In order to understand the genetic evolution of Penton protein of fowl adenovirus(FAdV),the specific primers of Penton were designed on the basis of published sequences of different genotypes on GenBank.Then Penton gene of 12 serotypes were amplified by PCR and constructed into pEASY-Blunt Simple Cloning Vector for sequencing.The nucleotide sequences of Penton protein of 12 serotypes were analyzed and compared with the nucleotide sequences of standard strains published on NCBI by ClustalW method to draw a genetic phylogenetic tree.The results showed that the nucleotide sequences of Penton protein of 12 serotypes had more than 99.2% homology with their respective standard strain,among which the homology of FAdV-1,FAdV-2,FAdV-3,FAdV-4,FAdV-6,FAdV-7,FAdV-8a,FAdV-8b,FAdV-10 and FAdV-11 were as high as 100%.This further confirmed the consistency between the laboratory preserved strains and the world standard strains.However,although Penton protein was highly conserved,there were still significant differences among different species.The nucleotide homology between FAdV-A1 and other species was 70.6%-73.3%,while that of FAdV-B5 was 70.6%-77.9%.The nucleotide homology between FAdV-C strains of different serotypes was 99.6%,while homology between FAdV-C4 and other species was 71.2%-73.4%.The nucleotide homology between FAdV-D strains of different serotypes was 95.6%-98.7%,while the highest homology between FAdV-D and other species was 85.3%.The nucleotide homology between FAdV-E strains of different serotypes was 98.2%-99.4%,while the highest homology between FAdV-E and other species was 85.3%.In conclusion,the homology difference between strains of the same species was small,and that of different species was significant.FAdV could be divided into A,B,C,D and E species according to nucleotide sequence of Penton protein.This study successfully cloned 12 serotypes Penton gene of FAdV and performed genetic evolution analysis,which laid the foundation for the diagnosis,monitoring and identification of FAdV in the future.

This study was aimed to efficiently and accurately edit the growth trait-related gene insulin-like growth factor 2 (IGF2) in porcine fetal fibroblasts cells (PFFs) of Bama pigs using base editors.The IGF2 gene sequences of Bama pigs and Large White pigs were identified by PCR amplification and sequencing,and the expression vector of sgRNA-IGF2 targeting Bama pigs was constructed.Then the editing efficiency and product types at IGF2 gene via different base editors were studied by cell transfection,detection of mixed cell editing efficiency,screening of single cell colonies and genotyping.The results showed that there was a single nucleotide polymorphism (SNP) site at 3 072 bp in intron 3 of IGF2 gene between Bama pigs and Large White pigs,and a single-stranded oligonucleotide sequence targeting the IGF2 gene was designed.The single-stranded oligonucleotide was annealed and ligated with the BsaⅠ-linearized pGL3-U6-sgRNA plasmid to construct the sgRNA-IGF2 expression plasmid.The sequencing results of the recombinant plasmid showed that the sgRNA sequence was accurately inserted between the U6 promoter and the sgRNA scaffold.Four different cytosine base editors (rA1-BE3,hA3A-BE3,hA3A-BE3-Y130F and hA3A-eBE-Y130F) were co-transfected with sgRNA-IGF2-expressing plasmid into PFFs,respectively.The editing efficiency in mixed cells showed hA3A-BE3-based CBEs could introduce higher efficiency of C-to-T mutation than rA1-BE3 (P<0.05).The results of flow cytometry,single cell culture and genotyping showed that 71.43% of single cell colonies were mutant in hA3A-BE3 group,but 42.86% of them had insertions/deletions (indels).The editing efficiency of hA3A-BE3-Y130F (56.86%) and hA3A-eBE-Y130F (40.38%) were lower than hA3A-BE3(71.43%),but the indels of them were also lower (31.37% and 21.15%).In addition,single cell colonies with homozygous mutation at IGF2 gene site were achieved by base editors in this study.Base editors,as new gene editing tools,could efficiently and accurately modify SNP associated with economic traits in pig genome and accelerate genetic improvement.

The purpose of this experiment was to enrich miRNA library of goat and explore the role of miRNAs in the regulation of cell growth.The expression of Saanen dairy goat fetal fibroblast miRNAs was analyzed by Illumina high-throughput sequencing and bioinformatics technology.Total miRNAs cDNA library from fibroblasts of Saanen dairy goat was successfully constructed,16 395 039 total reads and 16 150 181 clean reads had been obtained and among them were 205 857 unique sRNAs.From those clean reads,247 novel miRNAs were identified and 8 401 target genes were predicted with 10 832 target sites.Meanwhile,the first base pairs of candidate miRNAs were biased for U and A.GO analysis revealed that more than 69.6% of the genes were related to cells and cellular components,more than 54.1% of the genes were involved in organelle related activity,and approximately 65.0% of the genes were related with cells and cellular processes.KEGG pathway analysis showed that approximately 10.5% of the genes were related to a metabolic pathway,and that it was the pathway that accounts for the most.In conclusion,the data showed that miRNAs played an important regulatory role in fetal fibroblasts.The results provided a basis for further research on the growth regulation of fetal fibroblasts and miRNAs of donor cells in the breast bioreactor of dairy goats.

The purpose of the experiment was to study a new development method of brucellosis vaccine,and to successfully induce a rough Brucella abortus attenuated strain named RB71 by op inducer.Deletions of RB71-related genes were detected,The LPS integrity,growth characteristics,genetic stability,and viability in murine macrophage RAW264.7 cells were compared with those in smooth strains.The results showed that the mutant was successfully induced in the test,and the size of the missing fragment was 15 070 bp.The heat agglutination test was positive,which could be stained by crystal violet,and the acridine yellow agglutination test was positive.The extraction of lipopolysaccharide for silver staining showed that the O chain was deleted,and the induced strain RB71 lipopolysaccharide was incomplete.No gene mutation was detected by PCR after 30 consecutive passages.Under the same culture conditions in vitro,the growth rate of the induced strain RB71 was significantly lower than that of the parent strain A19.When infected with RAW264.7 macrophages in mice for 72 h,the intracellular survival rate was significantly reduced than that of the parent strain (P<0.01).In summary,this experiment successfully obtained an induced strain of Brucella RB71 with good genetic stability through the op inducer mutagenesis technique the survival ability of the induced strain in RAW264.7 cells was significantly weakened,which laid the technical foundation for the development of a new attenuated Brucella rough vaccine.

In this study,encephalomyocarditis virus 2A protein was prokaryotic expressed and purified.E.coli was used to express foreign proteins,the EMCV 2A gene was inserted into the prokaryotic expression vector pET-28a-sumo to obtain the recombinant plasmid pET28a-EMCV-2A and confirmed by PCR and sequencing.The plasmid was transformed into E.coli BL21(DE3).After ultrasound fragmentation,the expression of 2A protein was mainly in the form of inclusion bodies.By optimizing the protein expression conditions,the soluble expression of the target protein could be achieved.The recombinant protein was purified by beads and identified by SDS-PAGE and Western blotting.The results showed that the recombinant plasmid was successfully constructed.The molecular weight of the recombinant 2A protein was about 25 ku.The best condition was 16 ℃ with 1.0 mmol/L IPTG,about 50% of the proteins were soluble.The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and indicated that the purified protein was the recombinant protein.The study provided the basic theoretical basis for elucidating the molecular pathogenesis of EMCV and preparation of gene vaccines and antiviral drugs.

This study was aimed to explore the effect of short-term starvation treatment on the growth and development of lipid droplets in adipogenic differentiation C2C12 cells,provide the foundation for the interpretation and utilization of compensatory growth mechanisms.Adipogenic differentiation C2C12 cells were divided into two groups:Control group,high glucose treatment for 24 h,and starvation treatment group (FAST),serum-free and low-glucose medium starvation treatment for 24 h.The morphological changes of lipid droplets was observed by BODIPY staining and triglyceride determination,and Western blotting was used to determine the change of the expression level of perilipin family protein (perilipin 1-5) on the lipid droplet.The results showed that C2C12 cells induced adipogenesis for 12 days,which could make the cells reach a better fat-filling state.After treatment with serum-free and low-glucose starvation,the levels of triglycerides in adipogenic differentiation C2C12 cells decreased extremely significantly (P<0.01),the size of lipid droplets decreased extremely significantly (P<0.01),and the number of lipid droplets increased extremely significantly (P<0.01).The perilipin protein on the lipid droplets was changed,the expression level of perilipin2 and perilipin5 were extremely significantly up-regulated (P<0.01),and the expression level of perilipin3 was significantly up-regulated (P<0.05).In summary,serum-free and low-glucose starvation could induce the remodeling of lipid droplets and change the expression level of perilipin proteins on the lipid droplets.

