This study was aimed to understand the sequence information of pyruvate dehydrogenase kinase 4 (PDK4) gene CDS region and the structure and function of the encoded protein in Luchuan pigs,construct a eukaryotic expression vector of PDK4 gene,and analyze the expression of PDK4 gene in different tissues,which laid a foundation for elucidating the molecular mechanism of PDK4 gene in the growth and development of Luchuan pigs.The CDS region of PDK4 gene in subcutaneous fat of Luchuan pigs was amplified by RT-PCR technology,the structure and function of PDK4 gene were predicted and analyzed using bioinformatics software.It was inserted into a eukaryotic expression vector to obtain pEGFP-N1-PDK4,the recombinant plasmid was transfected into 3T3-L1 cells by the liposome method,and then the fluorescence was observed.The expression of PDK4 gene mRNA in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and subcutaneous fat in Luchuan pigs were detected by Real-time quantitative PCR.The results showed that the CDS region of PDK4 gene in Luchuan pigs was 1 224 bp in length,encoding 407 amino acids,and had 99.8% homology with the CDS region of PDK4 gene in Sus scrafa published on NCBI.Bioinformatics analysis of the protein encoded by PDK4 gene in Luchuan pigs revealed that its molecular mass was 46.144 ku,the total number of atoms was 6 509,the theoretical isoelectric point (pI) was 7.21,and the number of positively and negatively charged amino acids was 42,respectively.There were 2 N-glycosylation sites and 33 phosphorylation sites of PDK4 protein.Subcellular localization results showed that 34.8% of PDK4 protein was present in mitochondria,30.4% was present in cytoplasm,26.1% was present in nucleus,and plasma membrane and vacuole membrane each accounted for 4.3%.Compared with control group,the expression of PDK4 gene in experimental group extremely significantly increased (P<0.01).The expression of PDK4 gene was most abundantly expressed in subcutaneous fat,followed by liver,lung,heart,spleen and kidney,it had the lowest expression in longissimus dorsi muscle,and the expression of subcutaneous fat was extremely significantly higher than that of longissimus dorsi muscle (P<0.01).This experiment successfully amplified the CDS region of PDK4 gene and constructed a eukaryotic expression vector containing it,and successfully predicted its structure and function,providing a theoretical basis for studying the genetic improvement of subcutaneous fat deposition in Luchuan pigs.

The aim of the experiment was to clone the hormone sensitive lipase (HSL) gene of Guangling donkey and analyze its sequence,and further analyze the differential expression level of HSL gene in different tissues of Guangling donkey.In this experiment,RT-PCR method was used to amplify and clone the partial sequence of CDS region of HSL gene of Guangling donkey.After splicing the sequence,the full length sequence of CDS region of HSL gene could be obtained,and a series of bioinformatics analysis was performed on the sequence.Real-time quantitative PCR was used to detect the expression of HSL mRNA in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and subcutaneous fat in Guangling donkey.The results showed that the complete CDS region of HSL gene in Guangling donkey was 2 286 bp in length,encoding a total of 761 amino acids,and the sequence was successfully submitted to NCBI,accession No.MN231003.The homology of the nucleotide sequence of HSL gene in Guangling donkey with the corresponding sequences of Equus caballus,Vicugna pacos,Camelus ferus,Sus scrofa,Bos taurus,Capra hircus,Mus muqulus and Ovis aries were 99.6%,88.9%,88.6%,88.1%,86.9%,85.6%,80.8% and 79.1%,respectively.Phylogenetic tree prediction showed that HSL gene of Guangling donkey was closely related to the horse and the furthest to the mouse.Bioinformatics analysis found that the theoretical isoelectric point of HSL protein was 6.51,and the instability index was 56.83,and the total average hydrophilicity was -0.048,indicating that HSL was an acid-labile water-soluble protein.There were N-terminal domains and α/β hydrolase folding domains as well as regulatory domains in the conserved domains of proteins.There were 88 phosphorylation modification sites and 25 glycosylation modification sites in the protein sequence.There were strong hydrophobic regions in the protein,without signal peptides and transmembrane regions.The secondary structure showed that this protein was composed of alpha-helix,extended chain,beta-turn and random coil,which account for 45.33%,11.70%,5.65%,37.32%,respectively.Real-time quantitative PCR detection results showed that HSL gene mRNA was expressed in 7 kinds of tissues in Guangling donkeys,but there were differences.The highest expression was in subcutaneous fat and the lowest expression in heart,indicating that Guangling donkey HSL gene might be play a very important part in fat deposition in vivo.It provided a theoretical basis for further studying the function of HSL protein and its metabolic regulation mechanism of fat deposition in Guangling donkey.

The study was aimed to reveal the temporal and spatial expression of ACTC1 gene in Qinchuan cattle,which will lay the foundation for further research on the functions of ACTC1 gene in muscle development and fat deposition of beef cattle.Real-time quantitative PCR was used to detect the expression of ACTC1 gene in 13 tissues of 24-month-old adult Qinchuan cattle and 12 kinds of tissues of 4-day-old Qinchuan cattle.At the same time,the expression characteristics of this gene in myoblasts and preadipocytes of Qinchuan cattle at different differentiation stages (0,2,4,6 and 8 d) were studied and analyzed.The results showed that the expression of ACTC1 gene was the highest in heart of Qinchuan cattle,followed by skeletal muscle.Except for rumen and kidney,the expression of ACTC1 at 24-month-old adult cattle was significantly or extremely significantly higher than that at 4-day-old newborn cattle (P<0.05;P<0.01).The expression of ACTC1 gene in myoblasts increased at first and then decreased along with the myoblasts differentiation.On the 2nd,4th,6th and 8th day of myoblast differentiation,the expression of ACTC1 gene was significantly different from that on 0th day (P<0.01),which were 2.6,4.7,5.6,and 4.2 times of 0th day,respectively.This was consistent with the differentiation rate of myoblasts.The expression trend of ACTC1 gene in adipocytes decreased at first and then increased.The expression levels on 2nd day and 4th day were significantly different from those on 0th day (P<0.01).From the 2nd day of differentiation,the expression increased with the adipocyte differentiation.The expression of ACTC1 gene was the highest in muscle tissues (myocardium and skeletal muscle) of cattle,and its expression characteristics were consistent with the overall trend of muscle cell differentiation.In addition,there was also a certain rule of ACTC1 gene expression in adipocytes.In summary,it was speculated that ACTC1 gene might affect the growth and development of muscle tissue and fat deposition in cattle.

This study was aimed to clone and construct the adenovirus vector of pri-miR-16b gene in buffaloes,meanwhile the bioinformatics analysis of miR-16b and its predicted target genes were carried out to provide a basis for studying its role in oocyte maturation of buffaloes.pri-miR-16b gene was amplified from the ovary genome in buffaloes by RT-PCR,and then the pDC316-mCMV-EGFP-pri-miR-16b plasmid was constructed by homologous recombination.The homology and phylogenetic tree were analyzed and constructed by BLAST program and Mega 7.0,respectively,and the secondary structure of pre-miR-16 were predicted by ViennaRNA Web Services.The constructed vector was packaged with the adenovirus backbone plasmid pBHGloxdelE13cre to obtain the adenoviral particles named Ad-miR-16b,then three times of amplifications were performed and the virus titer was also determined.Ad-miR-16b adenovirus particles infected buffalo cumulus cells,and the expression of miR-16b in cumulus cells was detected by Real-time quantitative PCR.The target genes of miR-16b were predicted by TargetScan,and the KEGG pathway was enriched by the DAVID website for the predicted target genes.The results showed that the pri-miR-16b sequence in buffaloes was successfully cloned,and the sequence similarity with the bovine was found to be 100%.The high homology of pre-miR-16b in buffaloes with other species and the closest relationship with Bos taurus.The ViennaRNA Web Services prediction results showed that the secondary structure of pre-miR-16b had a typical single stem-loop structure,Ad-miR-16b adenoviral particles were obtained with a virus titer of 3.367×10¹⁰ GFU/mL.After the cumulus cells were infected,the expression of miR-16b was extremely significantly increased by Real-time quantitative PCR (P<0.01).The KEGG pathway enrichment analysis was conducted on 1 394 target genes predicted by miR-16b,and it was found that miR-16b might play a role in oocyte maturation mainly by regulating 74 signaling pathways such as MAPK,TGF-β and PI3K/AKT in cumulus cells.

