【Objective】 The aim of the present study was to screen and validate the interacting protein of Orf virus 129(ORFV129) using yeast two-hybrid experiment with the cDNA library from goat fetal turbinate cells (GFTCs).【Method】 The Smart^TM technology was used to construct the GFTCs cDNA library.A bait vector pGBKT7-129 was constructed, which was then transformed into Y2HGold yeast competent cells, to verify the activity of self-activating.The toxicity effect induced by pGBKT7-129 transfection was also evaluated by measuring the growth curve of the bacterial.Using ORFV129 as the bait vector, the host protein that interact with ORFV129 were screened and the positive colonies were identified by PCR method and sequencing.The GO databases was used for function annotation and pathway analysis, following which a Cytoscape v 3.8.0 software was used for protein-protein interaction visualization.The coding domain sequence of Complement C1q binding protein (C1QBP) gene was cloned by RT-PCR method from goat spleen tissue, which was then ligated to pcDNA3.1(+) to construct a eukaryotic expression vector pcDNA3.1-C1QBP, and it was transfected into GFTCs for subcellular localization analysis.【Result】 The GFTCs cDNA library was constructed successfully with a capacity of 6.0×10⁶ CFU/mL.The bait plasmid pGBKT7-129 was successfully constructed with no self-activation ability and no toxicity to yeast cells.A total of 14 cellular proteins interacted with ORFV129 were selected from yeast two-hybrid experiment and the positive clones were confirmed by PCR and sequencing.The CDS region of goat C1QBP gene was successfully cloned, with a length of 837 bp, encoding 279 amino acids.The phylogenetic tree showed that there was the closest genetic relationship between Capra hircus and Ovis aries.C1QBP protein was an unstable hydrophilic protein without signal peptide structure and transmembrane domain, mainly including three kinds of phosphorylation sites, including 18 serine, 4 threonine and 3 tyrosine sites.The secondary structure of C1QBP protein was composed of alpha helix (33.81%), random coil (46.40%), extended chain (16.91%) and beta turn (2.88%), and the tertiary structure was consistent with the secondary structure.Indirect immunofluorescence test showed that C1QBP protein was scattered in the cytoplasm.【Conclusion】 The host intracellular protein C1QBP gene that interacts with ORFV129 protein and plays a role in the natural immune response was screened.The indirect immunofluorescence test verified that C1QBP was located in the cytoplasm.It was speculated that ORFV129 interacted with C1QBP to induce inflammation, laying a foundation for further verifying the process of ORFV129 protein mediated ORFV inhibiting the immune response of the body.

【Objective】 This study was aimed to conduct a comprehensive and systematic investigation and analysis of Wnt gene family in chickens, and further clarify the expression patterns of Wnt gene family members in chickens during Newcastle disease virus (NDV) infection.【Method】 Based on chicken genome sequencing results, the whole genome sequence of Wnt gene family in chickens was obtained by querying the Pfam and UniPort databases, the phylogenetic tree was constructed, and its physicochemical properties, subcellular protein localization, chromosome distribution, conservative domain, and so on.The expression pattern of Wnt gene family members in chickens infected with NDV was analyzed.【Result】 Thirty-nine Wnt gene family members were identified.The phylogenetic tree analysis showed that there was a high similarity of Wnt members among chicken, human and mouse, there were 12 subfamilies, namely Wnt1, Wnt2(Wnt2a and Wnt2b), Wnt3a, Wnt4, Wnt5(Wnt5a and Wnt5b), Wnt6, Wnt7(Wnt7a and Wnt7b), Wnt8(Wnt8a and Wnt8b), Wnt9(Wnt9a and Wnt9b), Wnt10a, Wnt11 and Wnt16. However, the chicken lacked Wnt10b.Wnt members in chickens distributed on 11 chromosomes (Chr1, Chr2, Chr4, Chr6, Chr7, Chr12, Chr13, Chr21, Chr26, Chr27 and CHR34, respectively).There were 14 tandem repeats among the members, and no segment repeat occurred.There was 10 conservative motifs in 39 Wnt members of chickens, the 3'-end of Wnt gene family members were relatively conservative, except for rna-XM_040653060.2(Wnt6) and rna-XM_040650634.2(Wnt5a), the 3'-end of the other Wnt members were composed of motifs 2 and 5.In addition, 39 Wnt family members of chickens all contained conservative regions, namely motifs 1 and 3. Analysis of transcriptome data revealed that the expression of Wnt7a was significantly up-regulated during NDV infection.【Conclusion】 The Wnt gene family members in chickens presented non-uniform distribution on the chromosome, and the gene structure was relatively conservative.Tandem repeat was the primary amplification mode of Wnt gene family members.Wnt7a might be closely related to Wnt regulating macrophages to participate in microbial infection.

【Objective】 This study was aimed to construct Brucella BPE159 gene deletion strain, and study the in vitro growth characteristics and survival ability of the deletion strains in host cells and explore the effect of the secreted protein BPE159 on autophagy factor expression during Brucella infection.【Method】 The recombinant plasmid of Brucella BPE159 gene was constructed by homologous recombination.The Brucella BPE159 gene deletion strain S2308ΔBPE159 was constructed by electrotransformation of recombinant plasmid into Brucella S2308 competent cells.Specific primers were used to amplify the BPE159 gene, ligated and transformed to construct the pBBR1MCS-4-BPE159 vector, and the plasmid was extracted and electroporated to construct the BPE159 gene complement strain S2308ΔBPE159-C.Genetic stability of deletion and complement strains was determined by agarose gel electrophoresis.The Brucella-infected mouse macrophage RAW264.7 model was constructed, and the expression levels of autophagy cytokines ATG5, Beclin1, LC3a and LC3b genes after Brucella infection were detected by Real-time PCR.Mouse macrophages were infected with S2308, S2308ΔBPE159 and S2308ΔBPE159-C strains, and total RNA was collected.The effect of BPE159 gene deletion on the expression levels of autophagy cytokines after Brucella infection was detected by Real-time PCR.S2308, S2308ΔBPE159 and S2308ΔBPE159-C strains were cultured at the same initial concentration, and the trend of growth changes was observed, and the survival and reproduction ability of S2308ΔBPE159 at different time points was evaluated.【Result】 The BPE159 gene deletion strain S2308ΔBPE159 and the complement strain S2308ΔBPE159-C were successfully constructed, and they were stably inherited for 10 generations.The results of Real-time PCR showed that after 24 h infection of Brucella, compared with PBS, autophagy factor ATG5 gene was significantly decreased (P<0.05), Beclin1, LC3a and LC3b genes were extremely significantly decreased (P<0.01).Compared with S2308 group, the expression levels of autophagic factors Beclin1 and LC3a genes were significantly increased (P<0.05), and the expression levels of ATG5 and LC3b genes were extremely significantly increased (P<0.01) in S2308ΔBPE159 group.The growth curve results showed that, under the same culture conditions, S2308ΔBPE159 and S2308ΔBPE159-C had similar growth trends to S2308.The results of intracellular survival showed that, 8 h after infection, the number of S2308ΔBPE159 in cells was extremely significantly lower than that of parental strain S2308 (P<0.01), and the viability at 12 and 24 h was significantly lower than that of S2308 (P<0.05).【Conclusion】 In this study, we successfully constructed and obtained BPE159 gene deletion strain and complement strain of Brucella with good genetic stability.S2308ΔBPE159 had a similar growth trend as S2308, but its viability and reproduction ability was obviously reduced.After BPE159 gene deletion, Brucella could promote the expression of autophagic factors.The results of this study laid a foundation for studying the biological function and regulatory mechanism of secreted proteins of Brucella.

【Objective】 This study was aimed to analyze the proteomics of hepatic proteins in calves and screen the differential proteins related to colostrum and milk feeding.【Method】 Nine bull calves delivered by multiparity Holstein cows were randomly divided into 3 groups in this study, including 3 colostrum-fed calves slaughtered at 24 h postpartum (CI group), 3 milk-fed calves slaughtered at 24 h postpartum (M group) and 3 calves not fed any milk or colostrum slaughtered 2 h after birth (CT group).The expression of hepatic proteins in calves in 3 groups were detected using a Label-free approach, the differentially expressed proteins (DEPs) were screened with Q<0.05 as the threshold, and analyzed by GO function and KEGG pathway enrichment analysis.【Result】 The results showed that 3 901 proteins were identified, 3 521, 3 646 and 3 548 proteins were obtained from CI, M and CT groups, respectively.Of these proteins, 3 202 proteins collectively expressed in CI, M and CT groups.Moreover, 287 DEPs in liver of calves have been found to be differentially expressed between CI and M groups, and they were related to cellular metabolism, response to stress, growth and development and oxidation-reduction process, the most significantly enriched pathways were endocytosis, amino acid metabolism and oxidative phosphorylation;And 154 DEPs in liver of calves have been found to be differentially expressed between CI and M groups, which were involved in cellular metabolism, single-organism process and creatine biosynthetic process, the most enriched pathways were amino acid metabolic process, focal adhesion and endocytosis.【Conclusion】 The results revealed that the DEPs in liver of calves fed with colostrum were mainly involved in catabolism and oxidation-reduction process, which provided a new understanding of protein response mechanism in liver of calves after colostrum feeding.

【Objective】 The aim of this study was to establish immortalized equine fetal fibroblasts (EFFs) cell line in Mongolian horse, and lay a foundation for the study of horse reproduction and developmental biology.【Method】 Primary EFFs were isolated by digestion with 1 mg/mL collagenase Ⅳ.EFFs were infected with pLV-hTERT-IRES-hygro lentivirus expressing human telomerase reverse transcriptase (hTERT) and hygromycin B resistance.The third generation EFFs was used as the control, the positive cells of hTERT-EFFs were screened by hygromycin B, the expression of hTERT mRNA was detected by Real-time quantitative PCR.The growth curve was drawed to detect the cell proliferation by CCK-8 method.Cell apoptosis was detected by flow cytometry, the cell senescence was detected by β-galactosidase staining.DNAMAN software was used to compare and analyze the similarity of TERT nucleotide, protein and telomerase RNA component (TERC) sequences of Homo sapiens, Equus caballus, Mus musculus, Bos taurus, Ovis aries and Sus scrofa.【Result】 The primary EFFs could be passaged to 17 generations in the laboratory culture system, but the 17-generation cells experienced senescence.hTERT gene could be successfully transferred into EFFs and stably expressed for at least 20 generations.However, the proliferation ability of hTERT-EFFs was significantly lower than that of EFFs (P<0.05), and the apoptosis rate of hTERT-EFFs in early stage was significantly higher than that of EFFs (P<0.05).The detection of cell senescence proved the β-galactosidase positive staining was positive.Sequence alignment results showed that the mRNA conservation between horse TERT and hTERT was high, the similarity was 73.2%, and the protein conservation of horse TERT and hTERT was higher than that of other species, and the similarity was 76.3%, which indicated that horse TERT and hTERT were highly similar in structure and function.Horse TERC was highly similar to human TERC, and which were in the same branch on the homologous tree.However, there were mutations in the key nucleotides associated with TERT binding and telomerase activity in horse TERC.【Conclusion】 Overexpression of hTERT alone could prolong the number of in vitro passages of EFFs to less than 30 passages, but it was still not enough to immortalize EFFs cells of Mongolian horse with high quality, which might be related to the incompatibility between hTERT and eTERC.