The aim of the study was to investigate the changes of TNF-α,P-selectin,ICAM-1,NO and ET in porcine intestinal microvascular endothelial cells (PIMVECs) treated with Escherichia coli Shiga-like toxin type Ⅱ variant (SLT-Ⅱe).SLT-Ⅱe was extracted from Escherichia coli by using ultracentrifugation,and the concentration was determined by BAC kit.The purity of SLT-Ⅱe was detected by SDS-PAGE and Western blotting.The optimal incubation time (0,2,4,8,12,24 h) and concentration of SLT-Ⅱe (0.1,0.5,1,5,10 μg/mL) on PIMVECs were evaluated by WST-1.The levels of TNF-α,P-selectin,ICAM-1,NO and ET in cell culture supernatant of control group (0 μg/mL SLT-Ⅱe) and test group (1 μg/mL SLT-Ⅱe) were detected by ELISA.The results showed that using ultracentrifugation could extract pure SLT-Ⅱe with a concentration of 721.75 μg/mL.1 μg/mL SLT-Ⅱe could significantly reduce the activity of PIMVECs after 8 h (P<0.01) and significantly increase the TNF-α secretion of PIMVECs after 9 and 12 h treatment (P<0.05).The NO/ET values at 9 and 12 h were significantly higher than that in the control group (P<0.05).After 9 and 12 h treatment the content of P-selectin was significantly increased (P<0.05) and the levels of ICAM-1 was significantly increased at 12 h (P<0.05).In summary,1 μg/mL SLT-Ⅱe could induce the increase of TNF-α,P-selectin,ICAM-1 and NO/ET secreted by PIMVECs,which laid a foundation for studying the pathogenesis of piglet edema disease.

This experiment was aimed to explore a new way for microbial synthesis of Levan and optimize its synthesis conditions.Levan synthesis by whole-cell catalysis using recombinant Corynebacterium glutamicum that transferred into levansucrase,and the response surface method were used to optimize the synthesis conditions with Levan yield as the response value.Firstly,the Plackett-Burman design was used to screen out three main factors affecting yield (sucrose concentration,final concentration of ethanol in Levan extraction and Ca²⁺ concentration) from 7 related factors (sucrose concentration,bacterial concentration,transformation time,transformation pH,Ca²⁺ and Mg²⁺ concentration in PBS,and final concentration of ethanol in Levan fructan extraction).Then the three most important factors selected were used for the steepest climbing test to approach the region of maximum response value,and finally use the BBD response surface method to determine the interaction between the three main factors and the best synthesis conditions.The results showed that the average yield of Levan was 0.069 g/mL in the initial experiment.The response surface method was used to optimize the whole-cell biocatalysis of Levan by Corynebacterium glutamate.The most influential factor was sucrose concentration,the second was the final concentration of ethanol,and the last was the Ca²⁺ concentration in the transformation solution.When the sucrose concentration was 52.71%,the final ethanol concentration was 85.31%,and the Ca²⁺ concentration was 0.04 g/L,the average yield of Levan synthesized by recombination Corynebacterium glutamate was 0.238 g/mL,which was close to the model's predicted maximum value of 0.233 g/mL,and the conversion rate was about 44%.The conversion rate was 1.9 times higher than the initial test,and the yield was 3.4 times higher than the initial test.The results provided the basis for Levan industrial production,and laid the foundation for its research in animal medicine and nutrition.

The experiment was conducted to explore the effects of attapulgite nano zinc oxide (ANZ) on antioxidant and immune functions in spleen of weaned piglets.210 weaned piglets of similar weight were randomly divided into 7 groups with 6 replicates in each group and 5 piglets per repeat.The treatments were control group (CON group,basal diet),antibiotic group (ANT group,basal diet+antibiotic),zinc oxide group (ZO group,basal diet+3 000 mg/kg zinc oxide),nano-zinc oxide group (NZO group,basal diet+800 mg/kg nano-zinc oxide),low,middle and high dose attapulgite nano zinc oxide groups (LANZ group,basal diet+700 mg/kg attapulgite nano zinc oxide;MANZ group,basal diet+1 000 mg/kg attapulgite nano zinc oxide;HANZ group,basal diet+1 300 mg/kg attapulgite nano zinc oxide).The results showed that:Compared with CON group,spleen index in ZO,MANZ and HANZ groups increased significantly (P<0.05).Compared with CON and ANT groups,the area of spleen cells in NZO,LANZ,MANZ and HANZ groups increased significantly (P<0.05).Compared with other treatment groups,the size of spleen cells in MANZ group increased significantly (P<0.05).Compared with CON,ANT,NZO and HANZ groups,superoxide dismutase (SOD) activity in MANZ group increased significantly (P<0.05).Compared with ZO and NZO groups,catalase (CAT) activity in LANZ,MANZ and HANZ groups increased significantly (P<0.05).Compared with ZO group,the content of malondialdehyde (MDA) in NZO,LANZ,MANZ and HANZ groups decreased significantly (P<0.05).Compared with CON group,the content of interferon-γ (IFN-γ) in ANT,MANZ and HANZ groups decreased significantly (P<0.05).Compared with CON,ANT,ZO and NZO groups,the content of tumor necrosis factor-α (TNF-α) in MANZ and HANZ groups decreased significantly (P<0.05).Compared with ANT group,the content of interleukin-1 β (IL-1 β) in MANZ group decreased significantly (P<0.05).Compared with ANZ、ZO and NZO groups,the content of interleukin-10 (IL-10) in MANZ and HANZ groups increased significantly (P<0.05).Compared with CON,ANT and ZO groups,the content of interleukin-12 (IL-12) in LANZ and MANZ groups increased significantly (P<0.05).In conclusion,attapulgite nano zinc oxide could increase organ index and cell size of spleen,improved the antioxidant enzymes activity in spleen,decreased the content of proinflammatory factors,increased the content of anti-inflammatory factors,and enhanced the antioxidant and immune functions of weaned piglets.The optimum addition level of attapulgite nano zinc oxide was 1 000 mg/kg in this experiment.

The aim of this study was to explore the nutritional value of fermented peanut vine and its effects on the production performance,slaughter performance,serum biochemical indexes and feces' routine ingredient in Muscovy ducks.The feasibility of fermented peanut vine that applied to grain-saving feeding in Muscovy ducks was also explored.Three hundred and sixty 15-day-old male Muscovy ducks were randomly divided into 4 groups,6 repetitions in each group,15 ducks each repetition.The ducks in control group were fed with basic diet,while in group Ⅰ were fed with 90% basic diet+10% fermented peanut vine,in group Ⅱ were fed with 85% basic diet+15% fermented peanut vine and in group Ⅲ were fed with 80% basic diet+20% fermented peanut vine.After 7 days pre-feeding period and 48 days feeding period,production performance,slaughter performance and organ indexes were then measured.Meanwhile,serum,muscle and fresh feces were collected for measurement of the levels of biochemical indexes and feces'routine ingredients.The results showed that:① Compared to unfermented peanut vine,CP and Ca levels of fermented peanut vine were elevated by 31.82% (P<0.01) and 97.40% (P<0.01) respectively,while CF,P and H₂O levels were decreased by 2.35% (P<0.05),20.69% (P<0.05) and 53.23% (P<0.01) respectively.Nutritional value of fermented peanut vine was clearly increased.② Compared to control group, F/G of group Ⅰ and Ⅱ were decreased by 6.51% (P<0.01) and 6.84% (P<0.01) respectively.The leg muscle rate of groupⅠ,Ⅱ and Ⅲ were improved by 20.50% (P<0.05),25.55% (P<0.05) and 9.27% (P>0.05) respectively,and liver indexes were increased by 9.90% (P>0.05)、12.23% (P<0.05) and 3.18% (P>0.05) respectively,gizzard indexes were elevated by 17.70% (P<0.05),30.82%(P<0.05) and 36.66% (P<0.05) respectively,and glandular stomach indexes were increased by 1.37% (P>0.05),32.99% (P<0.05) and 36.43% (P<0.05) respectively.③ Fermented peanut vine could affect the levels of TP,ALB,GLB,A/G,ALT,AST,AST/ALT,UREA,UA and CRE in serum of Muscovy ducks.Compared to control group,N,P,K and H₂O levels in feces of ducks were decreased in the test groups.In conclusion,10% and 20% fermented peanut vine could markedly improve the production performance,reduce cost to gain ratio,and did not affect meat performance.But fermented peanut vine might affect the function of liver,kidney and immune in Muscovy ducks.

The experiment was aimed to investigate the effects of dietary alfalfa powder on the growth performance,body size,slaughter performance,organ index and meat quality of Siji geese.A total of 256 15-day-old geese (half male and half female) with similar body weight were randomly divided into four groups with 4 replicates of 16 geese each.The geese in control group were fed with corn-soybean meal-based diet (control diet),and geese in the experimental groups were fed the basal diet replaced with 10%,20% and 30% alfalfa powder,respectively.The results showed that,compared with the control group,dietary alfalfa powder supplementation had no significant effect on the average daily intake,average daily gain,feed conversion ratio,body oblique length,keel length,shin circumference,semi diving length,chest width and shin length (P>0.05),and also had no effect on the carcass ratio,half-eviscerated carcass ratio and pectoral muscle ratio (P>0.05).However,dietary 20% and 30% alfalfa powder supplementation significantly increased the chest depth (P<0.05),dietary 30% alfalfa powder significantly decreased the full-eviscerated carcass ratio (P<0.05),and dietary 10% alfalfa powder significantly increased the leg muscle ratio (P<0.05) of geese.In addition,dietary alfalfa powder significantly decreased the abdominal fat ratio (P<0.05),raised the growth index of liver,jejunum,ileum,and cecum (P<0.05),and increased the water loss rate of pectoral muscle and leg muscle (P<0.05) compared with the control group.Dietary 10% and 30% alfalfa powder significantly increased duodenal growth index (P<0.05) of geese.In conclusion,dietary alfalfa powder supplementation might improve the slaughter performance (increase chest depth and leg muscle ratio,reduce abdominal fat rate) and organ growth index (increase the growth index of liver,duodenum,jejunum,ileum and cecum).However,dietary alfalfa powder increased water loss rates in pectoral muscle and leg muscle,and 30% alfalfa powder reduced the full-eviscerated carcass ratio of geese.The appropriate supplementation level of alfalfa powder was recommended at 10% in the present study.