To investigate the expression and regulation of 4F2hc in mammary glands of dairy cows,and clarify the process of amino acids transport across plasma membrane of mammary epithelial cells,the expression of 4F2hc inmammary tissues of lactation cows and dry cows were detected by Western blotting and Real-time quantitative PCR.The mammary epithelial cells of lactating cows in vitro were treated with leucine,the effect of leucine on the expression of 4F2hc in the mammary epithelial cells of lactating cows was also detected by Western blotting and Real-time quantitative PCR.The expression of 4F2hc and the synthesis of milk protein in the mammary epithelial cells of dairy cows were detected by Western blotting.The results showed that the mRNA and protein expression levels of 4F2hc were significantly or extremely significantly higher in mammary tissues of lactating cows than that in dry cows (P<0.05,P<0.01).Adding leucine to cultured bovine mammary epithelial cells could extremely significantly increase the expression of 4F2hc mRNA and protein (P<0.01).Leucine stimulation could activate the mTOR signaling pathway (P<0.05),while rapamycin treatment could significantly inhibit the phosphorylation of mTOR signaling molecules and the expression of 4F2hc induced by leucine (P<0.05,P<0.01),and then extremely significantly inhibit the synthesis of β-Casein(P<0.01).These results revealed that the expression of 4F2hc was positively correlated with lactation activity in mammary glands of dairy cows.Leucine could regulate 4F2hc expression via mTOR signaling pathway in mammary epithelial cells of dairy cows,thus affecting milk protein synthesis.

This study was conducted to investigate the effects of fermented cottonseed meal (fermented by Candida tropicalis) on the growth performance,apparent digestibility,carcass traits,and lipid-related indices in broilers.One hundred and eight one-day-old Cobb broilers were randomly assigned to three groups with six replicates of 10 birds in each.The three diets consisted of a control diet supplemented with fermented cottonseed meal without bacteria (CSM,CON) and the experimental diets with fermented cottonseed meal with Candida tropicali (FCSM) and CSM+Candida tropicali (CT-USCM),respectively.The results showed as follows:①FCSM increased the average daily feed intake compared with CON (P<0.05).FCSM and CT-USCM had lower feed and gain ratio than CON group (P<0.05).②The body weight increased in response to dietary FCSM supplementation (P<0.05),the percentage of liver of CT-USCM group was higher than CON group (P<0.05),additionally,FCSM and CT-USCM supplementation decreased the percentage of abdominal fat and subcutaneous fat thickness (P<0.05).③The dietary nutrient digestibility of dry matter and crude protein significantly increased (P<0.05) with FCSM and CT-USCM supplementation.④Serum concentration of glucose,triglyceride,and low-density lipoprotein cholesterol decreased (P<0.05) in FCSM and CT-USCM fed broilers compared with CON group broilers.⑤The activity of hormone-sensitive esterase and the level of growth hormone in the liver and abdominal fat were higher (P<0.05) in FCSM and CT-USCM groups than CON group.The activity of acetyl CoA carboxylase and malic enzyme in the liver and abdominal fat in FCSM group were lower than CON group (P<0.05).The activity of lipoprotein lipase and fatty acid synthase in liver and abdominal fat in FCSM group were higher than the other two groups,respectively (P<0.05).In conclusion,both FCSM and CT-USCM treatment were beneficial for broilers as they positively affect their growth and digestibility in addition to altering their fat deposition.

To investigate the effects of dietary supplementation of wolfberry residue and liquorice mixed additives (LWRMA) on the production performance and meat quality of Yellow-feathered chickens,a total of 240 healthy one-day-old (half male and half female) Liangfenghua Yellow-feathered chickens were randomly divided into 4 groups according to the principle of similar body weight,and with 6 replicates per group (10 broilers per replicate).The control group (CS) was fed with a basal diet supplemented with 45 mg/kg chlortetracycline,while the groups Ⅰ,Ⅱ and Ⅲ were fed with basal diet supplemented with 1%,2% and 4% LWRMA,respectively.The study was lasted for 56 days.The results showed as follows:① Compared with CS group,the average daily feed intake (ADFI) of group Ⅲ was significantly increased (P<0.05),the ADFI showed a significant linear effects on the amount of LWRMA added in multiple periods (1 to 14,29 to 41,42 to 56,1 to 28,29 to 56,1 to 56)(P<0.05);There was no significant difference in average daily gain (ADG) and feed to gain ratio (F/G) among the experimental groups (P>0.05).②Compared with CS group,the breast muscle rate of group Ⅲ was significantly increased (P<0.05),and the breast muscle rate showed a significant linear effect on the amount of LWRMA added (P<0.05).The yellowness (b^*) of the breast muscle and leg muscle of groups Ⅱ and Ⅲ were significantly increased (P<0.05),the shear force of leg muscle of group Ⅲ was significantly reduced (P<0.05);The yellowness of the breast muscle and leg muscle,redness (a^*) values and water holding capacity (WHC) of the breast muscle and the shear force of the leg muscle showed a significant linear effect on the amount of LWRMA added (P<0.05).The average muscle fiber cross-sectional area and average muscle fiber density showed significant linear effects on the addition of LWRMA (P<0.05).③There was no significant difference in T-AOC,CAT,SOD and GSH-Px among the experimental groups (P>0.05).In conclusion,adding 4% LWRMA to the diet of Yellow feather chickens could not only achieve the same effects as chlortetracycline in production performance and muscle antioxidant performance,but also improve meat quality.

Fucoidan is a natural heteropolysaccharide,it is rich in natural resources,safe and non-toxic,it has a variety of physiological functions.Fucoidan can remove excess free radicals in the body,activate the antioxidant enzyme system,inhibit the signal transduction of oxidative stress pathways to exert antioxidant function,improve the body's immunity by inhibiting the production of inflammatory factors in the body,inhibiting the inflammatory pathway,promoting antibody expression,and improving the body's non-specific immunity,promote the apoptosis of cancer cells,and inhibit angiogenesis and improve the body's immunity to play an anti-tumor effect.Fucoidan also has various biological functions such as bacteriostasis,anti-virus and anti-thrombosis.Animal experiment shows that fucoidan can reduce the feed-to-gain ratio of piglets and improve the digestibility of nutrients,increase the total feed intake,total weight gain and feed conversion rate of chicks,increase significantly the weight,growth rate and PER of wild pheasants in South Asia,increase chicken's weight and breast muscle weight,improve the antioxidant capacity of pork,reduce intestinal inflammation and edema,increase intestinal villi height,improve intestinal flora balance,and adjust glucose and lipid metabolism,reduce body fat content.This article reviews the physical and chemical properties,physiological functions,and application status of fucoidan in animal production,in order to provide a theoretical basis for the application of fucoidan in animal production.

Feedstuff proteins have a directly influence on nutrition and growth of animals.Now,the evaluations of feedstuff protein are mostly based on the methods of biological value,while the study of protein scoring are rare.The authors summarized the six common international methods of protein nutritive value evaluation at present,using the nutritional requirements of 50-75 kg growing pigs(NRC,2012) as reference proteins,applying AAS,CS,EAAI,EAARR,SRCAA and FCM as evaluation criterion to evaluate the nutritional value of 9 common feedstuff (maize,wheat,barley,soybean meal,cottonseed meal,rapeseed dregs,corn gluten meal,fish meal and whey powder) protein.Furthermore,the application results of six protein nutrition evaluation methods in the evaluation and analysis of feed nutrition value were obtained.Through analysis and discussion,which showed that EAARR and FCM were more scientific and reasonable than other methods in protein evaluation,could provide reference for formula design of growing pig feed.

