【Objective】 The purpose of this study was to investigate the regulatory role and expression mechanism of TP53 apoptotic effector factor (PERP1) in follicular development.【Method】 Using ovarian tissue of laying hens as material,the full length sequence of the CDS region of PERP1 gene in laying hens was cloned,and an overexpression vector of the PERP1 gene was constructed.The relative expression of PERP1 gene,proliferation and apoptosis related genes BCL2 and c-Myc,and oxidative stress related genes SOD2 and CAT after transfection were detected using Real-time quantitative PCR.Three online bioinformatics analysis software were used to predict the core promoter region of PERP1 gene in chicken,and six promoter deletion primers were designed according to the prediction results.Using blood tissues of laying hens as materials,six promoter deletion fragments of PERP1 gene 5 '-UTR were cloned and recombinant plasmids were constructed,and the location of the promoter active region was verified by Luciferase reporter gene test.Use four transcription factor online prediction software to predict and analyze transcription factors in the promoter active region.【Result】 This study successfully obtained the full length sequence of the CDS region of PERP1 gene in chicken and constructed an overexpression vector for PERP1 gene.After overexpression of PERP1 gene, compared with control group, the anti apoptotic gene BCL2 and proliferative gene c-Myc were significantly downregulated (P＜0.01),while the relative expression of antioxidant stress genes CAT and SOD2 were not significantly different (P＞0.05).The results of bioinformatics software prediction and Luciferase report vector test showed that the core promoter of PERP1 gene was located upstream of the CDS region (―441/―71 bp).The transcription factor prediction results revealed 13 candidate transcription factors,including Sp1,ZEB1,Zic3,c-Jun,AP-2alpha,AP-1,NF-1,c/EBPalp,Adf-1,HNF-3,CACCC-bi,c-Myc,and Smad4.【Conclusion】 The PERP1 gene had the function of promoting granulosa cell apoptosis,and regulating its expression was of great significance for follicular development.

【Objective】 This study was aimed to analyze the expression pattern of a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) on PI3K/Akt pathway in ovary of twin and singleton Mongolian sheep at different stages of estrous cycle,and explore the mechanism of ADAMTS1 gene influencing reproductive traits in Mongolian sheep.【Method】 A total of 40 Mongolian sheep with twin and single births were selected for natural estrus,ewe estrus and diestrus periods were determined.Groups of 10 Mongolian sheep each in twin estrus,twin diestrus,single estrus and single diestrus groups. The granulosa cells of 2-3 years old Mongolian sheep were extracted by syringe,which were divided into control and experiment groups.No drug added to granulosa cells in control group,and 15 μmol/L LY294002 added to granulosa cells in experiment group,the cell viability were measured at 24,48 and 72 h.The relative expression of ADAMTS1,PI3K,PTEN,Akt,RPS6,Bcl-2 and BAX genes in ovary and granulosa cells of ewe were detected by Real-time quantitative PCR.The expression of ADAMTS1,PI3K and Akt proteins in ovary and granulosa cells were detected by Western blotting.【Result】 The results of Real-time quantitative PCR showed that the expression of ADAMTS1 and Bcl-2 genes in twin estrus group were significantly higher than that in other groups (P＜0.05).The expression of PI3K,Akt and PTEN genes in twin estrus and twin diestrus groups were significantly higher than that in single estrus and single diestrus groups (P＜0.05).The expression of RPS6 gene in single diestrus group was significantly lower than that in other groups (P＜0.05).Compared with estrus,the expression of BAX gene was significantly upregulated during diestrus (P<0.05).Western blotting results showed that the expression of ADAMST1 protein in twin estrus group was significantly higher than that in other groups (P＜0.05).Compared with single group,the expression of PI3K and Akt proteins in twin group were upregulated,the expression of PI3K protein in twin estrus and twin diestrus were significantly higher than that in single estrus and single diestrus groups (P＜0.05),and the expression of Akt protein in twin estrous was the highest,which were significantly higher than that in other groups (P＜0.05).Compared with control group,the granulosa cells in experiment group of Mongolian sheep had abnormal morphology and slow proliferation,and the relative expression of ADAMTS1, PI3K and Akt genes and proteins were significantly decreased (P＜0.05).【Conclusion】 The expression trends of ADAMTS1,PI3K and Akt genes and proteins were consistent and showed a positive correlation in ovary and granulosa cells of Mongolian sheep,suggested that ADAMTS1 gene played an important role in twin birth of Mongolian sheep mediated by PI3K/Akt signaling pathway.