The purpose of this study was to evaluate thermal environment of cowshed in four seasons in central plain of Hebei,and analyze the correlation between the temperature parameters and physiological indexes of dairy cows.Three dairy cow houses with different building structures were selected,the thermal parameters (ambient temperature (Ta) and relative humidity (RH)) and physiological parameters (respiratory rate,rectal temperature and body surface temperature) were detected.The results showed that the Ta,RH and index of temperature and humidity (THI) changed significantly in four seasons (P<0.05),the average daily temperature in summer was the highest (28.59 ℃),and the average daily temperature in winter was the lowest (1.55 ℃).In summer,dairy cows were suffering from mild heat stress for 15.5 h every day,and from moderate heat stress for 6.0 h.In winter,dairy cows were under mild cold stress for an average of 12.0 h per day.The Ta and THI of three cowsheds in each season had no significant differece (P>0.05),except for that in winter.Compared with the cowshed with low wall or roller blind,the average Ta in the cowshed with only roof was 0.80-1.27 ℃ higher in summer and 1.36-1.84 ℃ lower in winter.In addition,the physiological parameters of dairy cows were extremely significantly higher in summer than those in other seasons (P<0.01).The respiration frequency or rectal temperature of cows among cowsheds in summer were significantly different (P<0.05),and the seasonal difference in body surface temperature was significant (P<0.05).Correlation analysis of thermal parameters and cow physiological parameters showed that the respiratory frequency,rectal temperature and body surface temperature were positively correlated with THI and Ta (P<0.05).However,there was no significant correlation between physiological parameters and RH (P>0.05).The results provided scientific basis for environment evaluation of cows,and physiological status of dairy cows were inferred according to environmental thermal parameters,which provided data for occurrence and early warning of stress.

Allicin is a sulfur compound extracted from garlic,which has the advantages of high safety,no residue,no drug resistance,wide antibacterial spectrum,no incompatibility and so on.The physiological functions of allicin include reducing blood sugar and blood pressure,anti-oxidation,enhancing immunity and inhibiting harmful bacteria,and plays a role in maintaining animal intestinal health,improving intestinal flora and regulating fat deposition in animal production.Allicin,as a feed additive,is mainly of chemical synthesis type.Diallyl disulfide is synthesized at first,and then allicin is synthesized by oxidation reaction.Allicin can also be synthesized by hydrogen peroxide,magnesium peroxy phthalate or chlorobenzoic acid oxidizing diallyl disulfide in the laboratory.In actual production,allicin can promote the growth of ruminants,improve feed digestibility,improve rumen fermentation.Allicin can also reduce rumen methane production by inhibiting the activity of methanogenic bacteria in the rumen and reducing the number of methanogenic bacteria.The authors mainly introduced the synthesis,physical and chemical properties and physiological functions of allicin,and expounded the research results and prospects of the application of allicin in ruminants,in order to provide reference for the better application of allicin in ruminant production.

In recent years,with the intensive and large-scale development of livestock and poultry breeding,livestock and poultry are vulnerable to liver damage caused by feed mildew, antibiotic abuse,hepatitis virus and other factors.Resveratrol is a natural plant extract which is widely found in many plants.It belongs to polyphenols,which can improve liver damage caused by liver disease in the field of food science and medicine.At the same time, resveratrol can effectively protect the liver of livestock and poultry,relieve inflammation stimulation,oxidative stress,apoptosis and other physiological or pathological conditions of liver damage.In addition,resveratrol can improve the liver damage caused by non-alcoholic fatty liver that is prone to appear in the growth and fattening stage by regulating the lipid metabolism of livestock and poultry.The authors summarized the research progress of the protective effect of resveratrol on liver injury of livestock and poultry, briefly described it's physical and chemical properties and related biological functions, such as anti-inflammatory,anti-oxidation,anti-apoptosis.At the same time,the main types and pathologic ways of liver injury in livestock and poultry were introduced, and the protective mechanism and signal pathway of resveratrol to liver injury in livestock and poultry were analyzed,in order to provide the basis for resveratrol to improve the common liver injury of livestock and poultry and improve the quality of livestock products,and to provide theoretical support for exploring the possibility of resveratrol as a new high quality feed additive.

This study was aimed to explore the genetic variations in promoter region of adenylosuccinate lyase (ADSL) and its correlation with chicken freshness after freezing.161 Partridge Shank chickens were employed,the genetic variation of promoter region of ADSL gene was scanned by pool-based sequencing to discover candidate SNP,and the candidate SNP was genotyped using allele specific PCR.The association analysis was performed between the candidate SNP and four indexes (meat color,body weight,freshness (K value) after freezing of 60 days and pH) in both genotypes.The results showed that one SNP (c.-1670 C>A) was identified at 1 670 bp upstream of ATG in the promoter region of ADSL gene.The K value of muscle freshness of AA genotype (23.61%) was extremely significantly lower than that of CA (33.00%) and CC (33.56%) genotypes (P<0.01),indicating that the muscle of AA genotype was fresher than that of CA and CC genotypes after freezing for 60 days.No significant difference in meat freshness was observed between CA and CC genotypes (P>0.05).There was no significant difference in the body weight,meat color and pH among three genotypes (P>0.05).Bioinformatics prediction results showed that c.-1670 C>A caused the transcription factor site to change in the promoter region of ADSL gene,which might affect the expression of ADSL gene.This study concluded that there was a significant correlation between c.-1670 C>A in the promoter region of ADSL gene and freshness K value of breast muscle in Partridge Shank chickens after freezing for 60 days,c.-1670 C>A could be used as a candidate molecular genetic marker for meat freshness in Partridge Shank chickens.

This study was aimed to find the molecular markers significantly associated with litter size traits and uniformity of the piglet's birth weight,and provided a basis for genetic improvement of reproductive traits in pigs.This study was based on the genome-wide selective sweep analysis of pig conducted by the research group in the previous studies,two SNPs (rs338309012 and rs335824784) on porcine chromosome 3 were selected,and the genotyping-in-thousand by sequencing was used,the relationship between two SNPs and litter size traits (including total number born,total number born alive,number of stillborn pigs and number of mummified pigs,etc.) and the uniformity of the piglet's birth weight was association analyzed in Danish Yorkshire and American Yorkshire pigs.The results showed that rs338309012 were significantly associated with the total number born and the number of mummified pigs (P<0.05),and the total number born and the number of mummified pigs of AC genotype sows was significantly higher than CC genotype (P<0.05).rs335824784 was significantly associated with the effective number of born alive and the number of mummified pigs (P<0.05),and was also extremely significantly associated with the total number born alive and the number of stillborn pigs (P<0.01).The effect of different genotypes on the total number born alive and effective number of born alive were GG > CG > CC.On the contrary,the effect of different genotypes on the coefficient of variation of the birth weight of piglets,the number of dead and mummified fetuses were GG < CG < CC.Thus,the GG genotype sows with rs335824784 not only had more total number born alive,but also had regular piglet's birth weight and lower number of stillborn and mummified pigs,which could be used as a molecular marker to improve the litter size traits and uniformity of the piglet's birth weight.

The aim of this study was to investigate the regulatory effect and mechanism of C-type natriuretic peptide (CNP) on the proliferation,differentiation and lipolysis of intramuscular preadipocytes (IMPs) in breast muscle of chickens,and lay a foundation on further elucidating the molecular mechanism of intramuscular fat deposition in chickens.The IMPs of breast muscle in Yellow-feathered broilers were used as the model in vitro,the preadipocytes were induced by 10^-7 mol/L CNP.The proliferation,differentiation and lipolysis of IMPs were observed by CCK8,Edu staining,Oil Red O staining and isopropanol extraction,respectively.The concentration of cGMP and glycerin were detected by cGMP and glycerin kit,respectively.The expression of NPRA,NPRB and NPRC genes mRNA was detected by Real-time quantitative PCR.The results showed that compared with control group,the cell proliferation in CNP treatment group was enhanced significantly at 1 and 3 d (P<0.05).The cell differentiation and lipid deposition were extremely significantly decreased at 3 and 6 d (P<0.01),the intracellular cGMP concentration and glycerin concentration in the medium at 3 and 6 d were extremely significantly increased (P<0.01).The expression of NPRB and NPRC genes mRNA in CNP treatment group was extremely significantly or significantly higher than control group (P<0.01;P<0.05),and the expression of NPRA gene mRNA had no significant difference (P>0.05).The results revealed that CNP regulated the lipid deposition of intramuscular adipocytes through NPRB/NPRC-cGMP pathway in chickens.