The purpose of this study was to predict the ruminal effective degradability (ED) of dietary carbohydrate (CHO) by Cornell sheep net carbohydrate and protein system (CNCPS-S) and was measured to verify the accuracy of the predicted values of the CNCPS-S model.Three Kazakh wethers with (38.03±1.56) kg body weight and permanent ruminal fistula were chosen and three kinds of total mixed diet (TMR) were designed of which the concentrate/roughage ratio were 30:70,35:65 and 40:60,respectively.The outflow rate (Kp) of ruminal digesta and ruminal ED of three kinds of dietary CHO were measured by the ruminal perfusion test and nylon bag test.Meanwhile,the CNCPS-S model was used to predict Kp of ruminal digesta and ED of CHO in three TMRs,and the predicted and measured values of ED of CHO were analyzed by linear regression analysis so as to evaluate the accuracy of the model prediction.The results showed that:The Kp of ruminal digesta of three kinds of TMR increased sequentially with the proportion of concentrate (P<0.01),which were 3.58%/h,3.83%/h and 4.05%/h,respectively.And the ruminal ED of dietary CHO based on these calculations were 42.7%,43.1% and 42.3% (P>0.05),respectively.The model predictive values of ruminal digesta Kp in three TMRs were increased with the increase of concentrate proportion,which were 1.62,1.63 and 1.64%/h (P>0.05),respectively.The predicted values of ruminal ED of dietary CHO were 39.5%,39.1% and 39.6% (P>0.05),respectively.The average deviation between the predicted and measured values of ruminal ED of three dietary CHO was relatively small (mean deviation ≤ 4 g/100 g CHO),and the correlation was high (R² ≥ 0.86),the root mean square error was also lower (RMSE ≤ 1.93 g/100 g CHO).It was concluded that the prediction model of ruminal degradation in CNCPS-S had a good predictive ability for the ruminal effective degradability of dietary CHO.

In recent years, intensive farming has become the development direction of animal husbandry, with the larger and larger scale of breeding. Especially, as a pillar industry related to people's livelihood, pig breeding accounts for more than half of the whole animal husbandry. On the basis of ensuring the sustainable development of pig breeding industry, how to improve production efficiency and achieve resistance-free farming has become a current research hotspot. As a new type of feed probiotics, Bacillus coagulans has the properties of both lactic acid bacteria and bacillus, which can not only produce lactic acid, but also form spores. There are such functions as improving the intestinal health of pigs, building up the immunity and disease resistance, regulating the balance of intestinal flora and boosting the performance of animal production, so it has a great application prospect in pig breeding. The biological characteristics of Bacillus coagulans were reviewed in this paper. Meanwhile, the paper focuses on the effects of Bacillus coagulans on pig production performance in non-resistant diet, alternative antibiotics, diets containing antibiotics, mixed with other additives and other aspects. Combined with the existing experimental results, the mechanism of Bacillus coagulans on improving the growth performance of pigs was analyzed from the balance of intestinal health, digestion and absorption, immunity and intestinal flora. The problems and suggestions in the application of Bacillus coagulans in pig breeding were also put forward in order to provide reference for the further development and application of Bacillus coagulans preparation.

In order to further explore the application effects and mechanism of an amino acid blend (AAB),this study investigated the effects of dietary supplementation with AAB on liver energy state,free amino acids and relative expression of related genes in weaned piglets following our previous studies.Sixteen (21±3) days old weaned piglets with similar body weight were randomly assigned to control group and AAB group,with 8 replicates per group.The diet of the control group was basal diet supplemented with 0.99% alanine (isonitrogenous control),the diet of AAB group was basal diet supplemented with 1.00% AAB (glutamate:glutamine:glycine:arginine:N-acetylcysteine=5:2:2:1:0.5).On day 21 of the trial,the blood and liver samples were obtained from all piglets.The results indicated that:Compared to the control group,AAB group decreased plasma ALT and AST activity in piglets (P<0.05),increased the total protein content,RNA/DNA and total protein/DNA in the liver of piglets(P<0.05),reduced the MDA and H₂O₂ contents in the liver of piglets (P<0.05),increased the liver arginine and adenosine diphosphate (ADP) levels (P<0.05),increased mRNA levels of amino acid transporter b^0,+ AT and y⁺ LAT2 genes,glutathione S-transferase ω2 gene (GSTO2),interferon-γ gene (IFN-γ),lipoprotein lipase gene (LPL) (P<0.05),decreased mRNA levels of fatty acid synthase gene (FASN) (P<0.05).The above results showed that the addition of 1.00% AAB could improve the liver function,improve the antioxidant capacity,regulate the energy state and free amino acid metabolism of piglet liver,and might affect the fat metabolism and immune function of liver.

This experiment was conducted to study the effects of vitamin A levels of concentrate supplement on the digestion of dietary nutrients,blood biochemical indexes and antler yield of red deer.Eighteen Altai red deers,aged 5 to 7 years old with similar weight and good health were selected and randomly divided into 3 groups,with 6 replicates per group.And they were fed experimental concentrate supplements containing different vitamin A levels,which were 12 800 IU/kg (group Ⅰ),18 800 IU/kg (group Ⅱ) and 24 800 IU/kg (group Ⅲ) respectively.The deers were fed with roughage composed of alfalfa hay and straw freely.And the preliminary feeding period was 15 d and the trial period was 66 d.The results showed that:① The intake of dry matter (DM),organic matter (OM),calcium (Ca) and phosphorus (P) in group Ⅰ were extremely significantly higher than that in the group Ⅱ (P<0.01),and the intake of crude protein (CP) and crude fat (EE) were significantly higher than that in group Ⅱ (P<0.05).CP and P intake in group Ⅲ were significantly higher than that in group Ⅱ (P<0.05).② The digestibility of P in group Ⅰ was significantly higher than that in groups Ⅱ and Ⅲ (P<0.05),while there was no significant difference in the digestibility of other nutrients among the groups (P>0.05).③ The addition of vitamin A in concentrate had no significant effect on protein metabolism and glucose and lipid metabolism in Altai red deer (P>0.05).However,total blood protein (TP) and blood glucose (GLU) increased with the increase of vitamin A,while total cholesterol (TC) and UREA nitrogen decreased.④ AST,ALT and ALP decreased with the increase of vitamin A level,among which the activity of ALP in groups Ⅱ and Ⅲ was significantly lower than that in group Ⅰ (P<0.05).⑤ The blood immunoglobulin content increased with the increase of vitamin A level,among which the immunoglobulin G (IgG) content in group Ⅲ was significantly higher than that in groups Ⅰ and Ⅱ (P<0.01).⑥Malondialdehyde (MDA) content in group Ⅲ was significantly lower than that in group Ⅰ (P<0.05) and glutathione-peroxidase (GSH-PX) activity was significantly higher than that in group Ⅰ (P<0.01).Superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) in the three groups increased significantly with the increase of vitamin A level (P<0.01).⑦ Blood growth hormone (GH) contents of groups Ⅲ and Ⅱ were significantly higher than that of group Ⅰ (P<0.01),insulin-like growth factor 1 (IGF-1) and vitamin A contents of group Ⅲ were significantly higher than that of group Ⅰ (P<0.01),blood estradiol (E₂) level rises with concentrate vitamin A level (P<0.01).The blood testosterone (T) and 25-hydroxyvitamin D₃ (25-OH-VD₃) levels increased (P>0.05).⑧ There was no significant difference in velvet antler yield among the groups (P>0.05),with the highest antler yield in group Ⅰ and the lowest in group Ⅲ.To sum up,under the conditions of this experiment,Altai red deer had higher feed intake and antler yield when the vitamin A content of concentration was 12 800 IU/kg,while Altai red deer had stronger immune and antioxidant functions when the vitamin A content of concentration was 24 800 IU/kg.Comprehensive production benefits,recommended concentrate vitamin A level of 12 800 IU/kg was better.

The aim of this study was to investigate the enrichmen condition of Methanomassiliicoccales from buffalo rumen,methanol,monomethylamine,dimethylamine or trimethylamine were used as substrates in BRN culture medium respectively.Methane production,population and diversity indices of Methanomassiliicoccales and methanogen,wasmeasure by gas chromatography,Real-time quantitative PCR and high-throughput sequencing of the 16S rRNA gene.The results showed that:The methane production of trimethylamine group was higher than other group significantly (P<0.05).The copy of Methanomassiliicoccales and methanogen of methanol group and trimethylamine group were higher than control group significantly(P<0.05) in 1st generation.The copy of Methanomassiliicoccales and methanogen of trimethylamine group were higher than control group,monomethylamine group,dimethylamine group significantly (P<0.05) in 7th generation.The copy of Methanomassiliicoccales was detected in trimethylamine group and dimethylamine group only in 23rd generation,and the copy of methanogen of trimethylamine group was highest.The result of Alpha diversity indices showed that,the Ace and Chao1 index of trimethylamine group were higher than control group significantly (P<0.05).The relative abundance of methanogen at order level were more than 90%.The methane production,population and relative abundance of Methanomassiliicoccales were increased while trimethylamine was used as substratesduring transfers.