【Objective】 The aim of this study was to explore the regulatory effect of miR-142-3p on the lactation function of chemical induced mammary epithelial cells (CiMECs) of dairy goats in vitro, in order to promote the development of transgenic mammary gland bioreactor and lay a foundation and provide new ideas for the commercial production of "petri dish milk".【Method】 Milk goat ear fibroblasts (GEFs) were selected as the study material.After 8 days of induction by a single inhibitor of small molecule compound TGFβR-1/ALK5 (RepSox), the GEFs were identified by morphological changes, immunofluorescence staining of specific markers and oil red O staining.After that, liposome transfection technology was used to transfect miR-142-3p inhibitor (miR-142-3p inhibitor) and inhibitor control (inhibitor-NC) respectively in CiMECs of dairy goats with successful induction, and the cells without transfection were used as blank control (BC).24 hours after transfection, the expression of miR-142-3p was detected by Real-time quantitative PCR, the cell proliferation rate was detected by CCK-8 method, the expression of β-casein was detected by Western blotting, and the content of triglyceride was determined by triglyceride enzyme method.【Result】 After 8 days of GEFs induction, insular polynucleolus-like epithelioid aggregation was formed.Immunofluorescence staining showed that the induced GEFs expressed CK14 and CK18, the specific markers of mammary epithelial cells (MECs).The results of oil red O staining showed that lipid droplets of different sizes stained red were dispersed in the cytoplasm of induced GEFs, indicating that CiMECs of dairy goats with lactation function were successfully obtained.CiMECs transfection test results of dairy goats showed that compared with blank control group, the relative expression of miR-142-3p in miR-142-3p inhibitor group was significantly decreased (P<0.05), but there was no significant difference in miR-142-3p expression in inhibitor-NC group (P>0.05).The proliferation rate, β-casein expression and triglyceride secretion of CiMECs in dairy goats with miR-142-3p inhibitor group were significantly increased (P<0.05).There were no significant differences in the proliferation rate, β-casein expression and triglyceride secretion of CiMECs in dairy goats with inhibitor-NC group (P>0.05).【Conclusion】 GEFs could be successfully transformed into dairy goat CiMECs with lactation function after 8 days of induction by RepSox.Inhibition of miR-142-3p could significantly promote the proliferation of dairy goat CiMECs and increase the secretion of β-casein and triglyceride, thus affecting the lactation function.

Pig pluripotent stem cells are a kind of stem cells established in vitro with the capability of self-renewal and three germ layers differentiation, which can be used in developmental biology research, genome editing and disease modeling establishment.It has great value of application in animal husbandry production and regenerative medicine research.The culture system is of great significance for the successful construction of pluripotent stem cells, which contains a variety of components, including basic culture medium, amino acids and other nutrients, small molecular compounds (cytokines and signal pathway activators or inhibitors) to maintain cell pluripotency and inhibit its differentiation.There are many previous reports about porcine pluripotent stem cells, but effective culture system has not been obtained to maintain the long-term passage of porcine pluripotent stem cells and complete germline chimerism.Porcine pluripotent stem cells can be divided into three pluripotent states:Naïve state, Formative state and Primed state.And they can be classified into porcine expanded pluripotent stem cells, porcine embryonic stem cells, porcine pregastrulation stem cells and porcine induced pluripotent stem cells according to the derivation of the cell lines.Overall, the authors summarized the functional influences of current application of cytokines and signal pathway activators or inhibitors on the pluripotency and differentiation ability of porcine pluripotent stem cells.The present review could provide guidlines for the establishment of Naïve pluripotency porcine pluripotent stem cells with germline chimerism.

【Objective】 The study was aimed to investigate the effects of Astragalus polysaccharide (APS) on the intestinal morphology and local mucosal immune in chicks, in order to clarify the mechanism of APS in reducing intestinal mucosal injury in chickens with enteritis.【Method】 Fifteen 14-day-old SPF-grade chicks were randomly divided into three groups:Control group (Con), low-dose lipopolysaccharide (LPS) model group (D_L) and high-dose LPS model group (D_H), 5 chicks in each group.Control group was gavaged with normal saline, D_L and D_H groups were gavaged with 1 and 2 mg/kg BW LPS, respectively.After 3 days of continuous treatment, the small intestine were taken.HE staining method was used to observe the pathological changes of small intestine, and Real-time quantitative PCR was used to detect the changes in the expression of interleutin 1β (IL-1β) and tumor necrosis factor α (TNF-α), and screen the best LPS dose for constructing LPS-induced enteritis model.In the protection test of APS against intestinal mucosal injury, twenty 7-day-old chicks were divided into four groups:Control group (C), LPS inflammation group (L), APS group (A) and APS inhibited inflammation group (A+L), 5 chicks in each group.A and A+L groups were treated with APS (1.0 g/L) from 7-day-old to the end of this experiment, C group was fed water during this period.Chicks in L and A+L groups were given the selected optimal dose of LPS, after 3 consecutive days, the thymus, spleen, bursa of Fabricius and small intestine tissues were collected, the immune organ indexes of chicks were calculated in each group.HE staining was used to detect the pathological changes of chick small intestine, PAS staining was used to observe the number changes of goblet cells, Real-time quantitative PCR was used to detect the mRNA expression of tight junction proteins Occludin-1, Claudin-1 and ZO-1.【Result】 In the experiment of constructing LPS-induced enteritis model, the small intestinal mucosa lamina propria of chicks in D_Land D_H groups were congested and intestinal villi were injured, and the expressions of IL-1β and TNF-α mRNA were significantly higher than that of control group (P<0.05), which indicated that the intestinal tissues in D_L group had obvious pathological changes.Therefore, it was determined to feed 1 mg/kg BW LPS to establish enteritis model.In the protective test of APS against intestinal mucosal injury, compared with control group, for the chicks in L group, the villus of small intestine were broken, the lamina propria was congested, and the immune organ indexes were significantly decreased (P<0.05), the crypt depth of small intestine was significantly increased (P<0.05), the V/C was significantly decreased (P<0.05), and the goblet cells of small intestine were significantly decreased (P<0.05), the mRNA expression of tight junction protein Occludin-1, Claudin-1 and ZO-1 were all significantly decreased (P<0.05).Compared with L group, the immune organ indexes of chicks in A+L group were significantly increased (P<0.05), the intestinal mucosal damage was decreased, the intestinal villus height and V/C were significantly increased (P<0.05), the crypt depth was significantly decreased (P<0.05), the goblet cells were increased significantly (P<0.05), and the expression of tight junction proteins mRNA in jejunum and ileum were significantly increased (P<0.05).Compared with other groups, the number of goblet cells and the expression of tight junction protein in jejunum and ileum of chicks in A group were significantly increased (P<0.05).【Conclusion】 The enteritis model could be successfully established by feeding 1 mg/kg BW LPS to chicks.1.0 g/L APS could optimize the intestinal morphology, promote the development of local mucosal immune system of chicks, protect the intestinal tissue and intestinal mucosa of chicks with enteritis from damage, and finally achieve the effect of preventing bacterial enteritis in chicks.

【Objective】 The purpose of this experiment was to study the effect of low level of compound organic trace minerals (iron, copper, manganese, zinc and selenium) on serum biochemistry, immunity and liver antioxidant capacity of meat ducks, and to provide a certain scientific basis for the reduction and use of organic trace elements in meat duck feeding.【Method】 A total of 450 1-day-old Cherry Valley meat ducks(half male and half female) were randomly allotted into 5 groups with 6 replicates in each group and 15 ducks in each replicate.The blank control group (NC group) was supplemented with basal diet without additional trace minerals, and the ITM group was supplemented with NRC (1994) recommended amounts of inorganic elements (40, 10, 50, 60, 0.2 mg/kg of iron, copper, manganese, zinc and selenium, respectively).In 1/2(ITM+OTM) group, 50% NRC recommended amount of inorganic trace elements plus 50% NRC recommended amount of organic trace elements were added.In 1/2 ITM group, 50% NRC recommended amount of inorganic trace elements were add.In 1/2 OTM group, 50% NRC recommended amount of organic trace elements were added.The trial period was 42 days.On the 21st and 42nd days of the experiment, one meat duck was randomly selected form each replicate, and blood and liver were collected for the determination of serum biochemical related indexes, total protein and immunoglobulin levels in serum, and antioxidant indexes in liver.【Result】 ①Compared with NC group, the serum alkaline phosphatase (ALP) level of the meat ducks in ITM group was significantly increased (P<0.05) at 21st and 42nd day, meanwhile, at 42nd day, the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and the contents of uric acid (UA) in the serum of meat ducks were significantly decreased (P<0.05).Compared with ITM group, the activities of serum ALP of the meat ducks in 1/2 ITM group at 21 st day was significantly decreased (P<0.05).There was no significant differences in serum activities of ALP, ALT、AST、LDH and the contents of glucose (GLU), total cholesterol (T-CHO), triglyceride (TG) and UA of the meat ducks in 1/2 OTM and 1/2(ITM+OTM) groups at 21st day and 42nd day (P>0.05).②Compared with NC group, the serum levels of globulin (GLOB), total protein (TP), immunoglobulin A (IgA) and immunoglobulin M (IgM) at 21st and 42nd day and the level of immunoglobulin G (IgG) at 21st day in the meat ducks of ITM group were significantly increased (P<0.05).Compared with ITM group, the serum levels of TP, GLOB and IgG of meat ducks in 1/2 ITM group at 21st day and the serum levels of TP, GLOB, IgA and IgM at 42nd day were significantly decreased (P<0.05).The serum level of IgG in 1/2 OTM group at 21st day was significantly decreased (P<0.05), meanwhile, there was no significant difference in serum levels of albumin (ALB), GLOB, TP, IgA and IgM in 1/2 (ITM+OTM) and 1/2 OTM groups at 21st day and 42nd day (P>0.05).③Compared with NC group, the total antioxidant capacity (T-AOC) and the activities of catalase (CAT), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), copper zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in the liver of the meat ducks in ITM group at 21st day and 42nd day were significantly increased (P<0.05), the content of dialdehyde (MDA) was significantly decreased (P<0.05).Compared with ITM group, at 21st day and 42nd day, the liver MDA content in 1/2 ITM group was significantly increased (P<0.05), T-AOC and activities of CAT, GSH-Px, T-SOD and Cu/Zn-SOD were significantly decreased (P<0.05).Although the activities of some antioxidant enzymes decreased in 1/2 OTM group, T-AOC and MDA content did not change significantly (P>0.05).【Conclusion】 In summary, only feeding 50% of the NRC recommended dose of complex organic trace elements could achieve the effect of traditional commercial doses of inorganic trace elements to improve immunity and antioxidant capacity.And it was recommended to replace traditional commercial doses of inorganic trace elements with 50% of NRC-recommended doses of complex organic trace elements in meat duck diets.

【Objective】 The purpose of this experiment was to explore the structural composition and diversity of fecal flora in young suckling and adult female camels, and provide an important basis for healthy development of young camels.【Method】 12 adult female camels with 6-7 years old and similar physical condition, and 12 young suckling female camels with similar birth date (3 months old) were selected.The fresh fecal samples were collected by rectal fecal sampling method for V3-V4 region sequencing of 16S rDNA of fecal microbiota, and the sequencing data were analyzed accordingly.【Result】 The Chao1 and Shannon indexes of fecal flora in adult female camels were significantly higher than that of young suckling camels (P<0.05).At phylum levels, the top 10 bacteria in feces of adult female and young suckling camels were Firmicutes, Bacteroidetes, Verrucomicrobia, Proteobacteria, Tenericutes, Spirochaetes, Saccharibacteria, Actinobacteria, Cyanobacteria and Fibrobacteres.But the abundance of Proteobacteria and Tenericutes in feces of young suckling camels were extremely significantly higher than that of adult female camels (P<0.01).At family levels, the top 10 bacteria in feces of adult female and young suckling camels were Ruminococcaceae, Peptostreptococcaceae, Lachnospiraceae, Clostridiaceae, Christensenellaceae, Rikenellaceae, Verrucomicrobiaceae, Bacteroidaceae, Mogibacteriaceae and Paraprevotellaceae, and the abundance of Peptostreptococcaceae and Clostridiaceae in feces of adult female camels were extremely significantly higher than that of young suckling camels (P<0.01), while the abundance of Lachnospiraceae in feces of young suckling camels was extremely significantly higher than that of adult female camels (P<0.01).At genus levels, the top 10 bacteria in feces of adult female and young suckling camels were Clostridium, Oscillospira, Ruminococcus, Clostridiaceae_Clostridium, Akkermansia, 5-7 N15, CF231, Turicibacter, Clostridiaceae-SMB53 and Treponema, and the abundance of Clostridium, Clostridiaceae_Clostridium and Turicibacter in feces of adult female camels were extremely significantly higher than that of young suckling camels (P<0.01).【Conclusion】 The fecal flora diversity of adult female camels was significantly higher than that of young suckling camels, and show more stability.The dominant species of bacteria in feces of adult female and young suckling camels were consistent at the levels of phylum, family and genus, but there were difference in abundance.