【Objective】 This study was aimed to clone the Toll-like receptor 4 (TLR4) gene in Cygnus olor and analyze its encoded protein by bioinformatics,so as to provide an important reference basis for further exploring the immune mechanism of TLR4 protein against pathogenic microorganisms in Cygnus olor.【Method】 The sequence of TLR4 gene CDS in Cygnus olor was amplified by PCR and cloned.NCBI-BLAST and Mega-X were used for sequence comparison and phylogenetic tree construction.The structure and function of TLR4 protein and its interaction with host protein were predicted using ProtParam,SWISS-MODEL,etc.,GO functional and KEGG pathway enrichment analysis were conducted.【Result】 The sequence of TLR4 gene CDS in Cygnus olor was successfully cloned,the complete length of the gene was 2 550 bp,which was submitted to NCBI with the accession No.:OP908241.There were 79 rare codons of the TLR4 gene CDS sequence in Cygnus olor,including 6 consecutive rare codons,encoding 849 amino acids.It had high similarity with Anser anser and Anser cygnoides,and had the closest genetic relationship.The transmembrane domain of TLR4 protein was located at amino acids 647-667,and the N-terminal contained a signal peptide composed of 36 amino acids.The signal peptide cleavage site was between amino acids 36 and 37,with a confidence of 67.85%.TLR4 protein contained 10 N-glycosylation sites and 85 phosphorylation sites,which had similar tertiary structures to TLR4 protein in human,and the similarity was 45.48%.TLR4 protein had a total of 10 major interaction proteins,and 96 GO functional items and 7 KEGG pathways were enriched.GO functional entries included species interaction,innate immune response,stress response,signal transduction,etc.The KEGG pathway included influenza A,Herpes simplex virus type Ⅰ infection and Salmonella infection,etc.【Conclusion】 TLR4 gene in Cygnus olor was closely related to Anser anser and Anser cygnoides,with obvious codon species selectivity,and contained 6 consecutive rare codons.TLR4 protein had transmembrane domains and signal peptides,and was closely related to innate immune response,signal transduction and stress response.

【Objective】 This study was aimed to screen the differential microRNAs (miRNAs) in Xinji Fine wool sheep (XFWS) and Small-tailed Han sheep (SXW),and study the regulatory mode of miRNAs and target genes on the mechanism expression of wool fiber.【Method】 Two kinds of Xinji Fine wool sheep and Small-tailed Han sheep with significant differences in capillary were selected,exosomes in plasma were collected for transcriptome sequencing,libraries were constructed and miRNAs were identified,miRNAs were differentially expressed using edgeR packs,miRanda and Targetscan were used to predict miRNA target genes,GO function and KEGG pathway enrichment of target genes were performed,miRNAs-mRNA interaction regulatory network for differential miRNAs target genes were analyzed,and miRNAs in transcriptome data were validated using Real-time quantitative PCR.【Result】 There were 1 019 miRNAs in the plasma exosomes of Xinji Fine wool sheep and Small-tailed Han sheep,including 112 known miRNAs,907 newly predicted miRNAs,and 364 differentially expressed miRNAs after screening,including 62 genes positively correlated with wool fineness and 302 negatively correlated genes.After multi-database comparison,18 996 annotated target genes were obtained,and GO function enrichment analysis showed that 17 193 annotated target genes were enriched into 6 608 biological processes.KEGG pathway enrichment analysis was enriched in 276 pathways,and 5 miRNAs and 42 target genes related to the expression of wool fiber mechanism were found by protein interaction analysis.Real-time quantitative PCR verified differential miRNAs,the expression of oar-miR-218a,oar-miR-370-3p,oar-miR-133,oar-miR-29a,oar-miR-27a,oar-miR-485-3p,oar-miR-22-3p,oar-miR-23a,oar-miR-148a and oar-miR-412-3p in Xinji Fine wool sheep were significantly higher than those of Small-tailed Han sheep (P<0.05), the expression of oar-miR-26a,oar-miR-29b,oar-miR-329b-3p,oar-miR-409-3p and oar-miR-30a-3p in Small-tailed Han sheep were significantly higher than those of Xinji Fine wool sheep (P<0.05),which were consistent with transcriptome sequencing results.【Conclusion】 There were 364 differential miRNAs in Xinji Fine wool sheep and Small-tailed Han sheep,including oar-miR-218a,oar-miR-370-3p,oar-miR-133,etc.,among which differential miRNAs,oar-miR-133,oar-miR-150,oar-miR-127,oar-miR-23a,oar-miR-370-3p and their 42 target genes were involved in the mechanism expression of wool fiber,FGFR2,NOTCH1 and SHH participated in multi-channel regulation and played an important role in joint regulation.