This study was aimed to explore the correlation between the haplotypes of apolipoprotein A5 (ApoA5) gene and body weight,meat quality in Jinfen White pigs.180 Jinfen White pigs were selected,PCR was used to detect the single nucleotide polymorphism (SNP) of ApoA5 gene,and the linkage of these SNPs was analyzed by Haploview software.Non-fully linked SNPs were screened to form the haplotypes of ApoA5 gene,and the frequency of different haplotypes of ApoA5 gene were calculated.The correlation between the haplotypes of ApoA5 gene and body weight,meat quality was analyzed by SPSS software to identify the haplotypes of ApoA5 gene with excellent traits.The results showed that ApoA5 gene of Jinfen White pigs contained 21 SNPs and 5 completely linked regions which included 5,2,3,4 and 2 SNPs,respectively.Ten non-completely linked tag SNPs were selected to form the haplotypes of ApoA5 gene,Hap 1 had the highest gene frequency (46.7%).The haplotypes of ApoA5 gene had a significant correlation with the birth weight,weaning weight,6-month-old body weight,intramuscular fat content,shear force and drip loss,but no significant correlation with pH_{24 h} in Jinfen White pigs.Hap 11 of ApoA5 gene in Jinfen White pigs had the highest birth weight,weaning weight,6-month-old body weight and intramuscular fat content,the lowest shear force,and the lower drip loss.Hap 6 had the lowest birth weight.Hap 2 had the lowest weaning and 6-month-old weight.Hap 3 had the lowest intramuscular fat content and the highest shear force.Hap 10 had the highest drip loss.Hap 11 of ApoA5 gene in Jinfen White pigs had excellent traits,which could be used as a molecular marker to use in molecular breeding of Jinfen White pigs and improve the breeding efficiency.

The purpose of this study was to explore identification and division methods of Hu sheep family based on simple sequence repeats(SSR) polymorphism.Nine SSR loci of BM3051,HH64,TGLA137,MAF33,FCB48,VH72,INRA023,ETH152 and MCM527 were selected,which were distributed in several autosomes,with high polymorphism and high heterozygosity.DNA was extracted from the blood samples of 30 Hu breeding rams by blood DNA extraction kit.SSR primers were synthesized and labeled with the fluorescent group.The products were amplified by touch-down PCR.After genotyping,CERVUS3.0.7 was used for genotyping analysis.POPGENE1.32 was used to construct a dendrogram based on Nei's genetic distance to divide Hu sheep families.The results showed that the cumulative exclusion probability of single parent was 99.2094% when the genotypes of both parents were unknown,99.9688% when a single parent genotypes were known,99.9999% when both parents' genotypes were unknown.Exclusion probability could meet the requirements of paternity identification and pedigree analysis in genetic breeding.UPGMA clustering map was constructed according to Nei's genetic distance,and 30 breeding rams were divided into six families,which provided a reference for sheep breeding.The method could run the family identification and division of Hu sheep,which had application value for the breeding of Hu sheep.

Skeletal muscle is an important tissue of the vertebrate body,its embryonic development starts from the proliferation and differentiation of myogenic progenitor cells of the dermomyotome into myoblasts,and further develops to form myotubes and eventually form muscle fibers.The entire development process is subject to complex and tight regulation,any abnormal regulation may affect the normal development process of skeletal muscle.In recent years,studies have shown that,in addition to myogenic regulator factors,myocyte enhancer factors and paired box proteins,non-coding RNA includes microRNA (miRNA),long noncoding RNA (lncRNA)and circular RNA (circRNA) can be widely used as important regulators to participate in the regulation of skeletal muscle development.miRNA mainly acts on target mRNA,and regulates the expression of related genes at the post-transcription level by inducing the destabilization of target mRNA and/or inhibiting translation.lncRNA has long length and advanced structure,which can regulate the expression of related genes in both pre-transcriptional and post-transcriptional levels through a variety of action modes.circRNA is mainly used as a miRNAs ponge,competitively binds to miRNA,and participates in the regulatory network of skeletal muscle development.The author summarized the seven aspects of skeletal muscle development,miRNA mechanism,miRNA and skeletal muscle development,lncRNA mechanism,lncRNA and skeletal muscle development,circRNA mechanism and circRNA and skeletal muscle development,in order to provide reference for studying the regulation of non-coding RNA in skeletal muscle development.

The aim of the experiment was to study the effect of 3',5'-cyclic adenosine monophosphote (cAMP) adenylyl cyclase 2 (ADCY2) gene on adipogenic differentiation of preadipocytes in Yanbian Yellow cattle.The 3-day-old inguinal adipose tissue of Yanbian Yellow cattle was selected for the isolation,culture and induction differentiation of preadipocytes.The interference fragment of ADCY2 gene was designed and the pEX4 overexpression vector was constructed.Adipose cells of normal (Control),interference,and overexpression groups at 0,5,and 10 d were collected,respectively.The mRNA transcriptional levels of peroxisome proliferation activator receptor γ(PPARγ),CCAAT/enhancer binding protein (C/EBP) and ADCY2 in the process of cell differentiation were detected by Real-time quantitative PCR and the protein expression was detected by Western blotting.Oil red O staining was used to detect the change of lipid droplets,and triglyceride content was detected with triglyceride kit.The results showed that during adipogenic differentiation process,compared with the undifferentiated group,the expression of ADCY2 gene increased significantly at the 5th and 10th day (P<0.01),but decreased significantly at the 10th day compared with that of the 5th day (P<0.01).Compared with control group,the mRNA level of ADCY2 gene was increased about 4 000 000 folds in the overexpression group,the number of lipid droplets and the content of triglyceride in adipocytes was extremely increased (P<0.01),the expression of key lipid-forming genes C/EBPα and PPARγ were significantly up-regulated (P<0.01),but the mRNA levels of ADCY2 gene was significantly decreased in the RNA interference group,and the number of lipid droplets and the content of triglyceride in adipocytes decreased significantly(P<0.01),the expression of key lipid-forming genes C/EBPα and PPARγ were significantly down-regulated (P<0.01).Therefore,overexpression of ADCY2 gene could significantly increase the content of lipid droplets and triglyceride in mature adipocytes,and promote the expression of key lipid-forming genes C/EBPα and PPARγ,indicated that ADCY2 gene had a positive regulatory effect on adipocyte differentiation.

The purpose of this study was to understand the difference in the quality of alpaca wool in different regions and parts,and to provide reference data for alpaca breeding and alpaca wool production.In this study,a total of 636 wool samples of alpaca jaw,neck,shoulder,side,thigh,back and abdomen were collected from Xinjiang Wild Animal Park,Altay,Aksu,Shanxi,and Tianjin regions,and the traits such as mean fiber diameter,length,net wool rate,single fiber strength,medullated wool content and non-medullated wool content were measured.The differences of wool quality traits in different regions and parts were analyzed,and least squares analysis was performed.The results showed that there were extremely significant differences in alpaca mean fiber diameter,whiteness,brightness,fat content,net wool rate,wool length,single fiber strength,breaking elongation,and presence or absence of myelin wool content in different regions (P<0.01).There were extremely significant or significant differences in alpaca mean fiber diameter,raw wool grease,length,single fiber strength,and impurity content (P<0.01),and the content of medullated wool and non-medullated wool (P<0.05) in different sampling locations.According to the principle that the finer the alpaca wool was,the better the alpaca wool quality was,combined with the properties of single fiber strength and the length,the order of the alpaca wool quality of the different parts from high to low was:Back>side>thigh>shoulder>neck>jaw>abdomen.In breeding,the improvement of the non-medullated wool content could be regarded as the breeding goal of Xinjiang alpaca wool fineness,and the improvement of net wool rate and single fiber strength could be taken as the direction of the alpaca wool quality in Shanxi and Tianjin.At the same time,it could be considered to increase the non-medullated wool content of the alpaca's neck,jaw,and abdomen,in order to improve the overall wool quality of alpaca.