【Objective】 This experiment was conducted to study the effects of whole plant of triticale hay on the apparent digestibility of nutrients, serum biochemical indexes and rumen fermentation in breeding cattle, and provide data reference for the rational use of triticale hay in animal husbandry.【Method】 Under the same feeding conditions, 30 healthy, physiologically sound Holstein breeding cattle aged 10 months with an average weight of (325.0±20.0) kg were selected and randomly divided into 3 groups (control, Ⅰ and Ⅱ groups), with ten replicates per group, one cattle per replicate. The cattle in control group was fed the basal diet (alfalfa hay:wheat straw=1:1), and in groups Ⅰ and Ⅱ, 50% (alfalfa hay:wheat straw:whole plant triticale hay=0.5:0.5:1) and 100% (all whole plant triticale hay) hay of the basal diet were replaced by whole plant of triticale hay, respectively.The pre feeding period was 7 days, and the trial period was 66 days.The average daily gain and feed to gain ratio were calculated by weighing each breeding cattle before and after the trial period, and the feed intake was recorded daily. Manure and rations samples were collected in the last 4 days of the trial period to determine the nutrient apparent digestibility. Blood was collected before the morning feeding and rumen fluid was collected 2 h after the morning feeding on the 66th day of the trial period to determine the serum biochemical and rumen fermentation indexes.【Result】 ①The differences in weight and daily gain of breeding cattle between groups were not significant (P>0.05), the feed to gain ratio of group Ⅱ was significantly higher than that of the control group (P<0.05).②The apparent digestibility of dry matter (DM), organic matter (OM) and crude protein (CP) in group Ⅰ was not significantly different from that of control group (P>0.05), but was extremely significantly higher than that of group Ⅱ (P<0.01). The apparent digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in group Ⅰ were significantly higher than that of group Ⅱ (P<0.05), and was extremely significantly higher than that of the control group (P<0.01).③The levels of serum glucose (GLU), triglyceride (TG), total cholesterol (TC), total protein (TP), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in breeding cattle were not significantly different between groups (P>0.05). The concentrations of serum albumin (ALB) in group Ⅱ were extremely significantly higher than those in control group and group Ⅰ (P<0.01).The serum urea nitrogen (UN) content of cattle in group Ⅰ was significantly lower than that of the control group (P<0.05), but not significantly different from group Ⅱ (P>0.05).④Ruminal pH, total volatile fatty acid (TVFA), acetic acid, propionic acid, butyric acid content and acetic acid/propionic acid (A/P) of breeding cattle were not significantly different between groups (P>0.05);The concentration of ruminal NH₃-N of cattle in group Ⅱ was significantly higher than that in group Ⅰ (P<0.05), but not significantly different from that in control group (P>0.05).The content of ruminal microbial protein (MCP) of cattle in group Ⅱ was significantly lower than that in group Ⅰ (P<0.05).【Conclusion】 Replacing 50% of the hay in the rouhage of breeding cattle with whole plant triticale hay improved their serum biochemical indexes and rumen fermentation, and significantly improved the apparent digestibility of nutrients in the rations.

【Objective】 This study aimed to investigate the effects of dietary fermented soybean meal (FSBM) replacement on growth performance, digestive enzyme activity, and microbita composition in weaned piglets.【Method】 Forty-eight crossbred piglets (Duroc×Landrace×Large White, 6.88 kg±0.13 kg, 21 days±2 days of age) were allotted into 2 groups (control group and FSBM group) with 4 replicates per group and 6 piglets per replicate.Piglets in control group were fed a basal diet, and experimental diet was formulated with 6% FSBM at the expense of soybean meal in basal diet.The experiment lasted for 20 days.The daily feed intake and body weight were recorded.On 21th day of the experiment, 2 piglets were randomly selected from each replicate and slaughtered.The ileal digesta was collected to measure digestive enzyme activity and microbita composition, and the correlations between the relative abundances of OTUs and the digestive enzyme activity were analyzed.【Result】 ① Feeding FSBM significantly enhanced final body weight and average daily gain (ADG) while reduced feed/gain (F/G) of piglets (P<0.05).② The activities of trypsin and amylase in ileal digesta of piglets were increased by treatment with FSBM (P<0.05).③ Dietary FSBM replacement significantly improved the relative abundance of Bacteroidetes, Anaerovibrio, Prevotellaceae_NK3B31_group, Ruminococcaceae_UCG-005, Prevotella_2, Megasphaera, Parabacteroides, Solobacterium, Lachnospiraceae_UCG-004 in ileal digesta of piglets, while the relative abundance of Streptococcus was decreased (P<0.05).④ The trypsin activity was positively correlated with relative abundance of Oscillospira and Ruminococcaceae_UCG-005 (P<0.05), while was negatively correlated with that of Subdoligranulum and Sutterella (P<0.05).The amylase activity was positively correlated with relative abundance of Prevotellaceae_UCG003 and Faecalibacterium (P<0.05), while was negatively correlated with that of Succinivibrio and Romboutsia (P<0.05).The activities of lipase was negatively correlated with Citrobacter relative abundance (P<0.05).【Conclusion】 Dietary FSBM replacement could regulate structure of ileal microbiota composition, increase the activities of trypsin and amylase, and improve growth performance of weaned piglets.

【Objective】 The purpose of the experiment was to study the effect of supplementation with L-arginine on pregnancy rate and plasma parameters of re-service thoroughbred horses.【Method】 14 re-service thoroughbred horses were randomly divided into 2 groups, 7 in each group(n=7), as the control group and L-arginine group.The control group was fed with basic diet, and the L-arginine group was added with 50 g/d L-arginine on the basis of basic diet.The pre feeding period was 7 d, and the trial period was 30 d.When the returning mare was in estrus, L-arginine was supplemented on the day after the first artificial insemination.At the end of mating, the pregnancy was examined by rectum B-ultrasound, and the pregnancy rate was calculated.Blood was collected from the jugular vein of mares at 0, 15 and 30 d respectively.The activities of total nitric oxide synthase (TNOS), endothelial nitric oxide synthase (eNOS) and the contonts of nitric oxide (NO), vascular endothelial growth factor (VEGF), growth hormone (GH), insulin-like growth factor(IGF) were measured.【Result】 Compared with the control group, the pregnancy rate of the L-arginine group was significantly increased (P<0.05).There was no significant difference in plasma TNOS, eNOS, NO, ODC, IGF-Ⅰ, IGF-Ⅱ and VEGF levels/activities between the two groups at 0, 15 and 30 d (P>0.05).On the 30th day of the test, the plasma ammonia (NH₃) and urea nitrogen (UN) contents in the L-arginine group decreased by 13.49% and 26.91% respectively compared with the control group (P<0.05).Compared with the control group, the plasma GH content on the 30th day of the test was significantly increased by 50.26% in L-arginine group (P<0.01).And there was no significant difference in the contents of IgA, IgM and IgG of mares on the 0th, 15th, and 30th days of the test between two groups (P>0.05).【Conclusion】 Feeding L-arginine to the re-service thoroughbred horses could significantly increase the pregnancy rate and change the contents of UN, NH3 and GH in the circulating plasma of the mares.L-arginine could be used as a nutritional regulator to improve the pregnancy rate of thoroughbred mares.

【Objective】 This experiment was conducted to investigate the effects of dietary threonine(THR)level on growth performance, carcass quality and immune function of Yellow-feathered broilers aged from 22 to 42 days, and to determine their appropriate THR requirement.【Method】 A total of 1 600 22-day-old fast-growing Lingnan Yellow-feathered broilers were randomly divided into 5 groups with 8 replicates per group and 40 chickens (male and female half) per replicate according to the same body weight.Chickens in the 5 treatment groups were fed diets containing 0.55% (basal diet), 0.62%, 0.69%, 0.76% or 0.83% THR, respectively.A corn-peanut meal basal diet was adopted, and the experiment lasted for 21 days.At the end of the test, 2 chickens were selected for slaughter sampling in each repetition to determine slaughter performance, immune organ index and serum biochemical indicators.【Result】 Dietary THR level significantly affected body weight(BW), average daily gain (ADG) and feed/gain (F/G)(P<0.05).For female broilers, ADG of 0.88% THR group was the highest, and average daily feed intake (ADFI) of 0.81% THR level group was the highest.For male broilers, ADG of 0.81% THR group was the highest, and ADFI of 0.81% THR level group was the highest.There was a significant quadratic linear correlation between dietary THR level and ADG, ADFI of Yellow-feathered males and F/G of both males and females (P<0.05).According to the regression equation of ADF and ADFI, the optimal dietary THR level of Yellow-feathered male broilers was 0.81% and 0.83%, respectively.According to the regression equation of F/G, the optimal dietary THR level of Yellow-feathered male broilers was 0.83% and 0.86%, respectively.Dietary THR level had no significant effects on eviscerated percentage, half-eviscerated percentage, leg muscle percentage and breast muscle percentage of Yellow-feathered males and females (P<0.05).There were significant effects on eviscerated percentage and abdominal fat percentage of Yellow-feathered male broilers, the abdominal fat percentage in 0.81% THR group was significantly lower than that in 0.60% and 0.74% THR groups, and the abdominal fat percentage in 0.88% THR group was significantly lower than that in 0.60% and 0.67% THR groups (P<0.05).Dietary THR level significantly affected bursa of Fabricius index and thymus index of Yellow-feathered females, the bursa of Fabricius index in 0.60% THR group was significantly lower than that in 0.81% THR group, and the thymus index in 0.81% and 0.88% THR groups were significantly higher than that in 0.60% THR group (P<0.05), but had no significant effects on liver index and spleen index (P>0.05).Dietary THR level had significant effects on serum triglyceride and insulin contents of Yellow-feathered female broilers, serum insulin content of 0.60% THR level group was the lowest, the serum triglyceride content in 0.88% THR level group was the lowest, significantly lower than 0.60% THR level group (P<0.05).Dietary THR level significantly affected the triglyceride content of Yellow-feathered male broilers, and 0.88% THR level group had the lowest serum triglyceride content, significantly lower than 0.60% and 0.67% THR level groups (P<0.05).【Conclusion】 Appropriate dietary THR could improve the growth performance, carcass quality and immune organ development of Yellow-feathered broilers.The optimal THR level for Yellow-feathered male and female broilers aged from 22 to 42 days were determined to be 0.83% and 0.81% respectively based on growth performance, the daily requirement of threonine for male and female broilers were 0.59 g and 0.51 g, respectively.

【Objective】 The aim of this study was to compare the effect of feeding fresh alfalfa and full-price pellets on the antioxidant capacity of gastric mucosa and the structure of gastric muscle layer in rabbits, and provide basis for healthy feeding of rabbits and improving the income of farmers.【Method】 Twenty healthy weaned New Zealand rabbits of similar weight were randomly divided into two groups:Alfalfa and feed groups, with ten rabbits in each group.The rabbits in the two groups were fed with fresh alfalfa and full price pellet feed for 30 days, respectively.New Zealand rabbits were sacrificed by air embolization and dissected, and the length and weight of stomachs were measured.The antioxidant capacity of gastric mucosa, including the activity of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), and the contents of lipid peroxidation (LPO) and malondialdehyde (MDA), and the gastric pH and pepsin activity were determined by biochemical methods.At the same time, the histological section technique, hematoxylin and eosin staining and image analysis were used to determine the thickness of muscle layer of gastric cardia, fundus, corpus and pylorus.【Result】 The body weight and gastric juice pH of feed group were significantly higher than that of alfalfa group (P<0.05), while the relative gastric weight, the concentration of pepsin and the antioxidant capacity of gastric mucosa in alfalfa group were significantly or extremely significantly higher than those in feed group (P<0.05 or P<0.01).The thickness of total muscle layer in the gastric cardia, fundus and pylorus of alfalfa group were significantly greater than that of feed group (P<0.01).In the cardia, the thickness of longitudinal muscle in alfalfa group was significantly larger than that in feed group (P<0.05), and the thickness of circular muscle in alfalfa group was extremely significantly larger than that in feed group (P<0.01).The thickness of the oblique muscle at the bottom of stomach in alfalfa group was significantly larger than that in feed group (P<0.05).There was no significant difference in the thickness of muscle layer in other parts (P>0.05).【Conclusion】 The gastric chemical digestibility, antioxidant capacity and gastric muscle layer thickness of alfalfa group were significantly higher than those of feed group under different feeding conditions.