【Objective】 The purpose of this experiment was to analyze the changes of circularRNA (circRNA) expression profile in host cells of porcine small intestinal epithelial cells (IPEC-J2) at the beginning of enterotoxigenic Escherichia coli (ETEC) infection,and explore the regulation mechanism of circRNA.【Method】 Transcriptome sequencing was performed on the ETEC infected IPEC-J2 cells using Illumina PE150 sequencing platform,and differentially expressed circRNAs were screened using bioinformatics online software.After GO function and KEGG pathway enrichment analysis,circRNAs associated with ETEC infection were screened.Coding potential of circRNAs was predicted by IRESfinder and PFAM 33.1 software.Five differentially expressed circRNAs were randomly selected,and the expression of circRNAs and the digestion of RNase R were verified by Real-time quantitative PCR.【Result】Transcriptome sequencing results showed that 201 differentially expressed circRNAs were obtained,of which 95 were up-regulated and 106 were down-regulated.GO functional enrichment analysis showed that circRNA-derived genes at the early stage of ETEC infection mainly affected cell morphology,cell metabolism and ubiquitin-protein ligase activit.KEGG pathway enrichment analysis indicated that circRNAs might be involved in amino acid metabolism pathway,ubiquitin-mediated proteolysis pathway,adhesion junction and actin cytoskeleton regulation.Four circRNAs with coding potential were identified,among which novel_circ_0009996 and source gene TNFAIP3 were co-enriched in NF-κB and TNF signaling pathways.Real-time quantitative PCR results showed that the expression of the 5 circRNAs were consistent with the sequencing results,and the differentially expressed circRNAs were all circular,and the sequencing results were accurate and reliable.【Conclusion】 circRNAs and the source genes were mainly involved in inflammatory response,post-transcriptional regulation of gene expression,cellular immunity,cytoskeleton,cell proliferation,apoptosis and other related biological processes.novel_circ_0009996 had coding potential,which was mainly concentrated in the NF-κB and TNF signaling pathways.The results provided a theoretical basis for further exploring the regulatory mechanism of circRNA in the early stage ETEC infection.