Saccharomyces boulardii,a subspecies of Saccharomyces cerevisiae,has high temperature and bile salt resistance.Saccharomyces boulardii reduces intestinal pathogens by altering the surface of pathogens.In addition,the secretion of Saccharomyces boulardii can increase enzyme activity in animals,thereby promoting digestion and absorption of nutrients.Furthermore,the secretion of Saccharomyces boulardii can improve the body's anti-inflammatory effect by interfering with pro-inflammatory factors.Saccharomyces boulardii can prevent and cure diseases such as antibiotic-associated diarrhea,Clostridium difficile infection,traveler's diarrhea,irritable bowel syndrome,crohn's disease and other diseases.Saccharomyces boulardii has been certified in Europe and North America as a feed additive.In livestock and poultry applications,Saccharomyces boulardii can promote the proliferation of intestinal beneficial bacteria,produce lactic acid to reduce the content of intestinal pathogenic bacteria,promote the release of immune factors and the development of immune organs,thus decrease feed and gain ratio,improve the production performance,enhance immunity,reduce diarrhea,and regulate intestinal flora balance.In this paper,the authors reviewed the biological function,mechanism of action and application of Saccharomyces boulardii.It was expected to provide reference for its further research and wide application in the future.

This experiment was conducted to clarify the genetic diversity,genetic differentiation and phylogenetic status of yak in Karakoram-Pamir area.The mtDNA D-loop region sequence was selected as a molecular marker,and the sequence and genetic diversity of the mtDNA D-loop region of yak in Karakoram-Pamir area were analyzed by PCR direct sequencing and bioinformatics methods.The yak sequence in GenBank was used.The maximum likelihood method was used to construct the phylogenetic tree and the intermediary network relationship.The results showed that the mtDNA D-loop sequence of yak in Karakoram-Pamir area was rich in A and T bases,with AT content of 61.2%,and there were 63 polymorphic loci,accounting for 7.04% of the total number of nucleotides.The results indicated that A and T bases were rich in the mtDNA D-loop sequences at 61.2%.There were 63 mutation sites,accounting for 7.04% of all nucleotides,The average haplotype diversity (Hd) was 0.806,the average nucleotide diversity (π) was 0.01528,and the average nucleotide difference (K) was 13.509,indicating that the yak was rich in genetic diversity in Karakoram-Pamir area;Through phylogenetic analysis,there were two branches in yak in China,forming two branches and six small clades.The yak in Karakoram-Pamir area involved in this study had two different maternal origins.Additionally,yak in the Karakoram-Pamir area was less shared with other breeds of yak haplotypes.In the branch C,the yak group in the Karakoram-Pamir area accounts for a large proportion and was shared with wild yak.The yak population in Karakoram-Pamir area had a unique genetic background,which might be the result of early domestication of wild yaks.It was suggested to increase the identification of yak breeds and the formulation of breed standards in this area,and strengthen the protection of yak genetic resources in this area.According to the current situation of the population,wild blood yaks were introduced for purification and rejuvenation to prevent breed degeneration and decrease of genetic diversity.The introduction of foreign yak breeds and disorderly hybridization were reduced to ensure the characteristics of this breed of high-quality yak breed resources.

This study was aimed to investigate the association of the polymorphism of retinol-binding protein 4 (RBP4) gene with reproductive traits in Songliao Black pigs.132 Songliao Black pigs were selected,the single nucleotide polymorphism (SNP) of RBP4 gene in Songliao Black pigs were screened by PCR-HRM combined with DNA sequencing,and the correlation between SNPs and reproductive traits (total number born,number born alive,number of nipples,birth weight,3-week weight and weaning weight) were analyzed.The results showed that two SNPs (G45A and C170T) of RBP4 gene were detected,which were located in exons 3 and 5,respectively.There were three genotypes (GG,GA and AA) in the G45A site,and two genotypes (CC and CT) in the C170T site.The G45A site was uniform in Songliao Black pigs,and its variation and polymorphism were moderate,while it was not in Hardy-Weinberg equilibrium (P<0.01).The C170T site was uneven in Songliao Black pigs,and its variation and polymorphism were low,it was in Hardy-Weinberg equilibrium (P>0.05).In the G45A site,the total number born and number born alive were showed as GG > GA > AA,the total born and number born alive of GG genotype were significantly higher than AA genotype (P<0.05),the 3-week weight of GG genotype was significantly lower than GA genotype (P<0.05),the weaning weight of GG genotype was significant lower than GA and AA genotypes (P<0.05),there was no significant difference of birth weight and number of nipples in different genotypes (P>0.05).In the C170T site,there was no significant difference of all reproductive traits in different genotypes (P>0.05).In conclusion,the G45A site of RBP4 gene had a great influence on the litter size in Songliao Black pigs,it needed to further explore whether the G45A site could be used as a genetic marker for the litter size in Songliao Black pigs.The C170T site might not be a mutation site associated with reproductive traits in Songliao Black pigs,it needed to expand the number of pig for further verify.

In order to study the effect of myogenin (MyoG) and insulin-like growth factor-1 (IGF-1) genes expression on the growth and development of poultry,Yanjin Black-bone and Daweishan Mini chickens with large differences in body size were chosen.At the age of 300 days,the body weight,body size and slaughter traits of 20 chickens were measured for comparative analysis,and the relative expression of MyoG and IGF-1 genes mRNA in chest muscle,leg muscle and liver were detected.Finally,the correlation between growth and slaughter traits and gene relative expression were analyzed,respectively.The results showed that the body weight,body size and slaughter traits of Yanjin Black-bone chickens were higher than that of Daweishan Mini chickens.Moreover,the expression of MyoG and IGF-1 genes in chest muscle,leg muscle and liver of Yanjin Black-bone chickens were also higher than that of Daweishan Mini chickens,and the expression trend was as follows:Chest muscle > leg muscle > liver.Correlation analysis results showed that the expression of MyoG and IGF-1 genes in pectoralis muscle,leg muscle and liver of Yanjin Black-bone and Daweishan Mini chickens were significantly positively correlated with body weight,body size and slaughter traits (P<0.05).The relative expression of MyoG gene was extremely significantly correlated with all the traits except chest depth (P<0.01),but the relative expression of IGF-1 gene was significantly correlated with body weight,body length,chest depth and sternum length (P<0.05).Therefore,MyoG and IGF-1 genes could be used as candidate genes for growth traits of Yanjin Black-bone and Daweishan Mini chickens for breeding.

In order to explore the effect of SQLE gene on the apoptosis and proliferation of dairy mammary gland cells in vitro,the relative expression of SQLE gene,apoptosis and cycle-related genes of dairy mammary epithelial cells with SQLE gene knockdown were detected by Real-time quantitative PCR.Cells were screened by cycle,apoptosis detection kits and flow cytometry.The results showed that the relative expression of SQLE gene was significantly decreased after siRNA (SQLE siRNA) transfection.The cell proliferation was significantly inhibited (P<0.05).The number of cells in G1 phase significantly decreased (P<0.05),and the proportion of cells in S phase showed an increasing trend (P<0.05).No significant difference in cells in G2 phase.The relative expression of apoptotic gene Fas and proliferation and cycle related genes P27 significantly increased (P<0.05), Cyclin D1,Bcl-2,P21 and Bax genes decreased significantly (P<0.05).In summary,knockdown of SQLE gene could inhibit the proliferation of breast epithelial cells by regulating the expression of related genes.