【Objective】 The objective of this experiment was to study the effects of gastric and intraperitoneal injection of Lactobacillus rhamnosus (LGG) on the diversity of fecal flora in mice during pregnancy, and provide a theoretical basis for the physiological and intestinal health of animals during pregnancy.【Method】 45 SPF-grade pregnant Kunming mice with similar body weight (23.33 g±1.55 g) and the same breeding date were randomly divided into 3 groups, namely control group, test groups Ⅰ and Ⅱ, 15 in each group.Under the same feeding management and dietary nutrition level, starting from the second day of pregnancy, the control group was fed with basic diet, the test group Ⅰ was intraperitoneally injected 0.5 mL sterilized normal saline containing 0.125 g of LGG (continuous 14 days), and the test group Ⅱ was gavaged with 0.5 mL sterilized normal saline containing 0.125 g of LGG (continuous 18 days).The experiment lasted for 21 days. All mice were dissected 1 day before delivery, and large intestinal stool samples were collected.Genomic DNA was extracted from fecal samples of each group and amplified by PCR for library construction, the qualified libraries were sequenced using NovaSeq 6000 sequencing platform, Uparse software (v 7.0.1001) was used to cluster the sequences into operational taxa (OTUs) with 97% consistency and screen representative sequences.Species annotation analysis was performed using the Mothur method with the SSUrRNA database of SILVA 132, and community composition was counted at phylum, family and genus levels.QIQME 1.7.0 software was used for alpha diversity analysis, Chao1, ACE, Shannon, Simpson indexes were calculated, LEfSe analysis was performed, and FAPROTAX was used to predict the function of intestinal flora.【Result】 Compared with control group, Chao1 and ACE indexes in test group Ⅱ were significantly increased (P<0.05), the Simpson index of mice in test groups Ⅰ and Ⅱ was decreased (P>0.05).Compared with control group, at the phylum level, Firmicutes in test groups Ⅰ and Ⅱ were increased, Proteobacteria in test group Ⅰ was increased, and Proteobacteria in test group Ⅱ was decreased;At the family level, the levels of Lachnospiraceae, Lactate bacteria, Erysipelotrichaceae and Desulfovibrionaceae in test groups Ⅰ and Ⅱ were increased;At the genus level, Lactobacillus and undetermined Trichospiraceae were increased in test groups Ⅰ and Ⅱ. LEfSe analysis showed that there were two significant bacteria in control group.FAPROTAX predicted 35 functions for gut microbiota.【Conclusion】 Under the conditions of this study, LGG gavage could significantly improve Chao1 and ACE indexes, and the number of OTUs in LGG gavage group was higher than that in intraperitoneal injection group.The ratio of Firmicutes/Bacteroidetes, Proteobacteria, Desulvibriaceae and Lactobacillus in the gavage group were lower than those in the intraperitoneal injection group, while the undetermined Trichospiraceae were higher.A comprehensive comparison of the two supplementary feeding methods showed that gavage of LGG to pregnant mice could promote their health during pregnancy.

【Objective】 The purpose of this experiment was to study the association between single nucleotide polymorphism (SNP) in 3'-UTR, intron 1 and exon 7 of Z-DNA binding protein 1 (ZBP1) gene and lambing performance in goats, and provide new genetic markers for molecular breeding of high fertility goats.【Method】 Macheng Black goats, Black headed goats and Boer goats were selected as research samples, the blood samples were collected and DNA was extracted.Primers were designed according to the location information of SNP selected from transcriptome data in ZBP1 gene sequence of goats.Multiple single base extension SNP typing technique (SNaPshot) was used to analyze the genetic diversity of ZBP1 gene SNP.General linear model (GLM) was used to analyze the relationship between SNP of ZBP1 gene and lambing performance of 3 goat breeds.【Result】 There were 12 potential SNPs of ZBP1 gene in goats, of which 7 SNPs (g.9734 A>G, g.9772 T>C, g.352 C>T, g.955 C>T, g.1880 G>A, g.2566 T>C and g.7919 G>A) were polymorphic.There were 2 genotypes of AA and GA at g.9734 A>G in Macheng Black goats population, the other 6 SNPs showed 3 genotypes including CC(AA), CT(GA) and TT(GG) in Macheng Black goats, Boer goats and Black headed goats.g.9734 A>G and g.9772 T>C showed low polymorphism in Boer goats population (PIC<0.25), g.9734 G>A and g.7919 G>A showed low polymorphism in Macheng Black goats population (PIC<0.25), while g.9772 T>C, g.352 C>T, g.955 C>T, g.1880 G>A and g.2566 T>C showed moderate polymorphism in Black headed goats population (0.25<PIC<0.5), g.9734 A>G, g.352 C>T and g.7919 G>A showed low polymorphism in Black headed goats (PIC<0.25).The chi-square fitness test results show that g.9734 A>G, g.9772 T>C and g.352 C>T were in Hardy-Weinberg equilibrium in 3 goat populations (P>0.05), g.2566 T>C in Boer goats and Macheng Black goats population was in Hardy-Weinberg equilibrium (P>0.05).Correlation analysis results showed that g.9772 T>C was significantly associated with the first litter sizes in Boer goats (P<0.05), g.352 C>T and g.7919 G>A were significantly or extremely significantly associated with the overall litter sizes in Boer goats(P<0.05;P<0.01).g.352 C>T were extremely significantly associated with the overall litter sizes in Black head goat (P<0.01).g.9772 T>C was significantly associated with the first litter sizes in Macheng Black goats, g.9734 A>G and g.9772 T>C were significantly associated with the overall litter sizes in Macheng Black goats (P<0.05).Linkage disequilibrium (LD) results showed that there were strong linkage disequilibrium between g.352 C>T, g.2566 T>C, g.7919 T>C and g.9734 A>T in Boer goats, (D'>0.85, r²=1). g.352 C>T and g 2566 T>C, and g.2566 T>C, g.9734 A>T and g.9772 T>C in Black headed goats were in strong linkage equilibrium (D'>0.83, r²=1).There were strong linkage disequilibrium between g.352 C>T and g.9772 T>C in Macheng Black goats (D'>0.87, r²=1).【Conclusion】 There were 7 SNPs were found in this study, 5 fully linked SNPs were idetified on ZBP1 gene, which indicated that the 4 SNPs (g.352 C>T, g.7919 G>A, g.9772 T>C and g.9734 A>G) could be used as potential candidate genetic markers for lambing performance in the breeding process of specific goat breeds, it provided reference data for molecular marker saaisted breeding of goats.

【Objective】 The aim of this study was to investigate the changes of acrosin inhibitor (AI) expression and AI ubiquitination level before and after boar sperm capacitation in vitro, and to understand the relationship between AI and ubiquitin-proteasome system (UPS), so as to provide a reference for further study of the role of ubiquitin-proteasome pathway (UPP) in boar sperm capacitation.【Method】 Adult healthy Landwhite boars aged 18-24 months were selected, and boar semen was collected by hand-holding method, a part of sperm was capacitated, and a part of sperm was as control (fresh sperm).The kinetic parameters, quality parameters, tyrosine phosphorylation level and zinc ions (Zn²⁺) content of sperm were detected by computer assisted sperm analysis system (CASA), hypotonic swelling method (HOST), Coomassie brilliant blue staining, Western blotting and Zn²⁺ labeling, respectively.Western blotting was used to detect the expression of AI and ubiquitin (Ub) in sperm before and after capacitation.The localization of AI and Ub in sperm was detected by immunofluorescence.The binding of AI and Ub was analyzed by immunoprecipitation.【Result】 Compared with fresh sperm, the kinetic parameters VSL and BCF of capacitated sperm were extremely significantly increased (P<0.01), and the motility, plasma membrane integrity, acrosomal membrane integrity and viability of capacitated sperm were extremely significantly decreased (P<0.01).The tyrosine phosphorylation level of capacitated sperm protein was extremely significantly increased (P<0.01), and Zn²⁺ efflux was extremely significantly increased (P<0.01).The results of Western blotting showed that compared with fresh sperm, the expression of AI in capacitated sperm was extremely significantly decreased (P<0.01), and the expression of Ub was significantly increased (P<0.05).Immunofluorescence results showed that both AI and Ub were located in the acrosome region of sperm head.The results of immunoprecipitation analysis showed that AI was ubiquitinated in capacitated sperm, and the ubiquitinated AI was extremely decreased (P<0.01).【Conclusion】 During in vitro capacitation of boar sperm, the tyrosine phosphorylation level of sperm protein was extremely significantly increased and the ubiquitination level was enhanced, and AI was degraded, thereby releasing active acrosomal enzymes, in order to prepare for the subsequent sperm penetration through the zona pellucida (ZP) of oocytes to complete fertilization.

【Objective】 This study was aimed to investigate the phenotypic features of blood progesterone (BP) concentration and milk progesterone (MP) concentrations in Chinese Holstein cows based on the experimental data, the significant impact factors on the progesterone concentration and the relationship with milk composition.Then an attempt was made to find a way to obtain the progesterone phenotype on large population.【Method】 In August 2021, milk samples and tail root blood samples of Chinese Holstein lactating cows with different parity, lactation stage and pregnancy status were collected in two pastures in Beijing and Shandong, and progesterone concentration was measured.Finally, 402 MP concentrations and 298 BP concentration phenotypes were obtained for data analysis.After transforming the data of progesterone concentration to approximate following normal distribution, the fixed effect model was used to explore the influence of fixed effects on the progesterone phenotype of dairy cows, such as parity, lactation stage, pregnancy state, and pasture.Then, the R language cor function was used to calculate the correlation between progesterone and milk composition.The partial least squares method and individual and milk component information were used to predict progesterone concentration, so as to establish a high-throughput acquisition method for progesterone concentration phenotype.【Result】 Pregnancy state had an extremely significant effect on the concentration of transforming MP (P<0.01), parity and field effect had a significant effect on the concentration of transforming MP (P<0.05), while transforming BP concentration was only affected by pregnancy state (P<0.01).There was a significant or extremely significant positive correlation between milk solid, milk fat percentage, milk protein percentage, and transforming milk-blood progesterone concentration (r=0.14-0.37, P<0.05 or P<0.01).Based on the test data, the prediction accuracy of milk composition and individual information on transforming milk-blood progesterone concentration was not high (R²=0.030-0.17), but if the transforming progesterone concentration in blood or milk was increased to predict milk or blood transforming progesterone concentration, the prediction effect would be greatly improved (R²=0.40).【Conclusion】 This study showed that the factors affecting transforming progesterone concentration of Chinese Holstein cows in lactation period might include feeding conditions and parity in addition to pregnancy status.In addition, transforming progesterone concentration was significantly correlated with milk components such as milk fat percentage and milk protein percentage. Based on the relationship between transforming progesterone concentration and milk composition, the available strategy for predicting the transforming progesterone concentration in Chinese Holstein cows during lactation.Accordingly, it provided a supplement for the reproductive management in the farm and new ideas for developing new traits in dairy cattle breeding, a high-throughput method for collecting progesterone phenotype in Chinese Holstein cows.

【Objective】 The aim of this study was to explore melanocortin receptor-4 (MC4R) gene polymorphism and its correlation with growth traits in Qianbei Ma goats, in order to provide theoretical basis for breed selection and breeding of Qianbei Ma goats.【Method】 196 six-month-old Qianbei Ma goats were used as experimental materials, the single nucleotide polymorphism (SNP) of MC4R gene was screened by DNA mixing pool combined with Sanger sequencing, and the general linear model (GLM) in SPSS 22.0 software was used to analyze the correlation between SNP of MC4R gene and growth traits of Qianbei Ma goats.【Result】 There were 4 SNPs of MC4R gene in Qianbei Ma goats, g.59345469 C>A and g.59346773 T>C were located in the 5'- and 3'-untranslated region (UTR), respectively.The mutation of g.59345871 A>C resulted in the transformation of aspartic acid (Asp) to alanine (Ala) at position 37, and caused significant changes in the secondary structure of RNA and the secondary and tertiary structures of protein.The mutation of g.59346263 A>G resulted in the transformation of isoleucine (Ile) to valine (Val) at position 168, which had significant effects on the secondary structure of RNA and the secondary structure of protein.The results of association analysis showed that g.59345469 C>A had significant effect on the body weight, chest circumference and cannon circumference in Qianbei Ma goats (P<0.05). g.59345871 A>C had significant effect on the body weight, body height, chest circumference and cannon circumference in Qianbei Ma goats (P<0.05).g.59346263 A>G had significant effects on the body height, body weight and chest circumference in Qianbei Ma goats (P<0.05).g.59346773 T>C had significant effect on the body weight and chest circumference in Qianbei Ma goats (P<0.05).A total of 9 haplotypes and 21 diplotypes were detected by the combination of 4 SNPs, the combination had significant genetic effects on the body weight, body height, chest circumference and cannon circumference in Qianbei Ma goats (P<0.05), the growth traits of homozygous diplotype H5H5 (AAAAGGCC) individual was superior to that of other diplotypes.【Conclusion】 Four SNPs of MC4R gene were significantly associated with the body weight and chest circumference in Qianbei Ma goats, which could be used as genetic markers for molecular breeding of Qianbei Ma goats.