【Objective】 The CDS regions of cathepsin B (CTSB) and CTSS genes in New Zealand White rabbits were cloned and bioinformatics analysis was performed,and explore the expression regularity in different tissues of New Zealand White rabbits.【Method】 PCR was used to amplify and clone the CDS region of CTSB and CTSS genes,and Mega 7.0 software was used to construct the phylogenetic tree and analyze the amino acid similarity.Bioinformatics software was used to analyze the physical and chemical properties,cross-membrane structures,signal peptides and sub-cellular locations of CTSB and CTSS proteins.The expressions of CTSB and CTSS genes in different tissues of New Zealand White rabbits were detected by Real-time quantitative PCR.【Result】 The sequence length of CDS region of CTSB and CTSS genes in New Zealand White rabbits was 1 020 and 1 023 bp,encoding 339 and 340 amino acids,the molecular weight was 37.627 and 38.253 ku,the theoretical isoelectric point was 5.53 and 8.40,and the instability coefficient was 30.76 and 32.38,which were acidic and basic proteins respectively.The amino acid sequences of CTSB and CTSS genes of New Zealand White rabbits were closely related to pigs and horses,respectively,but were far related to chickens.The secondary structure of CTSB and CTSS proteins was mainly composed of alpha helix and random coil.The predicted tertiary structure of both proteins was consistent with the secondary structure,and both were hydrophilic proteins.CTSB signal peptide splicing sites was located at 17-18 amino acids,containing 1 N-end Propeptide-C1 domain and 1 Peptidase-C1A-C domain,while CTSS signal peptide splicing sites was located at 25―26 amino acids,which contained 1 Inhibitor-129 domain and 1 Peptidase-C1 domain.The results of subcellular localization showed that CTSB and CTSS proteins were mainly distributed in the cytoplasm (including the cell wall).Real-time quantitative PCR results showed that the expression of CTSB gene was higher in abdominal fat and liver,and the expression of CTSS gene was the highest in spleen,both of which were significantly higher than other tissues (P<0.05).【Conclusion】 The full-length CDS sequences of CTSB and CTSS genes in New Zealand White rabbits were successfully cloned,and the expression in different tissues of New Zealand White rabbits were revealed and the biological functions were initially analyzed,which providing a basis and reference for further research on the functions and regulatory mechanisms of CTSB and CTSS genes in rabbits.

Mastitis is the most common disease in dairy cows and can be caused by a number of factors.Due to the absence of obvious clinical signs during the development of subclinical mastitis status,it can result in more economic losses.In order to reduce the losses as more as possible,the appropriate approaches should be adopted to prevent subclinical mastitis.However,the traditional detection methods are limited in their ability to fully explain the mechanisms.The advances in bioinformatics have enabled the use of omics techniques to gain insight into the pathogenesis of subclinical mastitis and the resistance mechanisms of the cows.Metabolomics can reflect the nutritional status of the body,the effects of drugs and environmental pollutants,and other external factors,so it can be used to explain the pathologic changes in subclinical mastitis.Proteomics can analyze the expression of all proteins in the cells of diseased dairy cows and elucidate their properties and structural functions,providing potential biomarkers for the diagnosis of subclinical mastitis.The genome can examine the association between information like single nucleotide polymorphisms (SNPs) and trait of interest,enabling the localization of relevant genes.Transcriptomics can detect all RNA expression in a cell,tissue or organism under specific circumstances and physiological conditions.Epigenomics can alter gene expression without altering DNA sequences,thus acting as a regulator of traits.In order to understand the pathogenic mechanisms of subclinical mastitis and the resistance mechanisms of the dairy cows better,this paper reviewed the progresses of omics techniques regarding subclinical mastitis dairy cows,so as to provide references for further exploration of subclinical mastitis in the future.

【Objective】 This study was aimed to investigate the effect of astaxanthin extracted and purified from Rhodotorula mucilaginosa on the immune function of immunosuppressed mice,so as to provide theoretical basis for the development and application of astaxanthin in the prevention of immunosuppressed diseases or as a substitute feeding product for livestock and poultry.【Method】 Astaxanthin was extracted from Rhodotorula mucilaginosa ZTHY2 isolated from the coastal waters of Leizhou Peninsula.Sixty C57BL/6 mice were randomly divided into 6 groups:Blank control group,cyclophosphamide group,yeast β-glucan group and astaxanthin low,medium and high dose groups.From 1-14 days,the mice in yeast β-glucan group were gavaged with 100 mg/kg yeast β-glucan,the low,middle and high astaxanthin groups were gavaged with 30,60 and 100 mg/kg astaxanthin,respectively,and the blank control and cyclophosphamide groups were intragastrically injected with normal saline.On the 15-21 days,except for blank control group (normal saline),the mice in each group were intraperitoneally injected with 50 mg/kg cyclophosphamide.At the end of the experiment,the spleen and thymus of mice were collected,and the organ index and the proliferation of T and B lymphocytes in spleen were calculated.The histological changes of spleen and thymus were observed.The contents of interleukin 2 (IL-2),IL-4,IL-6,tumor necrosis factor α (TNF-α),interferon gamma (IFN-γ),immunoglobulin A (IgA),IgG,IgM,CD4,CD8,CD19,CD20,glutathione (GSH) and malondialdehyde (MDA) in serum,the activities of catalase (CAT) and superoxide dismutase (SOD) in serum,and the level of reactive oxygen species (ROS) in spleen were determined.