In order to study the growth and muscle quality of cross breed goat from Boer and Henan Huai goat,the body weight,slaughtering performance,conventional nutrients (water,crude protein,crude fat,crude ash,calcium,phosphorus,magnesium,iron and selenium),17 kinds of amino acids content and different parts (arm triceps,biceps femoris,longissimus dorsi muscle) of the carcass muscles of cross breed goat from Boer and Henan Huai goat were determined by routine growth performance test,national standard method and histochemical staining the fiber tissue characteristics (muscle fiber diameter,muscle fiber density) of meat were measured and analyzed.The results showed that the body weight,body length,body height and tube circumference of Boer and Henan Huai hybrid ram were significantly higher than those of ewe (P<0.05) at the new born stage;The body weight,body length,body height and chest circumference of ram at 1,3 and 6 months were significantly higher than those of ewe (P<0.05);At the age of one year and 1.5 years,the body weight and body size of ram were significantly higher than those of ewe (P<0.05).The carcass weight of 6-month-old Boer and Henan Huai hybrid ram and ewe was 12.94 and 11.33 kg respectively,and the slaughter rate was 49.01% and 48.34%,respectively.There was no significant difference in slaughter performance (P>0.05).The percentage of moisture,crude fat,crude protein and ash in carcass muscle was 71.00%,2.10%,19.17% and 2.31% respectively,the cholesterol content was 62.93 mg/100 g;The content of Mg,Ca,Fe,Se and P were 248.00,40.57,17.00,0.08 and 210.12 mg/kg,respectively.The proportion of Lys,His and Arg in FAO was 123.87%,140.42% and 102.88%,respectively,which were higher than the evaluation standard of FAO/WHO (ideal protein model);The proportion of Thr and Leu in FAO was 86.80% and 82.00%,respectively,which were not significantly different from that of FAO ideal protein model.Phe accounted for 36.90% of FAO index,which was significantly different from the amino acid content of FAO ideal protein model.The muscle fiber diameter of arm triceps was 39.98 μm and higher than that of the biceps femoris muscle (36.53 μm) and longissimus dorsi muscle (27.06 μm).The results showed that the slaughtering performance of Bohuai hybrid goat was better under the condition of house feeding,the content of amino acid,protein,fat and several main minerals in muscle was rich,and the fat in muscle appeared marbling distribution,which provided a reference for the next crossbreeding improvement of local goat breeds and the development of high quality mutton products.

Spermatogonial stem cells (SSCs) are located on the basement membrane of seminiferous tubules in the testis.Due to the abilities of self-renewal and differentiation into sperm,SSCs are the only adult stem cell in male mammals which can transmit genetic information to the next generation.Studies of SSCs also have become one of the hot topics in stem cell research.However,at present,the studies of SSCs mostly focus on rodents while the research progress of porcine SSCs (pSSCs) is relatively slow,which is largely due to the slow progress on isolation,purification,and in vitro culture technology as well as molecular marker identification of pSSCs.The main problems include:Extremely low amount of pSSCs in the testis,lack of specific molecular markers of pSSCs;unestablished techniques of in vitro isolation,purification,and culture of pSSCs;imperfect in vitro culture system of pSSCs.These problems lead to the low efficiency of pSSCs isolation and purification,failure for long-term culture and passage in vitro,which in turn results in lack of pSSCs materials for related mechanism research,and inconvenience of their in vitro study as well as application.In this review,we briefly summarize the research progress of pSSCs,including the molecular markers,the methods of in vitro isolation,purification,and cultivation,as well as transplantation technology,so as to provide theoretical references for pSSC study,accelerating the study and application of pSSCs in the fields of pig genetics,breeding and reproduction as well as male reproductive medicine.

The aim of this study was to investigate the expression and localization of osteopontin (OPN) in germ cells of Landrace boars,and their correlations with boar reproductive performance.The semen and testis in different age stages (3-day-old,3-month-old,6-month-old,and 12-month-old) from Landrace boars were collected,followed with the expression levels of OPN protein in semen and testis,and its location in boar testis cells detected by Western blotting and immunohistochemistry(IHC),respectively.At the same time,according to the standard of breeding parity ≥ 20 births and three times of breeding semen from the same boar,17 Landrace boars were selected along with their semen collected and evaluated.Further,productive performance data of the 1 388 boar-paired sows were calculated for evaluating the performance of those 17 breeding boars by total number born per litter (TNB),number born alive per litter (NBA),farrowing rate (FR) and fecundity.To evaluated the semen of those 17 breeding boars,sperm and seminal plasma were separated by low-temperature centrifugation of semen.Seminal plasma protein was extracted with acetone method,and sperm protein was extracted with Lysis buffer method.BCA (Bicinchoninic acid) and ELISA were used to detect OPN contention in sperm and seminal plasma,for subsequent correlation analysis between OPN protein and boar reproductive performance by SPSS software.Consequently,Western blotting results showed that two forms of OPN protein(67.4 and 33.7 ku) could be observed in sperm,seminal plasma and testis of Landrace boars at all age stages,and 67.4 ku form was highest expressed in testis of 3-month-old boars;IHC results showed that OPN were expressed in primary spermatocytes,secondary spermatocytes and sperm cells,but not in spermatogonia,sertoli cells and interstitial cells;BCA and ELISA results showed that the concentration of OPN protein in sperm was 7 times higher than that in seminal plasma (P<0.05).Additionally,correlation analysis between OPN protein and boar reproductive performance indicated the OPN protein in semen was significantly positively correlated with NBA (P<0.05).Collectively,our study suggested that OPN expresses in testis (at all age stages),sperm and seminal plasma of Landrace boars,and it might concern the reproductive performance of boars,thereby laying a foundation for the mechanism study of OPN protein in boar fertility.

The purpose of this study was to investigate the effects of reactive hydrogen peroxide (H₂O₂) on the expression of the protein ubiquitin SUMO-1 and the ability of sperm-oocyte binding of porcine oocyte during in vitro maturation (IVM).The test was divided into 5 groups:0 (control),10,50,75 and 100 μg/mL of H₂O₂ treatment group.Western blotting,flow cytometry,Real-time quantiative PCR,Hoechst staining and other methods were used to detect porcine oocyte in vitro maturation,content of protein SUMOylation SUMO-1,cell viability,expression of apoptotic gene mRNA,solubility of zona pellucida (ZP) and sperm-oocyte binding.The results showed that 75 μg/mL H₂O₂ group compared with the control,10 and 50 μg/mL H₂O₂ groups,the in vitro maturation rate and cell viability of oocytes decreased significantly,while 75 μg/mL H₂O₂ group compared with other H₂O₂ groups,the ZP lysis time prolonged significantly,and the number of sperm adhering to the ZP of mature oocytes was reduced (P<0.05).SUMO-1 protein markers appeared at 77 and 18 ku,and the content of SUMO-1 protein was significantly reduced in 75 μg/mL H₂O₂ group when compare with the control,10 and 50 μg/mL H₂O₂ groups (P<0.05).75 μg/mL H₂O₂ group compared with the control,10 and 50 μg/mL H₂O₂ groups,Bcl-2 gene was significantly down-regulated (P<0.05),while 50 μg/mL H₂O₂ significantly up-regulated Bax gene level when compared with the control and 10 μg/mL H₂O₂ groups (P<0.05).In summary,H₂O₂ could regulate the SUMOylation level and sperm-oocyte binding ability of porcine oocytes during in vitro maturation.

The purpose of this study was to investigate the effect of polyvinylpyrrolidone (PVP) on boar semen freezing.There were five groups in the experiment:Control group (not add PVP) and PVP treatment groups (add 0.25%,0.50%,1.00%,and 2.00% PVP to the frozen base fluid).Collecting the semen of Songliao Black pig by hand,diluted with five frozen base solutions,equilibrated for 1 h at 25 ℃,2 h at 17 ℃,3 h at 4 ℃,then filled in 0.5 mL straw,fumigated 3 cm above liquid nitrogen for 10 min,stored in a liquid nitrogen tank for 30 d and tested.Sperm viability,plasma membrane integrity,acrosome integrity,mitochondrial activity,DNA integrity,catalase (CAT) activity,superoxide dismutase (SOD) activity,glutathione peroxidase (GSH-Px) activity,reactive oxygen species (ROS) level and malondialdehyde (MDA) level were tested after thawing.The results showed that compared with the control group,adding 0.50% PVP to the frozen base fluid could significantly improve the sperm viability,plasma membrane integrity,acrosome integrity,mitochondrial activity,CAT activity,SOD activity,GSH-Px activity (P<0.05);and significantly reduced the sperm ROS and MDA level (P<0.05).Adding PVP was beneficial to improve DNA integrity,but had no significant effect on DNA integrity compared with the control group (P>0.05).In conclusion,adding PVP to boar semen in the frozen base solution could improve the quality of sperm after freezing and thawing,the best concentration was 0.50%.