【Objective】 The main purpose of this study was to evaluate the effects of ubiquitin activating enzyme(E1) inhibitor (TAK-243) on pig sperm capacitation, membrane remodelling and fertilization.【Method】 Semen was collected from 5 Landrace pigs, the effect of TAK-243 on the binding of ubiquitin (Ub) and E1 in sperm was evaluated by thiol ester method.Sperm were divided into fresh group(Fresh), DMSO group(DMSO), capacitated group (Capacitated)and 2.4, 4.8 and 7.2 nmol/L TAK-243 groups.The fresh group was resuspended in 2 mL PBS for sperm precipitation.Sperm precipitation was resuspended with 2 mL capacitation solution in capacitation group.In DMSO group and 2.4, 4.8 and 7.2 nmol/L TAK-243 groups, sperm precipitation was resuspended with 2 mL capacitated solution containing 0.07% DMSO and 2.4, 4.8 and 7.2 nmol/L TAK-243, respectively.The kinetic parameters of sperm were detected by microbial dynamic (static) image detection system.The tyrosine phosphorylation of sperm was detected by Western blotting.Zn²⁺ fluorescence probe was used to detect the content of Zn²⁺ in sperm.Acrosomal membrane remodeling was detected by peanut lectin staining.The expression of acrosomal surface sperm adhesion protein AQN-1 and AWN was detected by immunofluorescence assay.The effect of TAK-243 on sperm-egg binding and cleavage rate was detected by in vitro fertilization.【Result】 Thiol ester analysis showed that sperm in TAK-243 treatment group did not form Ub-E1 complex, which indicated that TAK-243 inhibited the activation of Ub by E1.Compared with fresh sperm group, the curve velocity (VCL) and mean whipping frequency of sperm in capacitated group were significantly increased (P<0.05).There were no significant differences in the kinetic parameters between the three concentrations of TAK-243 and capacitated groups (P>0.05).Compared with the capacitated group, the tyrosine phosphorylation level of the three concentrations of TAK-243 group was significantly decreased (P<0.05), and the tyrosine phosphorylation level of sperm protein gradually decreased with the increase of TAK-243 concentration.Therefore, In subsequent experiments, 7.2 nmol/L TAK-243 was used to investigate the Zn²⁺ level and acrosomal membrane remodeling in sperm.The results showed that the Zn²⁺ level, acrosomal membrane integrity, AQN-1 and AWN protein expression levels in 7.2 nmol/L TAK-243 group were significantly increased compared with those in the capacity group (P<0.05), and the adhesion rate and cleavage rate of sperm and oocytes were significantly decreased (P<0.05).【Conclusion】 7.2 nmol/L TAK-243 significantly decreased the protein tyrosine phosphorylation level, sperm-egg binding rate and cleavage rate during sperm capacitation, and significantly increased the content of Zn²⁺, acrosomal membrane integrity rate and acrosomal surface proteins AWN and AQN-1 expression, indicating that ubiquitin-proteasome signaling pathway was involved in sperm capacitation in vitro.

【Objective】 The purpose of this study was to study the effects of semen collectors, temperature and humidity, ammonia concentration and light intensity of pigpen on semen quality of boars, in order to improve semen quality.【Method】 2 966 semen determination records were collected from 347 Duroc boars aged (580±48) d from November 2021 to February 2022. The sperm collector made a daily record of the boar's sperm collection.Temperature and humidity, ammonia concentration and light intensity parameters in the pigpen were measured daily at 09:00 am. The semen traits were correlated with the records of semen collectors and environmental parameters by R software.【Result】 ① The sperm collector had a significant effect on semen quality (P<0.05). The semen collected by the sperm collector No.8 had the highest sperm motility and the lowest sperm abnormality rate. ② When the ambient temperature of pigpen was 22 to 25 ℃, the sperm motility was significantly higher than 18.5 to 22 ℃ (P<0.05), and the sperm abnormality rate was significantly lower than 18.5 to 22 ℃ (P<0.05). ③ When the relative humidity was 70% to 77%, the sperm motility was the highest and the sperm deformity rate was the lowest, but the total sperm count was the lowest, and the difference was significant compared with the relative humidity 46% to 60% (P<0.05), while there was no significant difference when compared with the relative humidity of 60% to 70%(P>0.05). When the relative humidity was 60% to 70%, the sperm motility, sperm deformity rate and total sperm count were all at high level. ④ With the decrease of ammonia concentration, sperm motility and total sperm count showed an upward trend, and sperm abnormality rate showed a downward trend, when ammonia concentration was 2 to 4 ppm, sperm motility, total sperm count and sperm abnormality rate were significantly different from that when ammonia concentration was 5 to 7.3 ppm (P<0.05). ⑤ When the light intensity was 90 to 110 lx, the sperm motility and sperm progressive motility were the highest, which was significantly different from that when the light intensity was 130 to 164.5 lx (P<0.05). Light intensity had no significant effect on sperm malformation rate (P>0.05).【Conclusion】 The semen quality of boar was higher when the temperature range was 22 to 25 ℃, humidity range was 60% to 70%, ammonia concentration was 2 to 4 ppm and light intensity was 90 to 110 lx. In production, the management and training of semen collectors should be strengthened to reduce the mechanical damage and bacterial contamination in the process of semen collection, so as to provide reference and theoretical basis for the future research and revision of the comfortable environment parameters of breeding boars and scientific regulation of the piggery environment.

【Objective】 This study was aimed to reveal the population characteristics, and estimate the genetic parameters of muzzle width in Chinese Holstein dairy cows.【Method】 The muzzle width, rump height, body height, body length and chest girth of 628 Holstein dairy cows was measured in two dairy farms from July 2021 to August 2021.The body weight was estimated by body length and chest girth.SPSS 26.0 software was employed to analyze the impacts of herd, month and lactation stage on muzzle width, and obtain the correlation coefficient between muzzle width and body size traits.The variance components of muzzle width were estimated using single-trait animal model based on DMU software, and its heritability was calculated.【Result】 The muzzle width of Holstein dairy cows was 17.28 cm with a small variation (variation coefficient of 5%).Herd and month had extremely significant effect on muzzle width (P<0.01), lactation stage had significant effect on muzzle width (P<0.05), and muzzle width increased with the increase of month.Muzzle width in early lactation was significantly smaller than that in other lactation stages (P<0.05).The extremely significant and positive correlations between muzzle width and body size traits were found in Holstein dairy cows (P<0.01).The estimated heritability of muzzle width in Holstein dairy cows was 0.28, which was a moderate to high heritability trait.【Conclusion】 The herd and month had an extremely significant effect on muzzle width, and the lactation stage had a significant effect.The muzzle width was weakly correlated with rump height.The muzzle width was a body size trait with moderate to high heritability in Holstein dairy cows.

【Objective】 The purpose of this study was to explore the lactation performance of East Friesen sheep, Hu sheep and East Friesen-Hu hybrid sheep.【Method】 A total of 197, 313, 346, 364 healthy East Friesen sheep (DF), Hu sheep (H), East Friesen-Hu hybrid F1 generation (F1) and hybrid F2 generation (F2) were selected from the same farm.The daily milk yield from 2020 to 2021 was collected, the lactation curve was fitted by sixth degree polynomial, and the parameters of lactation curve were estimated.Milk samples were collected and milk fat, milk protein, lactose, total solids and urea nitrogen were determined by milk analyzer.Morphological description of dairy sheep udders was taken and nipple morphological data was measured, including nipple length, nipples spacing and nipple diameter.After anesthesia, the breast tissue was collected during the nonpregnant period, and the tissue sections were stained with hematoxylin-eosin staining (HE)and examined under microscope.【Result】 The milk yield of East Friesen sheep was significantly higher than that of hybrid F1 generation (P<0.05).There was no significant difference in milk yield between hybrid F1 and F2 generations (P > 0.05).The milk yield of the hybrid F1 and F2 generations were significantly higher than that of Hu sheep (P<0.05).The six-degree polynomial model was used to fit the lactation curves of different hybrid dairy sheep, and the degrees of fit were higher than 0.90.The peak lactation days of East Friesen sheep, Hu sheep, hybrid F1 and F2 generations were 58, 18, 45 and 47 d, respectively, and the peaks daily milk yield were 2.31, 1.27, 2.24 and 1.97 kg, respectively.The milk fat content of Hu sheep was significantly higher than that of other hybrid dairy sheep (P<0.05).There was significant difference in milk protein content between East Friesen sheep and hybrid F1 generation (P<0.05), and between Hu sheep and hybrid F2 generation sheep (P<0.05).There was no significant difference in other milk components of each hybrid dairy sheep (P>0.05).The results showed that the nipple length of Hu sheep was significantly shorter than that of other hybrid dairy sheep (P<0.05), and the nipple distance of hybrid F2 generation was significantly higher than that of other hybrid milk sheep (P<0.05).There was no relationship between nipple size and milk yield.The udders groove of Hu sheep was shallow, and the nipple orientation was parallel to the ground.The udders groove of East Friesen sheep was deep, and the angle between the nipple orientation and the ground vertical direction was less than 45°.The udder morphology and structure of the hybrid F1 generation was similar to that of the hybrid F2 generation, and the nipple oriented in an outside camber shape, which was suitable for machine milking.The mammary gland tissues of East Friesen sheep, hybrid F1 and F2 generations were well developed, and the number of acinar ducts in the breast structure was denser than that of Hu sheep.【Conclusion】 After hybridization between Hu sheep and East Friesen sheep, the milk yield of hybrid F2 generation increased significantly.The results of breast morphology and breast structure showed that hybrid F2 generation had a good lactation foundation.

【Objective】 This experiment was conducted to study the host immune response to respiratory Infectious bronchitis virus virulent strain infection in layer and discover the antiviral effect of recombinant avian β-defensin (AvBDs).【Method】 70 7-day old White Leghorn chickens were randomly devided into two groups, thirty-five chickens in each group respectively.The challenge group was inoculated with respiratory IBV virulent strain (CK/CH/LSD/05I) by a combined intraocular and intranasal route, while the control group was inoculated with PBS by the same way.The clinical symptoms were regularly observed for 14 d and recorded.Five chickens from each group were randomly selected to kill by blood sampling at 12 h, 36 h, 3 d, 7 d and 14 d after the infection.Blood samples were collected to detect the level of serum antibody.Part of trachea and kidney were collected for histopathological examination.RNA was extracted from bursa of Fabricius, spleen, kidney and trachea, which was used to detect the viral load of IBV, the expression of Toll-like receptor (TLRs) and signal transduction molecules, AvBDs by Real-time quantitative PCR.Recombinant AvBD11 and AvBD12 were transfected into chicken embryo kidney (CEK) cells.The supernatant and cells were collected at 6, 12, 24, 36 and 48 h after the infection.RNA was extracted for detecting the viral load by Real-time quantitative PCR.【Result】 After being infected with respiratory IBV virulent strain, chickens showed slight respiratory symptoms, and there was no obvious pathological change in autopsy.The antibody level of the infection group turned positive at 14 d post infection, and the positive rate of respiratory IBV virulent strain group was 100%.The results of Real-time quantitative PCR detection showed that no virus was detected in control group, IBV antigen was only detected in the trachea at 14 d post infection in challenge group and the virus load was very low.Compared with control group, the expression of TLR1, TLR5, AvBD9 and AvBD12 genes in spleen were significantly up-regulated at 7 d post infection with respiratory IBV virulent strain (P<0.05).In challenge group, the expression of TLR1, TLR2, TLR3, AvBD5 and AvBD9 genes in bursa of Fabricius and the expression of nuclear factor-κB p65 (NF-κB p65), NF-κB c-Rel, AvBD5, AvBD9, AvBD11 and AvBD13 genes in trachea were significantly increased at 14 d after the infection (P<0.05).The results of recombinant defensin antiviral experiment showed that compared with the empty vector group, the viral load of CEK cells transfected with AvBD11 was significantly down-regulated at 24 and 48 h after the infection (P<0.05), and the viral load in the supernatant of CEK cells transfected with AvBD11 was significantly down-regulated at 48 h post infection (P<0.05).Compared with the transfected empty vector group, the viral load of CEK cells transfected with AvBD12 was significantly down-regulated at 12 and 24 h after the infection (P<0.05), and the viral load in the supernatant of CEK cells transfected with AvBD12 was significantly down-regulated at 12 and 48 h post infection (P<0.05).It showed that recombinant AvBD11 and AvBD12 had anti-IBV activity in CEK cells.【Conclusion】 The infection of the respiratory IBV virulent strain enhanced the gene expression of relative receptor and signal transduction molecules, upregulated the gene expression of defensins, and strengthened the body's natural immune response.The recombinant defensins AvBD11 and AvBD12 had antiviral effects, which provided a new idea for the preparation and research of non-antiviral feed.