【Result】 Compared with blank control group,the spleen and thymus indexes of mice in cyclophosphamide group were extremely significantly decreased (P＜0.01),the boundary between red and white pulp of spleen was unclear,the number of spleen lymphocytes decreased,the thymus cortex and medulla were unclear,and the structure was destroyed.The proliferation ability of T and B lymphocytes in spleen was extremely significantly decreased (P＜0.01).The contents of cytokines IL-2,IL-4,IL-6,TNF-α,IFN-γ,CD4,CD8,CD19,CD20 and immunoglobulin IgA,IgG and IgM in serum were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The activities of CAT and SOD and the content of GSH in serum were extremely significantly decreased(P＜0.01),while the levels of MDA and ROS were extremely significantly increased (P＜0.01).Compared with cyclophosphamide group,the structure of spleen and thymus in astaxanthin group became clearer with the increase of astaxanthin concentration,and the spleen and thymus indexes were extremely significantly increased (P＜0.01).Astaxanthin could extremely significantly promote the proliferation of spleen lymphocytes induced by concanavalin A (ConA) and lipopolysaccharide (LPS) (P＜0.01).Astaxanthin could extremely significantly or significantly increase the levels of IL-2,IL-4,IL-6,TNF-α,IFN-γ,CD4,CD8,CD19,CD20 and the contents of immunoglobulin IgA,IgG and IgM in serum (P＜0.01 or P＜0.05),and extremely significantly increase the activities of SOD and CAT and the content of GSH in serum of immunosuppressed mice (P＜0.01).At the same time,astaxanthin could extremely significantly reduce the levels of ROS and MDA (P＜0.01).100 mg/kg astaxanthin had the best effect.【Conclusion】 100 mg/kg astaxanthin could improve the immune function and antioxidant capacity of immunosuppressed mice,effectively improve the immunosuppression caused by cyclophosphamide,and could be used to prevent immunosuppressed diseases and develop as a substitute feed additive for livestock and poultry.

【Objective】 To isolate and culture adipose derived mesenchymal stem cells (AD-MSCs) from Holstein cattle in vitro, study their biological properties, and provide a new type of usable seed cells for stem cell clinical research.【Method】 The inguinal adipose tissue of 2-month-old Holstein bovine embryos was collected aseptically,and the cells were digested with type Ⅰ collagenase and cultured.Fluorescent antibody technology and RT-PCR were used to detect the specific markers (CD29,CD44,CD73,CD166,CD34 and CD45) on the cell membrane surface.The growth curves of P4,P10 and P16 generations were calculated using the purified cells and calculate population doubling time.The clone formation rate was calculated using the clone formation assay,the karyotype analysis was performed to determine whether the cells were genetically stable,and the differentiation potential to lipogenesis,osteogenesis and chondrogenesis were verified by adding induction solution to AD-MSCs culture dishes.【Result】 Cells isolated and cultured in vitro grew in an appressed swirling pattern with a long shuttle-shaped cell morphology.The results of fluorescent antibody technique and RT-PCR showed that CD29,CD44,CD73 and CD166 cell membrane surface antigens were expressed,CD34 and CD45 were not expressed,and the isolated cells were identified as AD-MSCs.The cell growth curve was plotted in S-shape,and the cell population multiplication time was 33,35 and 44 h for P4,P10 and P16 generations,respectively,and the clone formation rates were (51.67±1.53)%,(38.00±2.00)%,and (25.00±1.00)%,respectively.The two indicators showed significant differences between the three generations (P＜0.01 or P＜0.05).The karyotype results showed that AD-MSCs were normal diploids (2n=60,XX),and all chromosome morphologies were telomeric with no aberrations.Induced differentiation results showed that in vitro AD-MSCs had lipogenic,osteogenic and chondrogenic differentiation potential,with distinct lipid droplets,calcium nodules and cartilage clusters,respectively,and expressed lipogenic inducible genes lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor (PPAR-γ),osteogenic inducible genes type Ⅰ collagen (COL Ⅰ) and osteochondrogenic bridge protein (OPN),osteogenic chondrogenic gene SRY-like HMG cassette 9 (SOX-9) and chondroprotein glycan antibody (ACAN).【Conclusion】 Primary AD-MSCs could be successfully isolated in vitro using Holstein bovine embryos,and the cell morphology had typical mesenchymal stem cell morphology,with differentiation potential of lipogenic, osteogenic and chondrocytes,and the cells had fast proliferation ability and stability,which could provide germplasm resources for tissue and organ repair.