In order to investigate the molecular epidemiology and genetic variation of classical swine fever virus (CSFV) in China,this study used the RT-PCR method to amplify E2 and NS5B genes of 350 samples suspected of CSFV infection that collected from Henan,Hebei,Shandong,Heilongjiang and Liaoning provinces in 2018,and the PCR products were sequenced and analyzed.The results showed that 21 of 350 samples were positive for CSFV detection and a total of 14 CSFV E2 gene sequences and 7 partial CSFV NS5B gene sequences were obtained.The complete E2 and partial NS5B gene sequences analysis showed that the 21 positive samples belonged to the 2.1d subtype CSFV that domestic epidemic in recent years.It found that there was no difference in homology between the newly 2.1d subtype CSFV and the earlier 2.1d subtype strains.These new 2.1d subtype CSFV isolates had the same molecular characteristics on the 6 amino acids (R³¹,S³⁴,W¹⁸²,K²⁰⁵,K³⁰³,A³³¹) of E2 gene,and 15 cysteines of E2 protein had no variation.The 2.1d subtype CSFV isolated in South Korean had three unique amino acids (N⁹⁷,K¹⁵⁹,R²⁰⁵) of E2 protein.The Korean strain YC11WB was an intermediate strain between 2.1b and 2.1d subtype CSFV,and the 2.1d subtype CSFV being prevalent in China and South Korea might be derived from the earlier domestic 2.1b subtype CSFV,respectively.This study confirmed that the relatively active and epidemic CSFV strains were still 2.1d subtype in China and neighboring countries in 2018,which providing a basis for scientific prevention and control of CSFV in China.

This study was aimed to observe the effects of microencapsulated Saccharomyces boulardii (S.boulardii) and Enterococcus faecium (E.faecalis) on ulcerative colitis (UC) and discuss its possible mechanism in mice.Mice model with UC was established by 3% dextran sulphate sodium (DSS),the test was divided into 6 groups,the model mice were randomized into 5 groups,mice were treated with unencapsulated-free E.faecalis,microencapsulated E.faecalis,unencapsulated-free S.boulardii and microencapsulated S.boulardii solution,respectively,and saline solution in DSS group by oral gavage.UC model mice (DSS group) were fed with the same volume of saline as the drug every day,while the normal control group was fed with the same volume of saline solution.The symptoms and histological changes of UC mice were observed,the pro-inflammatory cytokines including IL-6,IL-10 and TNF-α were measured by ELISA,and the expression of the Occludin and Claudin-1 were measured by Western blotting.The results showed that compared with DSS group,the contents of TNF-α and IL-6 were significantly reduced in probiotics groups (P<0.05),and the content of IL-10 was significantly increased (P<0.05).The myeloperoxidase activities and histoligical scores (except for unencapsulated-free E. faecalis group) were significantly decreased in microencapsulated pribiotics groups after treatment (P<0.05).The expression of Occludin and Claudin-1 were significantly increased inunencapsulated-free S.boulardii and microencapsulated S.boulardii groups(P<0.05).E.faecalis and S.boulardii could reduce the inflammatory lesions in the colon of UC mice,and the microencapsulated pribiotics had a better effect,moreover,the mechanisms of the improvement were possibly related to the decrease of the contents of IL-6 and TNF-α,and the increase of the expression of Occludin and Claudin-1.

In order to study the effect of Melia azedarach on Haemonchus contortus,this experiment was to observe the effect of Melia azedarach extract on the activity and morphological changes of eggs and larvae,and to evaluate its repellent activity on Haemonchus contortus,so as to lay a foundation for the prevention and control of Haemonchus contortus disease in domestic animals and the research and development of plant repellents.In the experiment,5 concentrations (25,12.5,6.25,3.125 and 1.562 mg/mL) of water extract and ethanol extract of Melia azedarach were set for egg hatching test and larva activity test.The eggs and larva treated with different concentrations of drugs were counted,and the hatching state and morphological change of eggs and larva were observed after the action of drugs.The results showed that the activity of the eggs and larvae was good.48 h after the action of Melia azedarach extract,the eggs were in the state of non hatching and half hatching,and developed to the stage of mulberry,tadpole and larval death.The water extract and alcohol extract of Melia azedarach at different concentrations inhibited the hatching of the eggs,and the inhibitory effect of alcohol extract of Meliae on the hatching of the eggs was the best.At the concentration of 25 mg/mL,the alcohol extract of Meliae inhibited the hatching of the eggs,and the inhibition rate of hatching was 98.2%.At 12.5 mg/mL,the inhibitory rate of wormwood extract on worm eggs was significantly higher than that of oxydermal extract (P<0.01).The lethal effect of Melia azedarach seed extract on infective third stage larvae was poor,only at 50 mg/mL,it had a higher lethal rate to larvae,and the mortality rate was 82.6%.When the concentration of water extract of Meliae was 50 mg/mL,its lethal effect on larva was particularly prominent,the mortality rate of larva was 97.0%,and the dead larva was "linear" or "bow".By variance analysis,in the range of 12.5 to 50 mg/mL,there was a significant or extremely significant difference in the lethality rate between the water extracts of Melia azedarach at the same concentration (P<0.05;P<0.01).There were extremely significant differences in lethal rate of larva between the alcohol extract at the same concentration (P<0.01).In conclusion,Melia azedarach extract could inhibit the activity of eggs and infective third stage larvae of Haemonchus twist in vitro.Among them,25 mg/mL Meliae alcohol extract had the best inhibitory effect on egg hatching,and 50 mg/mL Meliae water extract had the best lethal effect on infective third stage larvae.

The purpose of this study was to isolate and classify Clostridium perfringens (CP) strains from cow viscera and tissue samples to provide materials for subsequent vaccine studies,in addition,sensitive drugs were screened out through drug sensitivity test to guide the pasture to treat cattle and effectively control and isolate cattle without disease.In 2019,some cows in 21 ranches in Shandong province reported diarrhea,hematochezia and short-term death.Some of the dead cows were dissected,and 64 viscera and tissue samples were sent to be cultured for CP preliminary screening,then the positive strains was identified by biochemical identification and PCR typing.The homology of strains was analyzed by 16S rDNA technique.The minimal bacteriostatic concentration of isolated strains against 7 kinds of antibiotics (erythromycin,levfloxacin,linezolid,vancomycin,clindamycin,tetracycline,rifampicin) was determined by E-test method,and sensitive drugs were screened out.8 samples showed gray,orange and black colonies on blood medium,CP chromogenic medium and TSC medium respectively.The biochemical identification results were consistent with the characteristics of CP.PCR typing results showed that 5 A-type strains,2 C-type strains,1 E-type strain.The homology of 8 isolates and Clostridium percapsulatum was up to 100% by 16S rDNA analysis;Among the 8 strains,2 were more sensitive to clindamycin and rifampicin,3 more sensitive to erythromycin and linezolid,1 more sensitive to levfloxacin.Lincoamides,rifamycin and macrolide were recommended for the prevention and control of two farms with diseased cattle.The effect of the prevention and control on the farms was tracked in time.The cattle without therapeutic value were immediately culled and treated innocuously,so as to improve the feeding conditions and reduce the feeding density.