【Objective】 The purpose of this study was to construct a recombinant Adenovirus with VP4 gene of Porcine rotavirus(PoRV) epidemic strain PoRV G9P[23] in China, and lay a preliminary foundation for the development of candidate engineering vaccine of PoRV.【Method】 PoRV VP4 gene was synthesized by referring to the VP4 gene sequence of PoRV G9P[23] strain in GenBank (accession No.:MH898990.1). The obtained target gene was recombined with Adenovirus shuttle vector pAdTrack-CMV and transferred into Escherichia coli Top10 competent cells to construct Adenovirus shuttle vector pAdTrack-CMV-VP4. The recombinant plasmid pAd-VP4 was obtained by homologous recombination with BJ5183 competent cells containing adenovirus backbone pAdEasy-1 after linearized by PmeⅠ endonuclease. The recombinant plasmid was digested by PacⅠ enzyme and transformed into Escherichia coli DH5α competent cells. The recombinant plasmid was transfected into HEK293A cells to obtain recombinant Adenovirus rAd-VP4. The recombinant Adenovirus was expanded and the half tissue culture infective dose (TCID₅₀) of recombinant Adenovirus was determined.RT-PCR was used to detect its expression in vitro, and its reactivity was detected by Western blotting. The prepared recombinant Adenovirus was inoculated mice intraperitoneally with different virus titers and different times of immunization. The serum was collected to determine IgG antibody levels by ELISA kit.【Result】 A recombinant Adenovirus rAd-VP4 with a size of 2 343 bp was amplified by RT-PCR, and the sequencing results were correct, indicating that the recombinant Adenovirus rAd-VP4 was successfully constructed. The viral titer of rAd-VP4 was 10^6.5 TCID₅₀. The recombinant Adenovirus rAd-VP4 was correctly expressed at the protein level and the molecular mass of the protein was about 87 ku. The results of antibody detection showed that the serum IgG antibody level of mice immunized with 10^6.5 TCID₅₀ rAd-VP4 was significantly higher than that of 10^5.3 TCID₅₀ TGE-PED-PRV triple live vaccine on the 35th and 42nd day(P<0.05). The antibody level of 10^6.5 TCID₅₀ rAd-VP4 on the 35th and 42nd day after immunization was significantly higher than that of 10^5.5 and 10^4.5 TCID₅₀ rAd-VP4 (P<0.05). The antibody level of 10^5.5 TCID₅₀ rAd-VP4 on the 28th day after immunization was significantly higher than that of 10^6.5 and 10^4.5 TCID₅₀ rAd-VP4 (P<0.05). There was no significant difference in IgG antibody produced by 10^6.5 TCID₅₀ rAd-VP4 immunizing 2 and 3 times at different immunization times (P>0.05).【Conclusion】 In this study, the recombinant Adenovirus rAd-VP4 was successfully constructed, and it's virus titer was 10^6.5 TCID₅₀. 10^6.5 and 10^5.5 TCID₅₀ rAd-VP4 produced higher IgG antibody levels on the 42th and 28th day, respectively. There was no significant difference in IgG antibody level between the second immunization and the third immunization. The results could provide reference for the development of PoRV recombinant Adenovirus vaccine candidate.

【Objective】 This study was aimed to express and purify the structural protein p22 of African swine fever virus (ASFV), and use it as a coating antigen to establish an indirect ELISA method for detecting ASFV antibodies and diagnosis of African swine fever.【Method】 The truncated KP177R gene encoding ASFV p22 protein (amino acids 24-145) was cloned into prokaryotic expression vector pET-32a(+) to construct the expressing plasmid pET-32a-p22.The recombinant plasmid was transformed into Escherichia coli (E.coli) BL21(DE3) competent cells and then induced by 0.1 mmol/L IPTG for 5 h.The corresponding p22 protein was purified by affinity chromatography on Ni ion and identified by Western blotting.BALB/c mice were inoculated with the purified p22 protein to prepare antiserum.Meanwhile, HEK293T cells were transfected with the eukaryotic expression plasmid containing the p22 full-length gene fragment (pCAGGS-EGFP-fp22) and used as antigenic matrix.Indirect immunofluorescence assay (IFA) was used to identify the reactivity of the antiserum.Based on the optimized parameters including the coating antigen concentration, serum dilution, blocking condition, reaction time of the first antibody, and working concentration of the enzyme-conjugated secondary antibody, an indirect ELISA method was established for ASFV antibody detection, and the clinical porcine serum samples were detected using this indirect ELISA method.【Result】 The truncated ASFV p22 protein was expressed in E.coli, and the protein concentration was 0.85 mg/100 g of bacteria.The prokaryotic p22 protein showed good immunogenicity, and the titer of the corresponding antiserum could reach 1:12 800.The optimized parameters of the ELISA (p22-ELISA)were as follows:The concentration of coating antigen was 0.125 μg/well, the dilution of serum was 1:800, and the dilution of the secondary antibody was 1:10 000.The cut-off value of p22-ELISA was 0.384.There was no cross reaction with CSFV, PRRSV, PCV2 and PRV antibody positive porcine serum, indicating good specificity.The coefficients of variation intra- and inter-batch were lower than 10%, indicating good repeatability.The p22-ELISA was used to detect 263 clinical pig serum samples, and the coincidence rate with thecommercial ASFV ELISA kit was 97.7%.【Conclusion】 This study established an indirect ELISA method for ASFV antibody detection based on the prokaryotic truncated ASFV p22 protein, the method had strong specificity, high sensitivity and good repeatability, and provided technical storage for the diagnosis and epidemiological investigation of ASFV infection.

【Objective】 The purpose of the test was to analyze the effect of Seneca virus A (SVA) infection on the expression profile of circular RNAs (circRNAs) in pig renal epithelial cells (PK-15), and to explore the potential regulatory role of circRNAs during SVA infection.【Method】 The circRNAs transcriptome of SVA-infected and control PK-15 cells were sequenced based on the Illumina HiSeq 2000 platform.The source genes of the differentially expressed circRNAs were subjected to GO function and KEGG pathway enrichment analysis, and the expression levels of the newly discovered circRNAs were identified by Real-time quantitative PCR.【Result】 Transcriptome sequencing results revealed that a total of 432 novel circRNAs were identified in SVA-infected and control PK-15 cells.In SVA-infected PK-15 cells, the expression levels of 87 circRNAs were significantly up-regulated and 74 circRNAs were significantly down-regulated compared to control PK-15 cells. GO functional analysis of the source genes of differentially expressed circRNAs indicated that the differentially expressed circRNAs source genes in SVA-infected PK-15 cells were mainly enriched in the nucleolus, organelle membranes, and physiological processes such as cellular macromolecular metabolic processes and development.The results of KEGG pathway enrichment analysis showed that the significantly differentially expressed circRNAs source genes in the SVA-infected PK-15 cells were mainly enriched in the cytokinesis process, cAMP signaling pathway, Rap1 signaling pathway and Ras signaling pathway.The results of Real-time quantitative PCR verification of the 6 significantly differentially expressed circRNAs demonstrated that their expression levels were consistent with the high-throughput sequencing.【Conclusion】 This was the first report of differential analysis of the circRNAs expression profiles of SVA-infected PK-15 cells.It was found that the differentially expressed circRNAs were widely involved in macromolecular metabolism, cytokinesis and cell proliferation, adhesion and antiviral processes, and this study provided a reference for the further exploration of the molecular mechanism of circRNAs during SVA-infection.

【Objective】 The purpose of the test was to prepare bivalent inactivated vaccine according to Guizhou epidemic strains of Riemerella anatipestifer (RA) serotype 1 and 2, and provide research data for the prevention and control of RA infection and vaccine development.【Method】 The local epidemic strains of serotype 1 Riemerella anatipestifer (RA-G06) and serotype 2 RA (RA-HS01) were used as strains, and the growth curves of the strains were determined by the smear method and the modified Coulomb method was used to calculate the median lethal dose(LD₅₀) of the strains to ducks.After culturing to a final concentration of 1×10¹⁰ CFU/mL, the two strains were mixed in equal proportions, and then the bivalent inactivated vaccine was prepared with carbomer as an adjuvant, which was used to immunize ducks after the vaccine quality inspection.The protection rate of the vaccine was evaluated by detecting the level of specific antibodies in the sera of the immunized ducks and the challenge protection test, and the histopathology of the heart, liver, spleen and brain tissues of the ducks in the challenge test were observed.【Result】 RA-G06 strain and RA-HS01 strain both reached the peak value after 12 h of culture, the numbers of viable bacteria were 2.1×10¹¹ and 3.3×10¹¹ CFU/mL, LD₅₀ were 1.44×10¹⁰and 2.63×10⁸ CFU/mL, respectively.The prepared vaccine was safe and the humoral immunity could be induced in ducks.The results of RA-G06 strain challenge protection test showed that the protection rates of the self-developed vaccine and commercial vaccine were both 90%.The results of the challenge test of RA-HS01 showed that the protection rate of the self-developed vaccine was 90%, and the protection rate of the commercial vaccine was 80%.Histopathological results showed that after immunization, each vaccine group could provide better protection to duck heart, liver, spleen and brain tissues, and the self-developed vaccine group had obvious advantages.【Conclusion】 It showed that the carbomer adjuvant inactivated vaccine prepared by using RA serotype 1 and 2 strains circulating in Guizhou had obvious immune effect, and could play an important role in the prevention and control of RA infection in Guizhou.

【Objective】 This study was aimed to obtain ASFV p37 protein and prepare polyclonal antibody against ASFV p37 protein to provide materials for structural and functional studies of ASFV p37 protein.【Method】 In this study, bioinformatics tools were used to analyze the p37 protein of ASFV HLJ/2018 (GenBank accession No.:MK333180.1), and the p37 gene was designed and synthesized, and the pET32a-p37 recombinant expression vector was constructed.The recombinant plasmid was transformed into of Escherichia coli BL21 (DE3) competent cells.After induced by IPTG, the soluble expression of the recombinant protein was analyzed by SDS-PAGE.The bacterial precipitate was denatured by 8 mol/L urea, centrifuged, filtered through a 0.45 μm membrane, then purified by nickel affinity chromatography, and their purification effect and specificity were verified by SDS-PAGE and Western blotting.The polyclonal antibody against ASFV p37 protein was prepared by immunizing mice with 50 μg of purified p37 protein per mouse.The titer of polyclonal antibody was determined by indirect ELISA. The specificity of the polyclonal antibody was detected by Western blotting and indirect immunofluorescence assay.【Result】 Bioinformatics analysis showed that the p37 protein was a stable hydrophilic protein with no transmembrane region or signal peptide.The secondary structure mainly contained alpha helix (45.28%), extended stran (15.09%) and random coil (31.54%).SDS-PAGE and Western blotting results showed that the prokaryotic expressed recombinant protein p37 was successfully expressed after induction by IPTG, with a size around 62 ku, mainly in the form of inclusion bodies, and could react with His monoclonal antibody.Indirect ELISA results showed that the titer of the prepared anti-mouse polyclonal antibody reached 1:256 000.Western blotting results showed a specific band at around 42 ku.Indirect immunofluorescence showed a specific red fluorescence, indicating that the polyclonal antibody reacted with the recombinant baculovirus-expressed p37 protein with good specificity.【Conclusion】 ASFV p37 protein was successfully expressed and purified, and a polyclonal antibody against ASFV p37 protein was prepared, indicating that the prepared recombinant protein had good immunogenicity and could generate strong immune response, which laid the foundation for the subsequent structural and functional study of ASFV p37 protein structure function, antibody and related vaccine development.