The effects of resveratrol on the quality of frozen-thawed sheep semen were studied in this study.Semen was collected from six Yunnan semi-fine wool sheep using artificial vagina.The semen was diluted with the Optidyl extender supplemented with resveratrol with a concentration of 0,0.1,1,10,or 20 μmol/L,followed by loading into plastic straws and equilibration at a low temperature.Then,the straws were pre-frozen in liquid nitrogen vapor,followed by storage in liquid nitrogen for 30 days.After thawing for 30 seconds in a 37 ℃ water bath,the parameters including sperm motility,acrosome integrity,membrane integrity,distribution of phosphatidylserine (PS) and ROS were measured.The results showed that the post-thaw sperm total motility,progressive motility and the rate of sperm with curved tail were 76.14%±0.97%,43.56%±0.91% and 43.24%±1.68% in the 10 μmol/L resveratrol group,respectively,which were significantly higher than those in the other groups (P<0.05).However,when the concentration of resveratrol in the freezing extender was 20 μmol/L,the post-thaw total motility,progressive motility and the rate of sperm with curved tail were 21.78%±0.79%,25.23%±1.34% and 4.84%±0.68%,respectively,which were significantly lower than those in the other groups (P<0.05).The acrosome integrity of sperm frozen in the 10 μmol/L resveratrol group was best,which was 50.47%±0.91% and significantly higher than the other treatments (P<0.05).The results of PS distribution showed that the percentage of viable sperm in the 10 μmol/L resveratrol group was 46.43%±2.95%,and significantly higher than that in the 20 μmol/L group (31.14%±3.56%,P<0.05),but there was no significant difference with the other groups (P>0.05).In addition,the PS labeling rate of sperm in the 20 μmol/L resveratrol group (39.82%±3.38%) was significantly higher than those of the other groups (P<0.05).In terms of ROS production,this study demonstrated that the rate of viable sperm (ROS and PI were negative) in the 10 μmol/L resveratrol group (63.57%±0.71%) was significantly higher than that of the other groups (P<0.05),while the rate of viable sperm (32.45%±1.42%) in the 20 μmol/L resveratrol group was significantly lower than those of the other groups (P<0.05).In conclusion,the post-thaw quality of sheep semen could be improved after the addition of resveratrol to the freezing extenders,which might be related to the antioxidant properties of resveratrol.But,the cryoprotective effects of resveratrol dependents on its used doses,and the optimal concentration was 10 μmol/L based on this present study.However,excessively high concentration of resveratrol could aggravate cryodamage on sperm.In addition,the protective effects of resveratrol on sheep sperm still needs to be verified by in vitro fertilization and artificial insemination.

Under the modern large-scale and intensive dairy farming production mode,the continuous supply of feed with high nutrition level is the material basis for maintaining the rapid growth and development of reserve cows and the high lactation performance of lactating cows.However,after the genetic quality reaches the "bottleneck period",although the milk yield of dairy cow can continue to maintain a certain high level with the continuous improvement of the nutrient concentration of feed and the increase of dry matter intake (DMI),the incidence of nutritional metabolic diseases of high-yield dairy cows also shows a rapid growth trend,especially ketosis caused by the negative energy balance in perinatal period,acidosis caused by high-precision diet and the increase of blood urea nitrogen caused by high-protein diet,negatively regulate the reproductive performance of dairy cows,resulting in inconspicuous postpartum estrus,decreased mating rate and conception rate of dairy cows,which directly affect the update speed of dairy cows production groups,the normal performance of high-quality cattle and the economic benefits of dairy farming.In this paper,the author introduced in detail the relevant research on the current situation and its mechanism influence of different nutritional metabolic diseases on the reproductive performance of dairy cows in recent years,and focused on analyzing the molecular mechanism of nutritional and metabolic diseases with high perinatal incidence such as ketosis,low blood calcium and moderate gastric acid,and put forward the prospect and thinking on the future research direction of nutritional and metabolic diseases and reproductive performance of high-yielding dairy cows,in order to provide some reference and theoretical basis for improving the reproductive efficiency of high-yielding dairy cows in large-scale pastures in China and the perinatal nutrition management level of dairy cows.

In order to evaluate the bactericidal activity of a novel armyworm antimicrobial peptide (AAP-1),the bactericidal effect of AAP-1 on Escherichia coli (E.coli) was investigated.Firstly,AAP-1 was synthesized by solid-phase chemically synthesis,and purified by high-performance liquid chromatography and tested by mass spectrometry.Then,the bactericidal effect of AAP-1 against E.coli was determined by the bacteriostatic circle method.The minimum inhibitory concentration (MIC) was detected by the double dilution method.The time kill curve was measured by bacterial plate counting method.Finally,the effect of AAP-1 on the nucleic acid leakage of E.coli was evaluation by ultra-micro spectrophotometer.The effect of AAP-1 on the intracellular DNA of E.coli was tested by nucleic acid gel electrophoresis.The overall effect of AAP-1 on E.coli was observed through transmission electron microscope.The results showed that the purity of the synthesized AAP-1 was more than 98%,and its molecular weight was 4 262.17 u.AAP-1 showed good antibacterial activity against E.coli,with a MIC of 7.8 μg/mL.AAP-1 had a concentration-dependent bactericidal effect on E.coli,and within 60 min the bactericidal effect gradually increased.AAP-1 could cause the nucleic acid leakage of E.coli,and resulted in a reduction in the total amount of intracellular DNA of E.coli with concentration-dependent.AAP-1 could also cause cell membrane destruction,significantly reduce intracellular electron density,resulted in cytoplasmic dissolution of E.coli,and so on.In summary,this study suggested that a novel antimicrobial peptide AAP-1 could be chemically synthesized with a purity of up to 98% and showed its good bactericidal effect on E.coli.This study could provide preliminary data for the in-depth study of the bactericidal mechanism of AAP-1 against E.coli and laid a theoretical foundation for the clinical application of AAP-1.

In order to investigate the therapeutic effect of Gardenia Sihuang powder on porcine reproductive and respiratory syndrome,45 pigs were randomly divided into five groups:Control group,virus group,low dose Chinese medicine prescription group,the middle dose Chinese medicine prescription group and the high dose Chinese medicine prescription group.Experimental pigs with porcine reproductive and respiratory syndrome virus (PRRSV) were added to each herb after 400 MTS of ultricro grinding,After continuous feeding for 14 days,clinical symptoms were observed daily.Five groups of serum samples were randomly collected for cytokine detection at 7,14 and 21 d,and pathological autopsy and lung samples were collected for pathological tissue section observation.The results showed that after adding traditional Chinese medicine for 7 days,the clinical symptoms were all relieved.At 14 days,the body temperature returned to normal,the symptoms of dyspnea disappeared,and the appetite recovered in middle and high dose groups.No meat-like changes were observed in the lungs of the traditional Chinese medicine treatment group,and there were bleeding spots or blood spots on the surface of the lungs.In the middle dose group,alveolar septa were thickened,blood congestion was mild,and lung parenchyma was mildly inflammatory cell infiltration.The levels of IgG and IFN-γ in the serum of the virus group decreased gradually,while the levels of IL-10 increased gradually.The contents of IgG in the serum of the three groups added with traditional Chinese medicine were higher than that of the virus group,and the content of IgG gradually increased.The content of IgG in the serum of the middle dose group was extremely significantly higher than that of the control group and the virus group (P<0.01).The content of IFN-γ in the three groups with traditional Chinese medicine gradually increased,and the serum content of IFN-γ in the middle dose group was extremely significantly higher than that of the control group and the virus group (P<0.01).The serum IL-10 content in the middle dose group gradually decreased,and the difference with the virus group was extremely significant (P<0.01),but not significant with the control group (P>0.05) at 14 and 21 days.The results of this study showed that fructus Gardenia Sihuang powder could effectively improve clinical symptoms,pathological changes and cytokine secretion in experimental pigs.It provided experimental basis for the prevention and treatment of porcine reproductive and respiratory syndrome.

In this study,the genome of Brucella Rev.1 strain was used as a template to amplify the BAB gene sequence,clone it into the prokaryotic expression vector pET-30a(+),obtain the recombinant plasmid pET-30a-BAB,and perform prokaryotic expression on the recombinant plasmid.The indirect ELISA method was constructed by using the expression products detected by Western blotting.The results showed that the BAB gene was successfully cloned and expressed in this experiment,and the purified expression product was analyzed by SDS-PAGE.This study obtained a relatively pure recombinant BAB protein;Western blotting test showed that the expressed protein could react specifically with Brucella sheep positive serum and had good reactogenicity;Using the recombinant BAB protein as the coating antigen,an indirect ELISA method for detecting BAB antibodies was established and optimized.The best determined coating conditions were as follows BAB protein coating amount was 0.25 μg/mL,serum dilution was 1:400;blocking solution was 3% pig-derived gelatin;secondary antibody dilution was 1:6 000;color development time was 10 min.The established method was used to detect 40 clinical sheep serums,and the cut-off value was calculated to be 0.607.That was,when the serum tested had P/N ≥ 1.5 and D_{450 nm} ≥ 0.607,it was judged as positive,when D_{450 nm} ≤ 0.561,it was judged as negative,and when 0.607<D_{450 nm}<0.561,it was judged as a suspect value,and retest was required.Compared with the Huhong plate test and the test tube agglutination test,the positive coincidence rate was 100%,the negative coincidence rate was 71.88%,and the total coincidence rate was 77.5%.