In order to improve and perfect the E.coli specific anti-O-antigen sera pool for micro-agglutination test in China,and further improve the preparation process of mono-specific anti-O-antigen serum,E.coli O50,O60,O117 and O131 serotypes were selected to investigate the method of preparing sera.First,inactivated antigens were prepared by reference strains of E.coli.Crude sera were extracted from rabbits after repeated immunizations by inactivated antigens.The method of micro-agglutination was used in the preparation process to determine the cross-lectins and agglutination value of different sera.The non-specific agglutination was eliminated by absorbing non-specific lectins and diluting the serum.Finally,E.coli specific anti-O-antigen sera for micro-agglutination test with good specificity were developed.The main cross-lectins of four kinds of E.coli specific anti-O-antigen sera were identified.Four kinds of sera with good specificity which could be used for E.coli O antigen micro-agglutination test were prepared,which agglutination titers were between 1:256 and 1:2 048.E.coli O50 and E.coli O60 serotypes sera were mono-specific anti-O-antigen sera.E.coli O117 and E.coli O131 serotypes sera contained 1-2 nonspecific cross-lectins,O14 and O107.This study was an important part of exploring the preparation process of mono-specific anti-O-antigen serum.The important theoretical bases were provided for improving the serum library of E.coli bacterial micro-agglutination test in China,and further improved the preparation method of E.coli O antigen serum for micro-agglutination test.

In order to investigate the bactericidal effect and mechanism of Plectasin-derived peptide NZ2114 against Streptococcus dysgalactiae (S.dysgalactiae) isolated from bovine mastitis,the drug resistance and minimum inhibitory concentration (MIC) were tested by broth dilution method,the MIC and the bactericidal kinetics curve in tryptic soy broth (TSB) and ultra-high temperature instantaneous sterilization (UHT) environment were determined for evaluating the antibacterial properties of NZ2114,the morphological changes of S.dysgalactiae treated with NZ2114 were observed by scanning electron microscope (SEM),transmission electron microscope (TEM) and flow cytometer.The effects of NZ2114 on the genomic DNA were further analyzed by gel retardation,fluorescence spectrum and circular dichroism spectrum.The results showed that all S.dysgalactiae were resistant to ofloxacin and tetracycline,but the clinical isolates showed more antibiotic resistance,the values of MIC were only 0.11 to 0.45 μmol/L.NZ2114 had bacteriostatic activity in TSB and UHT milk medium,and reduced the number of S.dysgalactiae colonies by 3 orders of magnitude in a short time (0.5 to 3 h).The results of SEM,TEM and flow cytometer showed that NZ2114 could cause the leakage of S.dysgalactiae content and kill the bacteria.Gel retardation analysis,fluorescence and circular dichroism all showed that NZ2114 could bind to the genomic DNA and destroy DNA.The above results showed that antibacterial peptide NZ2114 had strong bactericidal activity against S.dysgalactiae isolated from bovine mastitis,it could directly affect the genomic DNA and change its secondary structure after destroying the cell membrane.Therefore,antimicrobial peptide NZ2114 was a promising alternative antibiotic for the treatment of mastitis caused by S.dysgalactiae.

In order to explore the contamination,virulence genes carrying and antibiotic resistance of Escherichia coli(E.coli) in slaughtering process of free-range chicken.319 samples which came from 12 slaughter places of Wanzhou,Kaizhou,Wuxi,Fengjie district in Northeast Chongqing were collected in September 2018 to January 2019.These samples were analyzed by colony morphology,microscopic observation,biochemical test,PCR and drug sersitivity test.The results indicated that the isolation rate of E.coli was 22.57%(72/319).The isolation rates of restaurant,live poultry shop and slaughterhouse were 29.73%(22/74),25.00%(24/96) and 17.45%(26/149) respectively.The rates of samples of waste water,ground,feather,tools and carcass were 75.00%(18/24),21.62%(8/37),20.54%(23/112),17.65%(6/34) and 15.18%(17/112) respectively.All of the virulence genes were detected expect estA,estB and elt genes.The detection rate was 58.33%(42/72) and there were 14 combinations even 4 strains coexisted 4 virulence genes.The strains were most sensitive to amikacin and cefotaxime,and existed multi-antibiotic resistance seriously which most of them were from 7 to 10 folds.There was a high risk to be contaminated by pathogenic E.coli in slaughtering process of free-range chicken and existing multi-antibiotic resistance seriously.So the rational using of drugs and keeping a clean process of slaughter should pay more attention.

In this study,37 swabs were collected from a pig farm with suspected swine influenza in Guangdong province,inoculated into 9-day-old SPF chicken embryos and collected alluvial fluid,and a strain of swine influenza virus was isolated by hemagglutination (HA) test and hemagglutination inhibition (HI) test and RT-PCR identification.Eight gene fragments were amplified for sequencing and sequence analysis,and then compared with the reference strains in GenBank to construct the evolutionary tree.The results showed that the virus strain was H1N1 swine influenza virus.The isolate was designated as A/swine/Guangdong/2/2018 (H1N1).The complete genes were sequenced and the genetic analysis showed that the nucleotide sequences of the 8 segments of the isolate were similar to the corresponding sequences of A/swine/Guangdong/L3/2009(H1N1) by more than 99%,and were in the same branch as the classical swine influenza virus of the H1N1 subtype.The cleavage site of HA gene of the isolate was PSIQS↓GL,which was consistent with the molecular characteristics of low pathogenic influenza virus.HA gene receptor sites were 190D,225G and 226Q,which indicated that the strain could bind to both SA receptor of SAα-2,6-Gal human influenza virus and SA receptor of SAα-2,3-Gal avian influenza virus.There were six potential glycosylation sites at 28,40,104,304,498 and 557 amino acids.There were six potential glycosylation sites in amino acids of 50,58,63,68,98,146 and 235 of NA protein.The active sites of amino acid sequence of NA protein were 119E,199D,223I,275H,293R and 295N.There was no mutation in amino acid analysis site,indicating that the isolate was highly sensitive to neuraminidase inhibitors,but in M2 protein,31 amino acids changed from sensitive (S) to resistant (N),which might be resistant to amantadine drugs.The evolution analysis of swine influenza virus would provide important information for the prevalence and variation of swine influenza in Guangdong province.

In order to understand the effects of Chinese herbal medicine of Taishan Panshi san on the intestinal microflora in mice.45 SPF Kunming mice were selected and divided into control group A,test group B,and test group C.The mice in test group B and group C were administered with 0.86 g/mL Chinese herbal medicine of Taishan Panshi san for 0.3 and 0.4 mL respectively,while in group A were administrated with 4 mL distilled water,twice a day for 8 hours for 7 days.On the 8th day,feces samples from the rectum were collected from the control and test groups.Microbial genomic DNA was extracted for each sample.Illumina HiSeq sequencing technology were used to detects sample microbial community diversity.In the aspect of α diversity,the Chinese herbal medicine of Taishan Panshi san showed a fluctuant change in the microbial diversity index (Chao1 and Shannon) in the feces of mice,and the difference between the Chao1 index of the experimental group and the control group was extremely significant (P<0.01);In terms of β diversity,detecting the microorganisms in the feces of control and experimental group of mice,level on the phylum,compared with the control group,there were 9 dominant phylum,Proteobacteria and Verrucomicrobia in the experimental group decreased significantly (P<0.05),while Saccharibacteri and Tenericutes in the experimental group increased significantly (P<0.05;P<0.01);At the family level,there were 19 dominant bacteria families,Lactobacillaceae,Desulfovibrionaceae,Coriobactaceae,Porphyromonaceae,Family_XIII and Biofidobactriaceae in the experimental group were significantly lower than those in the control group (P<0.05);At the genus,there were 18 dominant bacteria,among which,Lactobacillilus,Desulfovibrio and Enterhorhabdus were significantly lower in groups B and C than in group A (P<0.05),while Alloprevotella,Bacteroides,Lachnospiraceae_UCG-006 and Faecalibaculum in the experimental C group increased significantly (P<0.05).The Chinese herbal medicine of Taishan Panshi san could selectively promote the growth of Bacteroidetes in the intestinal tract of mice,thereby significantly inhibiting the harmful effects of Proteobacteria,enhance the metabolism of sugar and fat,finally achieve a new balance of intestinal microbial diversity by improving the structure of intestinal microbial diversity.