【Objective】 Polyclonal antibody of interferon-inducible protein containing triangular tetrapeptide repeat 5 (IFIT5) in chickens was prepared to explore the effect of IFIT5 on the replication of J subgroup Avian leukemia virus (ALV-J) in chicken embryonic fibroblasts (DF-1).【Method】 Using cDNA obtained by reverse transcription of RNA extracted from spleen tissue cells in chickens infected with ALV-J as template, IFIT5 gene was amplified by PCR, and the purified PCR product and linearize vector were connected using recombinant enzymes to construct recombinant plasmids pET-28a-IFIT5 and pcDNA5-flag-IFIT5 and sequece.The correctly sequenced recombinant plasmid pET-28a-IFIT5 transformed Escherichia coli BL21 competent cells for induced expression.The fusion protein His-IFIT5 was purified and immuned 6-week-old BALB/c female mice to prepare polyclonal antibody.The specificity and titer of the antibody were determined by Western blotting and indirectly ELISA, respectively.The correctly sequenced recombinant plasmid pcDNA5-flag-IFIT5 was transacted in DF-1 cells, inoculated with ALV-J after 24 h, and the effect of overexpressed IFIT5 on ALV-J replication in DF-1 cells was detected by Western blotting.【Result】 IFIT5 gene with a size of 1 440 bp was successfully amplified, the recombinant plasmids pET-28a-IFIT5 and pcDNA5-flag-IFIT5 were successfully constructed.pET-28a-IFIT5 could express the soluble protein His-IFIT5 of about 56 ku.Western blotting results showed that the prepared polyclonal antibody of mouse anti-IFIT5 protein could specifically identify overexpressed IFIT5 protein in DF-1 cells with a titer of 1:32 000.pcDNA5-flag-IFIT5 could be highly expressed in DF-1 cells, and overexpressed IFIT5 in DF-1 cells could promote the replication of ALV-J.【Conclusion】 The polyclonal antibody of mouse anti-IFIT5 protein prepared in this study had high reactivity and specificity, overexpressed IFIT5 in DF-1 cells could promote the replication of ALV-J.The results provided a reference for in-depth study of the biological function of IFIT5 protein.

Parasitic protozoa, a group of unicellular eukaryotes, are pathogens that can cause disease in both human and animal, resulting in serious risk to public health and livestock industry development.DNA helicases, an important class of DNA repair enzymes, are involved in the DNA metabolism of almost all organisms.At present, the research of protozoal DNA helicase mainly focuses on Plasmodium falciparum, the reported DNA helicases of protozoa are mostly homologs of human or yeast, however, their conserved motifs are different from those of human and yeast, and it is an important potential target for studying drugs against protozoa.In this paper, we reviewed the conservative domain and functional characteristics of classical helicases, introducing the biochemical properties of each helicase such as polarity and preferred substrate, summarizing the families of reported DNA helicases of protozoa.At present, most of the reported DNA helicases are concentrated in Plasmodium falciparum.There are 18 species of Plasmodium falciparum, 3 species of Leishmania, 2 species of Trypanosoma brucei and Encephalitozoon cuniculi, and 1 species of Toxoplasma gondii.We also presented the progress of functional studies of the more interesting DNA helicases in protozoa:The RecQ family, DEAD-box family, UvrD family and RuvB family, among which three Plasmodium-specific helicases, PfPSH1/H2/H3 in the DEAD-box family had not found analogues in the host human.Furthermore, UvrD, shows no any homology to Homo sapeins, Mus musculus and Caenorhabditis elegans, and has significant similarity with bacteria and fungi.In this study, we reviewed the basic properties and functions of protozoan DNA helicases, described the current progress of research on protozoan DNA helicases and their potential as drug targets, in order to provide new research ideas for the screening of anti-protozoal drug targets and the prevention and control of protozoal diseases.

【Objective】 The aim of this study was to obtain Escherichia coli (E.coli)strains carrying virulence genes and multi-drug resistance from diarrheic piglets, and evaluate the antibacterial activity of aqueous extract of Chinese herbal medicine against multi-drug resistant E.coli, and explore the antimicrobial mechanism.【Method】 The enterotoxin genes and drug resistance of clinical E.coli isolates were measured with PCR and Kirby-Bauer (K-B) disk diffusion method, respectively.Antibacterial activity of Chinese herbal medicine against multi-drug resistant E.coli was evaluated through broth microdilution method, and then minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of aqueous extract of Chinese herbal medicine were obtained.After aqueous extract of Granati pericarpium exposure for different time, the conductivity meter and related detection kits were used to evaluated the changes of conductivity and alkaline phosphatase (AKP) content in bacterium solution, and intrabacterial ATP content.Intrabacterial soluble proteins changes were examined by SDS-PAGE and protein content detection, and then morphological changes of E.coli were visualized using scanning electron microscopy.【Result】 The prevalence of enterotoxin genes reaches 78.57% in 14 isolated clinical E.coli strains in PCR detection.Antibiotic-susceptibility testing results showed that all strains were resistant to at least two antibiotics, and all belonged to multi-drug resistant strains.The results of Chinese herbal medicine susceptibility testing showed the better antibacterial activity of aqueous extracts of Coptidis rhizoma, Scutellariae radix and Granati pericarpium against multi-drug resistant strains, among which the MIC and MBC values of Granati pericarpium were 31.25 and 62.5 mg/mL, respectively.Aqueous extract of Granati pericarpium could inhibit growth and reproduction of multi-drug resistant and quality control strains at concentrations of 1/2MIC or MIC.Within 3 h post-stimulation of aqueous extract of Granati pericarpium, conductivity of bacterial solution was extremely significantly increased compared with control group (P<0.01).During 6 h treatment of aqueous extract of Granati pericarpium, the results of AKP and ATP detections showed that AKP content in bacterial solution in treated group was extremely significant higher than control group (P<0.01), and intrabacterial ATP content was extremely significantly lower than control group (P<0.01).The results of SDS-PAGE and protein content detection showed that intrabacterial soluble proteins were decreased with the increase of action time.The results of scanning electron microscopy showed fragmentation and lysis in aqueous extract of Granati pericarpium-treated E.coli.【Conclusion】 The present study demonstrated that aqueous extract of Granati pericarpium could significantly inhibit growth of multi-drug resistant E.coli with enterotoxin genes, which provided theoretical reference and drug development basis for clinical treatment of piglet diarrhea infected by multi-drug resistant E.coli.

【Objective】 The purpose of this study was to discover the target and mechanism of curcumol against Encephalomyocarditis virus (EMCV) using network pharmacology and molecular docking technology.【Method】 The potential targets of curcumol against EMCV were obtained through PharmMapper and GeneCards databases.Cytoscape 3.7.2 software, STRING and DAVID databases were used to construct the target protein-protein interaction (PPI) network to screen the key targets.GO function and KEGG pathway enrichment analysis were carried out for the target proteins.Network model of curcumol anti-EMCV targets pathway was constructed.The binding energy and binding mode of curcumol with target protein were analyzed by AutoDock Vina 1.1.2.【Result】 The results of network analysis showed that there were 9 potential targets of curcumol anti-EMCV.Mitogen-activated protein kinase 14 (MAPK14), signal transducer and activator of transcription 1 (STAT1), albumin (ALB) and interleukin 2 (IL2) might be the core targets of curcumol anti-EMCV.The targets participated in metabolic pathways such as C-type lectin receptor, neurotrophin and JAK-STAT signaling pathways, and their function involved regulation of cytokine production in inflammatory response, protein kinase activity and drug binding etc.Molecular docking showed that there was strong binding energy between the four core target proteins and curcumol.There was hydrophobic binding between the core target proteins and curcumol, and there was hydrogen bond binding between ALB, STAT1 and IL2 with curcumol.【Conclusion】The results of this study indicated that, curcumol might play the anti-EMCV role by acting on the key protein of MAPK14, STAT1, ALB and IL2.This study provided theoretical basis and clues for the research and development of curcumol as an anti-EMCV drug.

【Objective】 This study was carried out to explore the intervention effect of N-butanol fraction of Polygonum hydropiper flavonoids (FNB) on oxidative stress in porcine lung-cell alveolar macrophages (3D4/2 cells) infected with Pseudoabies virus (PRV) in vitro, aiming to preliminarily explore and analyze the antioxidant effect and mechanism of FNB.【Method】 Porcine kidney cells (PK15 cells) were infected in vitro with PRV from 10^-10 to 10^-1 concentrations, and TCID₅₀ was calculated.The 3D4/2 cells were divided into BC, DMSO, PRV, FNB1, FNB2 and FNB3 groups. BC group was treated with DMEM medium only.The remain groups were treated with PRV which multiplicity of infection (MOI) was 0.1, after incubation for 2 h, DMEM medium was added to PRV group. DMSO group was supplemented with 0.05% DMSO, and FNB1, FNB2 and FNB3 groups were supplemented with 12.5, 25 and 50 μg/mL FNB, respectively.After cultured in vitro for 4, 8, 12 and 24 h, supernatants and cells were collected from each group separately, and then the content of nitric oxide (NO) in the supernatants, intracellular activity of reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), xanthine oxidase (XOD), myeloperoxidase (MPO) and intracellular content of malonaldehyde (MDA) at each time were determined by kits.【Result】 The results showed that the TCID₅₀ of PRV was 10^-8.25/0.1 mL.Compared with BC group, the level of NO in the supernatant of PRV group was extremely significantly decreased at 4 and 8 h and extremely significantly increased at 12 and 24 h (P<0.01), the content of intracellular ROS was significantly reduced at 4 h (P<0.05), and significantly or extremely significantly increased at 8-24 h (P<0.05 or P<0.01).The activities of iNOS and XOD were significantly or extremely significantly increased at 8-24 h (P<0.05 or P<0.01).The activity of MPO was significantly increased at 4-24 h (P<0.05).The content of MDA was significantly decreased at 4 h and significantly increased at 12-24 h (P<0.05).Compared with PRV group, the content of NO in FNB2 group was increased significantly or extremely significantly at 4-8 h (P<0.05 or P<0.01), and significantly reduced at 12 h (P<0.05), and was extremely significantly increased at 24 h in FNB3 group (P<0.01).The level of intracellular ROS was extremely significantly increased at 4 h (P<0.01), and extremely significantly decreased at 8-24 h (P<0.01).The activity of iNOS in FNB1 group was significantly increased at 4 h (P<0.05), and were significantly or extremely significantly increased at 8-24 h in the three FNB groups (P<0.05 or P<0.01).The activities of MPO and XOD were significantly or extremely significantly reduced at 4-24 h (P<0.05 or P<0.01).The content of MDA was significantly increased at 4 h and significantly increased at 8 h in FNB2 and FNB3 groups (P<0.05).The content of MDA were significantly reduced at 24 h in FNB1 and FNB2 groups (P<0.05).【Conclusion】 FNB could regulate the level of oxidative stress by interfering the secretion level of oxidative stress-related factors, and maintain the balance between oxidation and antioxidant.The results might provide a theoretical basis for the development and application of FNB.

【Objective】 This study was aimed to isolate Bacillus with antioxidant capacity and good tolerance from the rumen of dairy cows, so as to provide theoretical basis and reference for the use of bovine probiotics as antioxidant additives.【Method】 The rumen contents of 3 adult Holstein cows were collected and identified by bacterial isolation and purification, morphological observation, physiological and biochemical detection and 16S rDNA molecular detection.The scavenging rates of superoxide free radicals and hydroxyl free radicals, acid resistance, bile salt resistance and antibiotic resistance of the screened strains were also detected.【Result】 A total of 12 strains of Bacillus were isolated from rumen fluid of dairy cows, and 5 strains of Bacillus (X-1 to X-5) were screened by specific medium and Gram staining.Five strains of Bacillus were identified by molecular identification, and X-1 was identified as Bacillus thermoamylovorans, X-2 as Bacillus pumilus, X-3 as Bacillus proteolyticus, X-4 as Bacillus subtilis, and X-5 as Bacillus safensis.The results of antioxidant capacity test showed that all strains had certain antioxidant capacity, and strain X-5 had the highest antioxidant capacity, with the scavenging rates of superoxide free radicals and hydroxyl free radicals being 73.81% and 73.54%, respectively.When pH was 2.5, the survival rates of 5 Bacillus strains were all more than 25%.When the bile salt concentration was 0.6%, the survival rates of five Bacillus strains were all more than 60%, indicating that the strains had good tolerance.The results of drug sensitivity test showed that most of the isolates were highly sensitive to ofloxacin, nitrofurantoin, erythromycin and so on.It was resistant to cefuroxime, cefotaxime, streptomycin and so on.It was moderately sensitive to several drugs such as kanamycin, ciprofloxacin and ampicillin.【Conclusion】 The isolated Bacillus from cattle in this study had antioxidant capacity, and some of the strains had good acid resistance and bile salt resistance, which provided theoretical basis and reference for the use of Bacillus from cattle as antioxidant additives.

