs to the two bacteria.PMNs activity was determined by CCK-8 method,and the levels of inflammatory factors tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β),IL-8 and IL-10 were detected by ELISA.【Result】 The results showed that the purity of PMNs from peripheral blood of dairy cows isolated and cultured in vitro reached 99%,and the survival rate decreased gradually with the extension of culture time,and the survival rate reached more than 85% within 24 h.The logarithmic growth periods of Staphylococcus aureus and Staphylococcus epidermidis were 4-7 h and 4-8 h,respectively.When MOI was 10,the survival rate of Staphylococcus aureus at 2,4 and 6 h was extremely significantly higher than that of Staphylococcus epidermidis (P ＜ 0.01).Compared with control group,the contents of TNF-α and IL-8 in PMNs infected by Staphylococcus aureus and Staphylococcus epidermidis were extremely significantly or significantly increased at different time points (P＜0.01 or P＜0.05).The content of IL-10 in cells was extremely significantly or significantly increased at 3,6,12 and 24 h after infection (P＜0.01 or P＜0.05).The content of IL-1β in cells was extremely significantly or significantly increased at 6,9,12 and 24 h after infection (P＜0.01 or P＜0.05).Compared with Staphylococcus aureus group,the content of TNF-α in Staphylococcus epidermidis group at 3 h of infection was extremely significantly decreased (P＜0.01),and the contents of IL-10 and IL-8 were extremely significantly or significantly increased (P＜0.01 or P＜0.05).After 6 h of infection,the contents of TNF-α and IL-1β in Staphylococcus epidermidis group were extremely significantly decreased (P＜0.01).The contents of TNF-α and IL-8 in cells were extremely significantly or significantly increased at 9 h after infection (P＜0.01 or P＜0.05).【Conclusion】 Both Staphylococcus aureus and Staphylococcus epidermidis could evade phagocytic and killing effects of PMNs in peripheral blood of dairy cows, and the cell viability was significantly reduced after infection. Staphylococcus aureus and Staphylococcus epidermidis could mediate the innate immune response by regulating the secretion of inflammatory factors in PMNs of dairy cows.This result laid a foundation for screening candidate factors related to Staphylococcus aureus mastitis resistance by targeting PMNs in dairy cows.

【Objective】 The aim of this study was to explore the distribution characteristics of ammonia (NH₃),carbon dioxide (CO₂) and airborne particle concentration in a broiler house with vertical tiered cages in winter,and provide basic parameters for the control of environmental uniformity in three-dimensional breeding chicken houses.【Method】 From November 22 to December 30,2022,a broiler house with vertical tiered cages (Cobb White feather broiler) in Northern Henan was used as the research object.At 7,14,21,28 and 35 days of age,the concentrations of NH₃,CO₂ and airborne particle at 15 different positions in the house were measured at 06:00,09:00,12:00,15:00 and 18:00,respectively.【Result】 ①The average NH₃ concentration in the broiler house was 1.72 mg/m³,with significant differences in daily variation (P＜0.05).In the changes of stage,the NH₃ concentration at 14 days of age was significantly higher than that at 21 and 35 days of age (P＜0.05).In longitudinal comparison,the NH₃ concentrations in the middle and rear ends were significantly higher than that in the front end at 7,14 and 28 days of age (P＜0.05).In the vertical direction,the NH₃ concentration increased significantly from top to bottom at 21 and 35 days of age (P＜0.05),and the NH₃ concentrations in the middle and lower equal height positions were significantly higher than those in the upper equal height position at 28 days of age (P＜0.05).In the three-dimensional distribution,there were significant differences among the 15 measurement points (P＜0.05),and the NH₃ concentration difference at 14 days of age was the largest,which was 0.81 mg/m³.② The average CO₂concentration in the broiler house was 1 987 mg/m³,and the 14-day-old was significantly higher than other days (P＜0.05).In the diurnal variation,the CO₂ concentrations at 06:00 and 09:00 were significantly higher than those at 15:00 and 18:00 (P＜0.05),and 12:00 was the lowest.In longitudinal comparison,the CO₂ concentration in the rear end was significantly higher than that in the front end and the middle at 7 days of age (P＜0.05),the middle and rear end were significantly higher than the front end at 14 days of age (P＜0.05),and the rear end was significantly higher than the front end at 35 days of age (P＜0.05).There was no significant difference in vertical direction (P＞0.05).In the three-dimensional distribution,there were significant differences among the 15 measurement points (P＜0.05),and the concentration difference was the largest at 35 days of age,which was 471 mg/m³.③The average concentrations of fine particulate matter (PM_2.5),inhalable particulate matter (PM₁₀) and total suspended particulate matter (TSP) were 125.28 μg/m³,155.24 μg/m³ and 2.17 mg/m³,respectively.There were significant differences in diurnal variation (P＜0.05).The phase change increased significantly with the increase of day age (P＜0.05),and there were no significant differences in longitudinal,vertical and three-dimensional distribution (P＞0.05).【Conclusion】 In winter,the concentration distribution of NH₃,CO₂ and airborne particle in the broiler house with vertical tiered cages were uneven,and the concentration of CO₂ exceeds the standard,so it was necessary to strengthen ventilation.