This study was aimed to isolate a mutant strain of porcine epidemic diarrhea virus and prepare a PEDV inactivated vaccine with high valence by suspension culture process for immunizing against PEDV effectively in China.200 small intestines and theirs contents of diarrhea piglets died of diarrhea,collected from many large-scale pig farms in China,were detected by RT-PCR and sequenced,a mutant strain of porcine epidemic diarrhea virus was selected and put on the suspension-cultured Vero cells in a 2 L reactor for virus isolation and continuous cell culture,the harvested virus suspension,which was identified and determined TCID₅₀,was inactivated by formaldehyde and mixed with aluminum hydroxide gel adjuvant to prepare PEDV inactivated vaccine.After its physical behavior,stability viscosity,sterility test were checked out,the safety and immune efficacy were studied by immunizing the pregnant pigs and theirs piglets.The results were as follows:86 samples were detected positive in 200 samples,cytopathy occurred after the mutant strain samples screened were passaged to 5th generation,the virus suspension was harvested in 10th generation and identified as a mutant strain of PEDV,named PEDV-GF10 strain.The virus titer of harvested virus suspension was measured up to 1×10^8.0 TCID₅₀/mL after concentrated.After the vaccine was checked out,the sows,40 and 25 days before delivery in experimental groups,were injected into Xuehai acupoint with 4 mL vaccine and the pigs in blank group were free of immunifications.The results showed that there were no obvious differences in the production status of the sows in experimental groups and blank group and the temperature of theirs 3-day-old healthy piglets injected different doses of vaccine,and the vaccine was safe to the sows and piglets.Forty 3-day-old piglets producted by pregnant sows in experimental groups and blank group were randomly selected and taken 4 mL 1×10^8.0TCID₅₀/mL F10 virus culture.The PEDV morbidity of piglets in blank group was 100% after injection and the antibodies were negative;10% piglets in blank group had mild diarrhea symptoms,the protection rate was up to 90%,antibody of passive immunity in piglets lasted for more than 35 days.Virus titer of mutant strain of PEDV-GF10 improved a lot by suspension cell culture,the PEDV-GF10 inactivated vaccine was safe,and could effectively prevent and control the variation strain of PEDV in China.

In order to investigate the genetic variation of porcine reproductive and respiratory syndrome virus (PRRSV) strain GZ-R and the characteristics of NSP2 gene,in this study,two specific primers were designed based on NSP2 gene for nested RT-PCR,cloning and sequence analysis of the target gene.The results showed that the second round of nested PCR not only amplified the expected band of about 3 000 bp,but also amplified another band of 1 200 bp.The two DNA fragments were cloned into the pMD19-T vector,and two fragments were obtained by sequencing.The large fragment was 2 980 bp long and named as GZR-NSP2-L.The small fragment was 1 131 bp long and named as GZR-NSP2-S.The homology comparison results of the two fragments showed that GZR-NSP2-S had GZR-NSP2-L N-terminal nucleotide 1-945 and C-terminal 2 795-2 980 nucleotide,and lack of nucleotide between bits 946-2 794 in GZR-NSP2-L.The nucleotide homology of GZR-NSP2-L with VR-2332 strain and Lelystad virus strain was 81.2% and 48.6%.The results of homology comparison of encoded amino acids were consistent with the results of nucleotide homology,revealing that the PRRSV GZ-R strain belonged to the American-type strain.Comparison of amino acid deletion sites revealed that GZ-R NSP2 had 30 discontinuous amino acid deletions at positions 481 and 533-561.The deletion sites were consistent with PRRSV highly pathogenic strains.In summary,the PRRSV GZ-R strain could be identified as American-type highly pathogenic strain.The experimental results could provide some references for further exploring the epidemic trend of PRRSV in Guizhou province and the mechanism of NSP2 in PRRSV replication.

Avian influenza (AI) is a kind of avian virulent syndrome caused by avian influenza virus (AIV),which threatens animal and human public health and seriously affects the development of poultry industry in China.Vaccination has always been the most effective means to control the spread of avian influenza virus.Based on the continuous development of genetic engineering technology,a variety of new vaccines have been developed and put into use.Among them,avian influenza DNA vaccine has many advantages,such as high safety,simple preparation,easy storage and transportation.Common HA DNA vaccine,NA DNA vaccine,M DNA vaccine,NP DNA vaccine.Avian influenza DNA vaccine introduces a recombinant plasmid containing the target gene sequence into animal cells to induce a humoral and cellular immune response.In order to improve the immune effect of avian influenza DNA vaccine,researchers at home and abroad have made some achievements in enhancing the transfection efficiency and gene expression level of DNA vaccine by adding appropriate adjuvants,introducing target genes into ideal plasmid vectors and optimizing antigen sequence.Since the development of DNA vaccines,many subtypes of avian influenza DNA vaccines,including H1,H3,H5,H7 and H9 subtypes,have been gradually developed.In 2018,the H5 subtype DNA vaccine developed by Harbin Veterinary Research Institute of The Chinese Academy of Agricultural Sciences obtained the National Class Ⅰ Veterinary Medicine certificate,which is the first DNA vaccine of avian influenza to be approved in China,greatly promoting the development of DNA vaccines.This review mainly discusses the development and innovation of avian influenza DNA vaccine in terms of vector construction,immune mechanism,adjuvant and vector selection,and vaccine research and development,and briefly analyzes its application prospect,in order to provide new ideas and references for researchers to develop new avian influenza vaccine.

The purpose of this study was to explore the effects of probiotics on chickens' resistance to Escherichia coli infections,and to provide a basis for the development of microecological agents.Based on the Oxford Cup test and the bacterial adhesion inhibition test,three kinds of probiotics and their equal proportion mixed bacterial liquid with good effect of inhibiting Escherichia coli in vitro were obtained initially:Lactobacillus plantarum ZN-3,Lactobacillus rhamnosus QC,Clostridium butyricum HYCB and mixed bacteria preparation X.The 7-day-old SPF chicks were continuously orally fed with probiotics for 30 days and infected with Escherichia coli XT-13 once a day from the 26th to 30th day,and the survival rate and weight change were recorded.Through the same oral and infection protocols,the chicks were necropsied after infection with XT-13,and the results of necropsy and pathology were analyzed,and the amount of bacteria in the liver and intestines was detected by immunohistochemistry.In addition,in order to analyze the effect of oral administration of mixed probiotic preparations on cytokines in chickens infected with Escherichia coli,non-lethal doses of XT-13 were selected to infect chickens,and the levels of interleukin-10 (IL-10),interleukin-17 (IL-17) in serum and avian defensin-β1 (AvBD1) in the liver were measured on the 4th,8th and 14th days after infection.At the same time,the bactericidal ability of the serum was tested on the 14th day after infection.Finally,the broad-spectrum protection ability of probiotics to O78 serotype Escherichia coli AV006 infected chickens was evaluated.The results showed that the antibacterial effect of the supernatant of each probiotic fermentation broth was obvious,and the mixing of probiotics could reduce the adhesion rate of XT-13 to DF-1 cells (P<0.05).The weight gain effect of mixed probiotics was better than that of single probiotics,which had a good protective effect on chickens infected with XT-13.The survival rate was 87.50%.The mixed probiotics significantly reduced the load of XT-13 in liver and large intestine,alleviated the pathological damage,and regulated the levels of IL-10 and IL-17 in serum and the levels of AvBD1 in the liver,while improving the bactericidal ability of the serum.In addition,the broad-spectrum protection test showed that the mixed probiotics reduced the mortality of chickens infected with O78 serotype Escherichia coli.Therefore,the mixed preparation of Lactobacillus plantarum ZN-3,Lactobacillus rhamnosus QC and Clostridium butyricum HYCB could promote chick weight gain,reduce bacterial load,reduce tissue lesions,regulate the host's immune response,and had a good prevention effect on avian colibacillosis,which had a great potential to develop into a probiotic preparation against avian colibacillosis.

This study was aimed to evaluate the efficacy of the fermented traditional Chinese medicine on treating colibacillosis and immune regulation in chickens.240 healthy 1-day-old AA White-feathered broilers were selected to carry out the control test of Escherichia coli O78 LD₅₀ and compound Chinese medicine fermentation broth on colibacillosis.The therapeutic effect was evaluated at 28 days old,and the immune organ index,antioxidant index and intestinal flora were measured at 28 and 42 days old.The results showed that the infection time of Escherichia coli was delayed from 4 to 24 h after the treatment of fermentation liquid of traditional Chinese medicine fermented by Lactobacillus plantarum and Bacillus subtilis.The effective rates of high dose group (1.0 g/mL),medium dose group (0.5 g/mL) and antibiotic group (neomycin sulfate) were 80.0%,76.7% and 80.0%,and the cure rates were 80.0%,70.0% and 70.0%,respectively.Compared with infection and antibiotic groups,the thymus index and bursa index in high dose group at 28 days old were increased by 165.6%(P<0.05),25.4%(P>0.05) and 82.6%(P<0.05),94.3% (P<0.05),respectively.The thymus index and bursa index at 42 days old were increased by 97.4%,82.0% and 43.4%,27.5% (P<0.05),respectively.The T-AOC,TP,GSH-Px and T-SOD antioxidant indexes were significant difference (P<0.05),and significantly increased the content of lactic acid bacteria in intestine and reduced the content of Escherichia coli at 28 and 42 days old (P<0.05).In conclusion,the medium dose group of fermented traditional Chinese medicine (0.5 g/mL) had obvious antibacterial effect and provided substitute for antibiotics,the high dose group of fermented traditional Chinese medicine (1.0 g/mL) was significantly better than antibiotics for broilers.