This experiment was aimed to find out the cause of death of Rhizomys sinensis in a Rhizomys sinensis farm in Guizhou.A strain of bacteria (GZ-S1) was isolated from the submitted Rhizomys sinensis in this study,which was identified through morphological observation,biochemical test,16S rRNA gene sequence analysis,antibiotic sensitivity tests,resistance genes detection and animal infection experiment.The results showed that the isolates was Gram-positive with α-hemolysis reaction and could grow smooth and white colonies on the blood plate.Biochemical identification showed that the isolates had positive reactions to sucrose,fructose,glucose,nitrate reduction,indigo matrix and citrate,but negative reactions to galactose,mannose,hydrogen sulfide test,VP test,MR test and lysine decarboxylase contact test.After 16S rRNA gene sequencing,BLAST alignment and phylogenetic tree analysis,it was found that the isolates GZ-S1 was on the same branch as 12 reference strains of the balloon strain 201707CJKOP-Y31 (MG593595.1),Aerococcus viridans strain Mnlv2 (GQ246745.1),and so on.The results of bacterial drug sensitivity test showed that the isolates were sensitive to oxacillin, carbenicillin and ceftriaxone, was moderate sensitivity to amoxicillin,ofloxacin,sulfamethoxazole,bacteresulf polymyxin B,florfenicol and cefradine, and was resistant to enrofloxacin, kanamycin, tobramycin, neomycin, doxycycline and tetracycline.The result of resistance genes detection showed that the isolates carried resistance genes including tetA(1 031 bp),tetB(513 bp),tetD(326 bp),aadd(146 bp) and aac(6')Ⅰb(655 bp),which was consistent with the antibiotic sensitivity phenotypes.The results of artificial infection test showed that the isolates had pathogenicity.This study made an accurate diagnosis of the causes of the disease in a Rhizomys sinensis farm,and provided a reference for the prevention and rational drug use of bacterial diseases for Rhizomys sinensis.

In order to select the candidate strain of live attenuated vaccine with gene deletion for equine rhinopneumonitis(ER).The primers were designd according to the target gene sequence in GenBank of EHV-1 (accession No.:KF644579.1),using the DNA of the popular strain XJ2015 in this region as a template,the left and right homology arms gEH1,gEH1 of gE gene were amplified by PCR,EGFP expression cassette (CMV+EGFP+polyA) as the marker gene,after enzyme digestion,the genes were ligated to the vector pUC-19 in turn,and the plasmid pUC-gEH1H2-EGFP was successfully constructed.Co-transfection of XJ2015 genome and plasmid pUC-gEH1H2-EGFP into RK-13 cells for homologous recombination,screening for gE-deletion strains with a markered gene EGFP,and then determining the titer of recombinant strain after purification.The results showed that,a recombinant strain XJ2015-△gE-EGFP with EGFP gene was successfully obtained after 5 rounds of fluorescent plaque purification,PCR and sequencing identification,and the titer of the recombinant strain (10^7.1TCID₅₀/0.1 mL) decreased by about 10^1.7TCID₅₀/0.1 mL compared with the original strain (10^8.8TCID₅₀/0.1 mL).A gE gene deletion mutant of equine herpesvirus type 1 strain was successfully constructed with homologous recombination technology which provided a foundation for future screening of weak virus vaccine with gene deletion in equine rhinopneumonitis.

The purpose of this study was to prepare porcine circovirus type 3 (PCV3) capsid protein by construct a prokaryotic expression vector pGEX-4T-1-Cap.Recombinant plasmid pGEX-4T-1-Cap was constructed by inserting codon optimized PCV3 Cap gene into pGEX-4T-1 vector,and then transformed into Escherichia coli BL21 (DE3) competent cell.The optimal induction conditions were screened out,and the recombinant Cap protein was purified by glutathione agarose resin.The results showed that the recombinant plasmid pGEX-4T-1-Cap was successfully constructed.A large amount of soluble recombinant capsid protein could be obtained under the condition that the final concentration of IPTG was 0.1 mmol/L and induced at 16 ℃ for 20 h.SDS-PAGE results showed that the molecular mass of the recombinant capsid protein was 51.6 ku with the same size as expected.Western blotting analysis showed that the purified Cap protein reacted positively with mouse anti-GST monoclonal antibody and PCV3 rabbit-derived positive serum,further confirming the expression of the recombinant protein.Therefore,the recombinant protein PCV3 Cap expressed and purified in this study,it laid a foundation for the development of new gene engineering subunit vaccine and the establishment of ELISA method.

The effects of Lactobacillus plantarum (L.plantarum) culture on the growth performance,oxidative reaction and MAPK signaling pathway of broilers infected with Listeria monocytogenes (L.monocytogenes) were investigated in this study for exploring the efficacy of L.plantarum culture against listeriosis and possible mechanism.A total of 480 one day old AA male broiler chicks were randomly allocated into 4 groups including 6 replicates per group of 20 chicks each replicate.Chicks in control and infection groups were offered a basal diet,whereas other two groups were supplied antibiotic enrofloxacin hydrochloride at 40 mg/kg or inactivated L.plantarum culture at 1.6 g/kg.Feeding trial lasted for 28 days.At 5 days old,chicks in infection,antibiotic and L.plantarum culture groups were orally administrated 1 mL L.monocytogenes (10⁵ CFU),whereas chicks in control group were offered the same liquid without the pathogen.The results showed that L.plantarum culure significantly improved ADWG,ADFI and final body weight of 7 and 28 days old broilers (P<0.05),significantly decreased feed/gain ratio (P<0.05),significantly decreased serum and duodenal mucosa concentrations of diamine oxidase,malondialdehyde and protein carbonyls (except for 14 d in duodenal mucosa) of 7 and 28 days old broilers (P<0.05),and significantly decreased mRNA levels of MAPK3,MAPK10 and DUSP6 (except for 7 d) genes of 7 and 28 days old broilers (P<0.05).The results suggested that dietary L.plantarum culture could inhibit L.monocytogenes infection,and improve antibacterial,antioxidative ability and anti-inflammatory ability through MAPK signaling.L.plantarum culture had a good therapeutic effect on L.monocytogenes infected broilers,and could be used as an alternative for antibiotics.

The aim of this paper was to detect recessive mastitis more accurately and to provide a reference for evaluating the change of recessive mastitis milk.The methods of California mastitis test (CMT) and bromine vanilla phenol blue test (BTB) were used in the present experiment to detect the recessive mastitis of 35 cows,and the pH,milk serum amyloid A (SAA),milk composition and blood cells composition were also detected and compared between two methods.The results showed that the positive rate detected by CMT method (40%) was higher than that of BTB method (34.3%),while the difference was not significant (P>0.05),and the match rate of CMT method (35.71%) was lower than that of BTB method (41.67%).The pH of milk in two positive groups were significantly higher (P<0.05) than that of negative group.The logarithms of somatic cell count of positive group detected by BTB test was significantly higher than that of negative group (P<0.05),which was numerically lower than that positive group detected by CMT test.The SAA content in the milk of positive group detected by BTB test was significantly lower than that of the negative group (P<0.05).There was no significant difference in the blood cell components among the two testing positive and negative groups (P>0.05).It was concluded that the CMT test was more sensitive than that of BTB test,while the BTB test was more accurate.

In order to reduce the odor pollution caused by livestock and poultry manure,four species of microbes with deodorization function such as Bacillus velezensis, Bacillus subtilis,Candida utilis and Lactobacillus casei were selected as 4 factors of experimental design in this study.The corresponding viable counts used in pig manure were 1×10⁴,1×10⁵,and 1×10⁶ CFU/g as the three coding levels of the response surface design.In order to obtain an ideal microbial inoculation proportion for fecal deodorization,4 factors and 3 levels were used to construct 29 combinations of microorganisms in Box-Behnke design for removing indole,NH₃ and H₂S in feces.The results showed that the optimal ratio of Bacillus velezensis,Bacillus subtilis,Candida utilis and Lactobacillus casei obtained by response surface optimization were 0.47%,0.05%,0.01% and 1.00% at the concentration of 1×10⁸ CFU/mL,respectively.After 7 d microbial fermentation,the removal rates of indole,NH₃ and H₂S in pig feces were 73.59%,63.60% and 70.29%,respectively.It could be concluded that an ideal compound microbial preparation for pig fecal innocent treatment had been obtained in this study,which provided the high application value to control the odor pollution caused by livestock and poultry manure.