【Objective】 The aim of this experiment was to investigate the prevalence and drug resistance of Mycoplasma gallisepticum (MG) in different areas of China, so as to provide a scientific basis for the surveillance, prevention and control of MG disease.【Method】 The prevalent strains were isolated and purified from the 43 infected air sacs from suspected MG infected chickens in poultry farms of Guangdong, Fujian and Anhui provinces.The isolates were identified by culture characteristics test, biochemical characteristics test, Giemsa staining, serological methods and then PCR sequencing analysis, pathogenicity and sensitivity to common antibiotics were tested.【Result】 3 isolates were obtained successfully, and could change the broth medium from red to yellow.They showed typical "fried egg"-like colony in the solid medium and could ferment glucose, not hydrolyze arginine, and not decompose urea, which were consistent with the culture characteristics of MG.Results of Giemsa staining and serological specific identification further confirmed the isolates to be MG. The sequence analysis of the mgc2 gene revealed that the 3 isolates were more closely related to the typical virulent strains.Animal challenge tests showed that the clinical symptoms of the 3 isolates were consistent with those of MG infection, and the incidence was 70%-90%, the 3 isolates had high pathogenicity.The results of antimicrobial susceptibility test showed that the 3 isolates were resistant to enrofloxacin, temicoxin, oxytetracycline and flufenicol, and were sensitive to tylvalosin and warnimilin.The sensitivity of the 3 isolates to spectinomycin, aureomycin, tyloxacin and tiamulin was different in different regions.The Fujian strain showed obvious resistance to spectinomycin, the Anhui strain showed resistance to tiamulin, and the Guangdong strain was sensitive to spectinomycin, aureomycin and tiamulin.【Conclusion】 3 MG isolates with high pathogenicity were isolated, and they were drug-resistant, their sensitivities to drugs were regional.In the future, it was still necessary to strengthen the drug sensitivity monitoring of MG in various regions to reduce the emergence and spread of drug resistance.

【Objective】 This study was aimed to predict the potential targets and mechanism of WU Shen venation smooth decoction in the treatment of atherosclerosis, so as to provide reference for the clinical application of WU Shen venation smooth decoction.【Method】 The anti-atherosclerosis active components and targets of WU Shen venation smooth decoction were screened through the database of Traditional Chinese Medicine System Pharmacology Analysis Platform (TCMSP), the atherosclerosis targets were obtained by GeneCards and OMIM databases and standardized by UniProt database, and the intersection targets were obtained by R language script.Cytoscape 3.9.1 software was used to establish the drug-component-target network diagram and analyze the topology properties.Upload the intersection target protein-protein interaction (PPI) relationship network was obtained from STRING database, and GO function and KEGG pathway enrichment analysis were performed in DAVID platform.AopE^-/- male mice were used for experimental verification, HE staining was used to observe the aortic lesions.The levels of monocyte chemokine 1 (MCP-1), interleukin-6 (IL6), IL1B and tumor necrosis factor-α (TNF-α) were determined by ELISA.【Result】 There were 124 active ingredients in WU Shen venation smooth decoction, 112 target genes, 4 711 atherosclerotic targets, and 98 overlapping targets.Topology analysis showed that the key components of WU Shen venation smooth decoction in the treatment of atherosclerosis were quercetin, beta sitosterol, stigmasterol and mignonette.The core targets were prostaglandin-endoperoxide synthase 1 (PTGS1), steroid receptor coactivator 2 (NOCA2), monkey serine protease 1 (PRSS1), etc.In the GO enrichment analysis, biological processes obtained 321 items (P<0.01), mainly related to the response to lipopolysaccharide, tumor necrosis factor, fatty acid metabolism, etc.;Molecular function obtained 93 items (P<0.01), mainly related to membrane rafts, membrane microdomains, synaptic membranes, etc.;Cell component obtained 45 items (P<0.01), which mainly involved DNA-binding transcription factor binding, RNA polymeraseⅡ-specific DNA-binding transcription factor binding, and nuclear receptor activity.A total of 129 signaling pathways (P<0.01) were identified by KEGG enrichment analysis, such as cancer pathway, lipid and atherosclerosis pathway, fluid shear stress and atherosclerosis pathway, etc., and most of them were related to the regulation of immune inflammation and atherosclerosis.The results of four items of blood lipid showed that, compared with model group, the levels of cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in serum of mice in the drug group were decreased significantly (P<0.05), while HDL-C were increased (P>0.05).The results of HE staining showed that the aortic plaques, lipid infiltration and the number of foam cells in the treatment group were significantly reduced.The results of ELISA showed that under the action of drugs, the inflammatory factors MCP-1, IL6, IL1B and TNF-α in aortic tissue homogenate were significantly decreased (P<0.05).【Conclusion】 WU Shen venation smooth decoction could treat atherosclerosis through multiple components, multiple targets and multiple pathways, among which the active ingredients quercetin, β-sitosterol, stigmasterol and luteolin might have inflammatory control and immune regulation effects by acting on PTGS1, acetylcholinesterase(ACHE), adenosine (AR) and other targets.

As an important part of vaccines, adjuvants have received extensive attention for their safety and their immune-enhancing effects on vaccines.There are many kinds of adjuvants for animal vaccines, including aluminum adjuvant, oil emulsion adjuvant and liposome adjuvant.Due to the limited adjuvants available and the increasing demand for vaccines, the search for safer and more effective vaccine adjuvants has become a new upsurge.Traditional Chinese medicine has the characteristics of easy access, low toxicity and strong immune activity.It has the potential to become a new vaccine adjuvant.This article reviewed the adjuvant activity of the active ingredients of traditional Chinese medicine, the immune regulatory effect and the mechanism of action after the combined vaccine, expounded on the possibility and research progress of the active ingredients of traditional Chinese medicine as adjuvants for animal vaccines, and provided a theoretical basis for the development of new adjuvants.

【Objective】 The purpose of the experiment was to explore the biological characteristics of potential microorganisms in the dead barking deer in the Jiaozi Snow Mountain Nature Reserve, Kunming, reveal whether there was a correlation between them and the death of the barking deer, and to provide data support for microbial diversity in Kunming Jiaozi Snow Mountain Nature Reserve.【Method】 Part of the muscle tissue of the dead barking deer was collected aseptically in the experiment.The bacteria were isolated and cultured.After identification by Gram staining microscopy, biochemical test and 16S rRNA gene amplification, sequence comparison was conducted and phylogenetic tree was constructed to determine the type of the isolated strain, and drug sensitivity test and animal regression test were conducted on the isolated strain.【Result】 The colony morphology of the isolated strain on TSA and blood agar medium was round, with neat edges, smooth surface and milky white color, it did not grow on MAC medium, Gram staining microscope showed purple bacilli.The biochemical test results showed that, the isolated strain could not ferment sucrose, raffinose, sorbitol, etc., phenylalanine, ornithine, lysine, V-P test, and glucose gas production were positive.The 16S rRNA gene sequence and phylogenetic tree analysis showed that the strain was 100% similar to the Carnobacterium maltaromaticum (C.maltobotulinum) strain MMF-23 (GQ304931.1) in the GenBank.Combined with morphological, biochemical features, the isolate was identified as C.maltaromaticum, named BJT86, and the 16S rRNA sequence was submitted to GenBank to obtain the accession number ON966116.1.The results of drug sensitivity test showed that the isolated strain was resistant to 6 kinds of antibiotics such as penicillin, amoxicillin and piperacillin, and sensitive to 18 kinds of antibiotics such as cefazolin, cefotaxime and cefsulbactam.Animal regression test showed that the isolated strain was not pathogenic.【Conclusion】 In this experiment, a strain of C.maltaromaticum was successfully isolated for the first time from the muscle tissue of the dead barking deer in the Jiaozi Snow Mountain Nature Reserve in Kunming, which could provide a new reference direction for food safety and basic data for the preparation of probiotics.

【Objective】 The purpose of the experiment was to study the active components and potential mechanism of hawthorn in regulating oxidative stress by using network pharmacology and molecular docking methods.【Method】 The active chemical ingredients and potential targets of hawthorn were screened by TCMSP database, and the potential targets were standardized in Uniprot database.Targets related to oxidative stress were screened out by GeneCards and OMIM databases.The overlapping targets of hawthorn and oxidative stress were gained via Venny 2.1.0.STRING database and Cytoscape 3.8.0 software were used to construct the hawthorn-main active ingredients-target network and protein-protein interaction (PPI) network to screen the core targets.AutoDocks Tools 1.5.7 and AutoDock Vina 1.1.2 were used to perform molecular docking.DAVID database was used for GO function enrichment analysis and KEGG pathway annotation.【Result】 Active ingredients of hawthorn were screened, including quercetin, kaempferol, isorhamnetin, stigmasterol, sitosterol, and ent-epicatechin.185 and 9 647 potential targets were identified respectively based on the main active ingredients of hawthorn and oxidative stress.A total of 171 overlapping targets of hawthorn and oxidative stress were screened, including AKT1, TP53, TNF, IL6, HMOX1, MPO, NQO1, et al.The results of molecular docking showed that the 6 active ingredients of hawthorn showed high binding activity with these targets, with NQO1 to stigmasterol and sitosterol, and AKT1 to quercetin, kaempferol and epicatechin being the most.GO function analysis showed that the prevention of oxidative stress by hawthorn involved 987 items, biological processes mainly included positive regulation of transcription from RNA polymeraseⅡ promoter, cytokine-mediated signaling pathway, positive regulation of transcription DNA-templated.While KEGG annotation identified several signal pathways, including TNF, IL17 and MAPK.【Conclusion】 This study revealed that hawthorn might regulate oxidative through multiple ingredients, targets and pathways, which would provide a scientific basis for analyzing the precious anti-oxidative mechanism.

【Objective】 This study was aimed to explore the environmental parameters and their distribution pattern under two ventilation modes in large-scale piggery farms of Hangzhou, and screen out the suitable ventilation modes for promotion in this region.【Method】 The fattening houses in Hangzhou with two representative ventilation modes, transverse ventilation and longitudinal ventilation, were selected as the research objects, and the thermal environmental parameters, concentrations of harmful gases at different time points in the morning, noon and evening and at three different locations of the wind inlet, the center of the piggery and the wind outlet were monitored for a week.The effects of different time points and different positions on the thermal environment parameters such as temperature, relative humidity, wind speed, and the concentrations of harmful gases ammonia (NH₃) and hydrogen sulfide (H₂S) were analyzed, and the thermal environment parameters and harmful gases concentrations under the two modes were compared and analyzed.The air precipitation method was used to collect the bacteria in the environment of fattening house, and the bacteria were cultured and statistically analyzed to compare the difference in the number of bacteria in fattening house under the two modes.【Result】 The relative humidity in fattening house under the two ventilation modes were lower than those of the national standard and were less affected by location and time points.The relative humidity of fattening house under longitudinal ventilation mode was significantly higher than that of the transverse ventilation mode (P<0.05).There was no significant difference in the average temperature between the two ventilation modes (P>0.05), and the temperatures of fattening house in the noon were significantly higher than those in the morning and evening (P<0.05).The temperatures in piggery were affected by the location under the longitudinal ventilation mode.The average wind speed of fattening house under the longitudinal ventilation mode was (1.09±0.42) m/s, which was in line with the national standard and extremely significantly higher than that of the transverse ventilation mode (P<0.01).The wind speed in piggery was affected by the location under the ventilation mode, and the time points had little effect on it.The concentrations of harmful gases in the air environment of the pigpen under the two ventilation modes were within the national standard range, but the concentrations of NH₃ and H₂S in the longitudinal ventilation mode were significantly lower than those of the transverse ventilation mode (P<0.05).There were no significant differences in H₂S and NH₃ concentrations at each time points (P>0.05).The distribution of harmful gas concentration was significantly affected by different positions in fattening house, and the concentrations of harmful gases at the wind inlet were significantly lower than those at the center of the piggery and the wind outlet(P<0.05).The total number of bacteria in the air under the longitudinal ventilation mode met the national standard and were significantly lower than those of the transverse ventilation mode (P<0.05).【Conclusion】 The overall environment of fattening house under longitudinal ventilation mode was better than that under transverse ventilation mode.The design of fattening pig house tended to promote the longitudinal ventilation mode.