The purpose of this study was to optimize the antifreeze protectant of attenuated Salmonella Cholerae C500 and improve the survival rate of C500.The effect of four types of cryoprotectant (permeating,semi-permeating,non-permeating cryoprotectants and antioxidants) on the survival rate of C500 before and after cryopreservation was investigated by single factor test.Each cryoprotectant was set at different concentrations and mixed in equal volume of C500.After two weeks of cryopreservation in liquid nitrogen,becteria plate count was carried out to calculate the survival rate,and the best one of the permeating cryoprotectant,semi-permeating cryoprotectant,non-permeating cryoprotectant and antioxidants and their optimal concentrations were beneficial to the preservation of bacteria was initially selected.Finally,the four protectors selected were tested by orthogonal test of four factors and three levels to get the optimal combination formula.The results of single factor test showed that the best permeating,semi-permeating,non-permeating cryoprotectants and antioxidants were glycol,sucrose,PVP and glycine,and the corresponding preservation concentrations were 15%,6%,6% and 0.25%,respectively.The orthogonal test showed that each factor had a significant impact on the survival rate of C500.The optimal combination formula was 17% glycol,9.0% sucrose,6.0% PVP and 0.35% glycine.The optimal combination had the optimal effect at -80 ℃,the survival rate was 88.34% in 6 weeks,followed by liquid nitrogen group,-20 ℃ group and 4 ℃ group.The results showed that the formulation of the protective agent obtained in this experiment greatly improved the antifreeze ability of C500 and its survival rate.

The purpose of this study was to investigate the dynamic of mink enteritis virus (MEV) maternal antibody and the immune effects of subunit vaccines.The offspring of immunized female mink were studied,serum of 21,30,45 and 60 days old mink were collected to determine the HI titer of maternal antibody of MEV.25 healthy minks aged 47~52 days were selected to be inoculated with MEV genetic engineering subunit vaccine,and then blood samples were collected 14 d before and after immunization to determine MEV HI titer.The clinical symptoms and the titer of feces HA of mink enteritis virus were observed 14 d after immunization.Dying and surviving minks were euthanized 14 d after challenge,the duodenum,jejunum and ileum were collected for histopathological observation and immunohistochemical detection.The results showed that the MEV HI of the offspring of immunized female mink decreased gradually with the increase of day age,it was higher at 21 d of age,the MEV HI titer of some minks at 45 d was <1:32 and ≤ 1:4 at 60 d.The MEV HI titer of the control mink was not higher than 1:4 on 14 d after the preparation of qualified vaccine,while it was increased to 1:64~1:512 in immune group.Challenge protection tests showed that the immuned minks were 100% resistant to the attack of mink enteritis virus,and there was no abnormality in the mink's mental state,diet and feces,the HA titer of feces matter after challenge was 1:8~1:16.Histopathological and immunohisto-chemical tests showed that MEV genetic engineering subunit vaccine could well prevent the replication of virus in intestinal mucosal epithelial cells and the damage to intestinal epithelial cells.Therefore,the antibody titer was highest at 21 day-old after immunizing female mink,and the prepared vaccine could break through maternal antibody interference and produce high levels of antibodies when it immunized minks at about 50 d of age,and could resist the attack of mink enteritis virus virulent strain.

The aim of this study was to clone the duck interferon-induced protein with tetratricopeptide repeats 5(IFIT5) gene,express duck IFIT5 protein and prepare the polyclonal antibodies against the duck IFIT5 protein.Primers specific for duck IFIT5 gene were designed according to Mallard duck reference sequence(accession No.:KF956064)available in GenBank.The complete ORF of duck IFIT5 gene was amplified by PCR.Recombinant prokaryotic expression plasmids pET-30a-IFIT5,pET-32a-IFIT5 and eukaryotic expression plasmid pcDNA3.1-IFIT5 were constructed.Recombinant duck IFIT5 proteins were expressed and analyzed by SDS-PAGE.Recombinant duck IFIT5 proteins were purified by Ni-NTA column affinity chromatography.Rabbit anti-duck IFIT5 polyclonal antibodies were prepared from rabbits which had immunized by recombinant protein pET-30a-IFIT5.The titer of polyclonal antibodies was detected by indirect ELISA,and the specificity was identified by Western blotting.Eukaryotic expression recombinant plasmid pcDNA3.1-IFIT5 was transfected into BHK21 cells and the reactivity of polyclonal antibodies was verified by indirect immunoinfluscence assay.The results showed that recombinant duck IFIT5 proteins were expressed in the form of inclusion bodies.The titer of polyclonal antibodies was approximately up to 1:49 600.Rabbit anti-duck IFIT5 polyclonal antibodies could identify recombinant protein pET-32a-IFIT5.Indirect immunofluorescence assay (IFA) confirmed that the purified polyclonal antibodies could identify duck IFIT5 protein by eukaryotic expression system specifically.These results demonstrated that the duck interferon stimulating gene IFIT5 was cloned successfully,rabbit anti-duck IFIT5 polyclonal antibodies could be used in the detection of duck IFIT5 protein,and also provided material support for the further study of duck IFIT5 protein.

The aim of this study was to evaluate the combined toxicity of enrofloxacin with each of three antimicrobials (ciprofloxacin,florfenicol and sulfamethazine).MRC-5 cells were used as a cell model to simulate the damage of lung cells caused by mixed contamination of antimicrobials.Multiple concentration gradients and mixing ratios were set.CCK-8 method was used to determine the inhibition rate of cell growth caused by four antimicrobials and the inhibition rate of cell growth caused by enofloxacin mixed with three antimicrobials,respectively.Then Chou-Talalay method was used to fit the median effect plot and to calculate the combination index (CI) value.The results showed that the growth inhibition rates of MCR-5 cells caused by four single drugs went up in a step-like manner with the increase of drug concentration in the tested concentration range,among them,the growth inhibitory rate of MCR-5 cells by florfenicol was low (<4.5%).The combined toxicity of the three binary combinations showed a concentration-dependent and mixing-ratio dependence.Mixing enrofloxacin and ciprofloxacin showed synergistic toxicity (CI<1) on MRC-5 cells at high and middle concentration groups,and antagonistic toxicity (CI>1) at the very-low-concentration groups.Mixing ciprofloxacin and florfenicol mainly showed an inhibited toxicity (CI>1).Binary combination enrofloxacin and sulfamethazine might present either a synergistic joint toxicity (CI<1) or an antagonistic joint toxicity (CI>1) as the concentration and mixing ratio changing.This study showed that it was necessary to assess the combined toxicity of antimicrobials in the toxicity evaluation of antimicrobials.Using Chou-Talalay method,the joint toxicity of multiple antimicrobials could be quickly and efficiently determined at cellular level.

The aim of the study was to observe the preventive and therapeutic effect of Clostridium butyricum-longan polysaccharide fermentation broth on mouse model with ulcerative colitis (UC).30 SPF male BALB/c mice were randomly divided into blank group,model group and fermentation broth group,10 mice/group.According to the dosage standard of 10 mL/kg body weight,sterile deionized water was administered to the blank group and model group,and Clostridium butyricum-longan polysaccharide fermentation liquid was administered to the fermentation broth group for 7 days.From the 8th day,the model group and the fermentation broth group were allowed to drink 5% dextran sulfate sodium (DSS) solution for 7 consecutive days to induce the UC model,and the blank group was free to drink sterile water.On the 14th day,all mice were weighed,and DAL scores and clinical scores were scored.Then the mice were dissected after eyeball collection and blood sampling.The length of the colon was measured and scored for histological damage of the colon,sections were made after formalin fixation.The content of IL-6,IL-10 and TNF-α in serum were detected by ELISA.The results showed that UC model was established successfully and symptoms were obvious in the model group.Compared with the model group,the weight loss of the mice in the fermentation broth group was significantly reduced (P<0.05),DAL score,clinical score,colon shortening degree and histological injury score were extremely significantly reduced (P<0.01).The colon tissue of mice in the fermentation broth group showed a relatively complete epithelial structure and crypts,and only part of the inflammatory cells invaded the epithelial tissue.ELISA results showed that the IL-6 and TNF-α levels in the model group were significantly higher than those in the fermentation broth group (P<0.05),and the IL-10 content was significantly lower than that in the fermentation broth group (P<0.05).It showed that Clostridium butyricum-longan polysaccharide fermentation broth had a certain preventive effect on DSS-induced UC in mice,and its mechanism might be related to reducing the content of IL-6 and TNF-α,increasing the content of IL-10,and inhibiting inflammation.

Salmonella Kentucky is another Salmonella serotype associated with human intestinal diseases following Salmonella Typhimurium and Salmonella Enteritidis.It is gradually attracting attention because of its strong ability to acquire and spread drug resistance.Among the many subserotypes of this serotype divided according to the sequence of multiple loci,ST198 subserotype has the characteristics of genomic islands that are easy to obtain drug resistance mutations,and has been evolving since the 21st century.In addition,resistance to fluoroquinolones and carbapenems have been acquired and spread worldwide,causing clinically difficult cases and the concern of the World Health Organization.This article summarizes the evolution of Salmonella Kentucky and highlights the resistance status and mechanism of Salmonella Kentucky with regard to fluoroquinolones and carbapenems,so as to provide a theoretical basis for the laboratory control and monitoring system of Salmonella in China.

The aim of this study was to provide theoretical basis for regulatory departments to formulate risk management measures for bacterial resistance of animal origin.The point assessment method was applied to assess the risk of bacitracin resistance in animal origin foods of Chinese residents,according to the model of risk assessment analysis,based on the data from the monitoring report of the citizen's physiques in China in 2014,the fifth Chinese total diet study and the official website of European Medicine Agency.Dietary exposure to bacitracin was calculated by estimated daily intake,and hazard quotient (HQ) was used in risk characterisation to indicate risk level of health damage exposed to bacitracin.Firstly,through the way of hazard identification,the toxicity of bacitracin by oral ingestion was very low.But the pathogenic bacteria such as Enterococcus and Clostridium perfringens had developed resistance to bacitracin,with the resistance genes easily transferring to the human intestinal flora through bacteria commonness.Secondly,the hazard characterization indicated the toxicological ADI was 0.055 mg/(kg·BW),and the microbiological ADI was 3.9 μg/(kg·BW).Thirdly,exposure assessment showed that the maximum dietary exposure of bacitracin was in 2 to 7 age group among Chinese residents.And that of women through intake of dairy products was higher than men.Among 2 to 7 age group,dairy products contributed most to the dietary exposure of bacitracin,while meat was the largest contributor above 8 years old.Fourthly,risk characterization hazard quotient was 1.4901 for males and 1.4121 for females among Chinese residents aged 2 to 7 by calculation,and <1 among other age groups.Finally,the uncertainty analysis showed that the lack of monitoring data of bacitracin residues in animal foods,the lag of authoritative data collection and release,the absence of consumption data of aquatic products brought uncertainty to the results.In total,the risk of bacitracin resistance as a medicated feed additive through animal foods exposure was high in children aged 2 to 7 and moderate in other age groups.The maximum residue limit standard should be revised appropriately and risk management of bacitracin resistance need to be strengthened in imported animal foods.

The aim of this study was to evaluate the effects of Lactobacillus salivarius TCSL1 on the growth performance,hematology and serum biochemical indexes and intestinal health of puppies,which was to lay a foundation for the research and development of probiotics for puppies.24 healthy six-week-old Chinese garden dogs were randomly divided into 4 groups,6 dogs were in each group.The experimental groups were fed with low (5×10⁶ CFU/mL),medium (5×10⁸ CFU/mL) and high dose (5×10¹⁰ CFU/mL) Lactobacillus salivarius TCSL1 orally for 28 days,respectively.At the same time,the control group took saline orally for 28 days.During the experiment,the behavior,diet and diarrhea were observed and recorded regularly.After 28 d,dogs were weighed respectively to calculate the gained weight.Blood samples were collected to detect hematology,serum biochemistry,intestinal barrier and inflammatory cytokines.Feces were collected to detect the content of secretory immunoglobulin sIgA and the number of main intestinal bacteria.The results showed that compared with the control group,all the experimental groups could promote the weight growth of puppies,reduced the diarrhea rate,and had a positive correlation with the oral dose.The number of lymphocyte,erythrocyte,immunoglobulin and alkaline phosphatase content increased,while alanine transaminase and creatinine decreased,but there were significant differences between the groups (P<0.05).With the increase of oral Lactobacillus salivarius dose,the content of intestinal barrier indicator factors such as diamine oxidase (DAO),D-lactate and lipopolysaccharide (LPS) in the serum of the experimental groups showed a significant decrease (P<0.05).The contents of TNF-α and IL-6 in serum of the experimental groups decreased significantly (P<0.05),while the contents of TGF-β1 and IL-10 increased significantly (P<0.05).The content of secretory of immunoglobulin sIgA in the feces of the experimental groups increased significantly (P<0.05),the number of coliform and Enterococcus in the feces of the experimental groups decreased significantly (P<0.05),the number of Lactobacillus and Bifidobacterium increased significantly (P<0.05).The above results showed that the oral Lactobacillus salivarius TCSL1 could improve the growth performance of puppies,enhanced the intestinal health and reduced the frequency of diarrhea.5×10¹⁰ CFU/mL per day of the oral Lactobacillus salivarius TCSL1 had the best effect.

The present study aimed to determine the role of ClpS gene,and to analyse the impact of ClpS mutation on the virulence of Brucella.A ClpS gene mutant strain,named ΔClpS was constructed by homologous recombination technology.The bacterial growth kinetics,the LPS synthesis ability and the survival ability of bacterial within macrophages as well as the virulence in mouse model were measured.In addition,the difference between parent strain 2308 and the mutant strain ΔClpS were compared.The results showed that under the same culture conditions,no difference in bacterial concentration was observed between 2308 and ΔClpS strains.The silver staining examination showed that the expression level of LPS extracted from two strains were similar,indicating ClpS gene mutation did not alter the growth rate and LPS synthesis ability of Brucella. In the cell infection assay,the survival ability of ΔClpS strain in cells was extremely significantly lower than that of 2308 strain at 72 h after infection (P<0.01).The results of mouse infection experiment showed that in the first week after infection,no significant difference in spleen weight and bacterial concentration between 2308 and ΔClpS strains infected mice was observed.However,at 4 weeks after infection,the bacterial concentration in spleen of ΔClpS infected mice was 10^3.93 CFU/g spleen,which was significantly lower than that of 2308 strain (10^6.68 CFU/g spleen,P<0.01).The spleen weight of ΔClpS infected mice was also remarkably lower than that of 2308 strain (P<0.01).In summary,the results suggested that the ClpS gene of Brucella did not play a role in Brucella growth rate and ability of LPS synthesis,whereas ClpS gene mutation decreased the ability of Brucella colonization in mouse spleen.

To determine the pathogen and related symptoms caused by renal failure,and to provide a reasonable diagnosis and treatment plan,a case of disease on Welsh Corgi dog was tested by the method of symptoms,blood routine examination,blood biochemical test,blood gas analysis and identification of pathogen.The results showed that these data of W-SCR,W-MID,MID and RDW-CV rose than normal reference index in blood routine examination before the hospitalization,respectively.Meanwhile,data of W-LCR,PCV,MCV,MCH and RDW-SD had reduced separately.These data of Ca²⁺,GLU,BUN,PHOS,AMYL and CRE were up than normal reference index in blood biochemical test,respectively.These data of GLU,BUN and K⁺ had risen respectively,nevertheless,these data of Na⁺,Cl^- and PCO₂ had lowered separately.The result was positive by the antigen testing with the Leptospira detector plate.After treatment with synthetical therapy,WBC and SCR had risen than normal references index,and data of RBC,HGB and PLT were down respectively.By the Hotelling T² analysis of the difference between before and after the therapy on blood routine index,it was significant difference in the multivariate test (F=1 905 045.32,P=0.001).These data of WBC,W-LCR,SCR,MID,HGB,PCV,MCV and RDW-SD were extremely significant differences in single variable analysis (P<0.01),and other data with significant differences were W-MID,RBC,MCH and PDW(P<0.05).But,in blood biochemical test,most of data were in normal reference values.The mental state of sick dog was recovering gradually normal than before.Multivariate test was no significant difference by the Hotelling T² analysis (F=85.21,P=0.081),but other data of GLU,BUN,PHOS,AMYL and CRE were extremely significantly different in single variable analysis (P<0.01).These data of GLU,BUN and K⁺ were up than after therapy in blood gas analysis,and data of Na⁺,Cl^- and PCO₂ reduced respectively.There were extremely significant differences in the multivariate test (F=7 978.48,P=0.008).These data with extremely significant differences were GLU and BUN in single variable analysis(P<0.01).The sick dog was recovering normal after treatment,and mental state went back to normal after 7 days therapy using the drugs of diuretics,antibiotics,rehydration electrolyte,energy medicine,vitamins and CoA.It should be noted that the part of high index of reexamination was a normal manifestation when the body was recovering to normal functioning.The method of synthetical fluid therapy was provided the correctness to treat the renal failure caused by canine Leptospira.

Chicken embryo has long been an important experimental model in basic and applied science because of its clear development process,especially in the early development of chicken embryo chorioallantoic membrane stage,due to its abundant blood vessels,it is a natural immunodeficiency host and can be used as an ideal experimental model for pathology,pharmacology and oncology research.The authors briefly described the tissue structure of early stage of chicken embryo,the application of chick embryo chorioallantoic membrane model in tumor,angiogenesis,organ transplantation,burn and other diseases,and the application of anti-cancer drug screening based on the pathological model of chicken embryo.The advances in the application of chicken embryo and avian cell lines in virus reproduction,vaccine production,therapeutic protein production and monoclonal antibody production were reviewed.A variety of human viruses,avian viruses and mycoplasma can proliferate in chicken embryos and avian cell lines and be used in vaccine production.In this paper,the development and characteristics of commonly used avian fibroblasts and pluripotent stem cells were described,and the source of commercial avian cell lines and some susceptible viruses were summarized.Chicken embryo expression system can produce human glycosylates at specific sites of target proteins,reduce the allergic reaction of the target protein to human,and poultry eggs are cheap and easily available,so it can be used as a suitable donor for the production of human monoclonal antibodies and therapeutic proteins.In this paper,the recent progress in the application of chicken embryos and avian cell lines in the field of biomedicine was introduced,and the future application of chicken embryos as animal models was forecasted.