【Objective】 The purpose of this study was to explore the protective effect and mechanism of taraxasterol on oxidative damage of chicken primary hepatocytes induced by aflatoxin B₁ (AFB₁), and provide theoretical basis for the prevention and treatment of AFB₁ poisoning with taraxasterol.【Method】 Chicken primary hepatocytes were isolated by tissue block enzyme digestion and identified by PAS glycogen staining.The growth curve of hepatocytes was drawn by CCK-8 method.The toxicity of AFB₁ on hepatocytes was determined by MTT method,and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the supernatant of hepatocytes were measured to determine the modeling concentration of AFB₁ in vitro.MTT method was used to find out the safe concentration of taraxasterol.The test was divided into 6 groups: Normal group,AFB₁ group,taraxasterol groups (high,medium and low doses) and Sil group.After the establishment of the primary hepatocyte injury model induced by AFB₁ and drug administration,the contents of reactive oxygen species (ROS) and malondialdehyde (MDA),superoxide dismutase (SOD) and glutathione (GSH) in the cells were determined by reagent kits.The expression of heme oxygenase-1 (HO-1),NADPH quinone oxidoreductase 1 (NQO1),Kelch like ECH associated protein 1 (Keap1),and nuclear factor E2 related factor 2 (Nrf2) of the key genes in Keap1/Nrf2 signal pathway were determined by Real-time quantitative PCR.【Result】 The cells were successfully isolated and identified as chicken primary hepatocytes. The cell viability was higher after 24-72 h culture; 0.05 μg/mL concentration was used as the modeling concentration of AFB₁ which induced primary hepatocyte injury in vitro; 20, 10 and 5 μg/mL concentrations were used as taraxasterol administration concentrations of the high, medium and low dose groups. Compared with AFB₁ group, taraxasterol could extremely significantly reduce the contents of ROS and MDA (P＜0.01), extremely significantly increase the activity of antioxidant SOD in AFB₁-induced chicken primary hepatocytes (P＜0.01), and extremely significantly or significantly increase the expression of HO-1, NQO1 and Nrf2 (except for 5 μg/mL taraxasterol) genes (P＜0.01 or P＜0.05), and reduce the expression of Keap1 gene in chicken primary hepatocytes induced by AFB₁. 【Conclusion】 Taraxasterol could improve the antioxidant capacity of chicken primary hepatocytes by regulating the Keap1/Nrf2 signal pathway,thus protecting against the oxidative damage of chicken primary hepatocytes induced by AFB₁.

【Objective】 This experiment was conducted to investigate the effects of complex enzymes and Bacillus subtilis on growth performance,meat quality and liver function of Yellow-feathered broilers.【Method】 A total of 1 200 1-day-old fast large Yellow-feathered cocks were randomly divided into 5 groups with 6 replicates per group and 40 chickens per replicate.The birds in control group were fed basal diet,and the birds in other four groups were fed the basal diet supplemented with 20 mg/kg virginiamycin (antibiotic group),500 mg/kg complex enzymes (complex enzymes group),200 mg/kg Bacillus subtilis (Bacillus subtilis group) and 500 mg/kg complex enzymes＋200 mg/kg Bacillus subtilis (combined group),respectively.The experiment lasted for 63 days.At 21,42 and 63 days of age,broilers were weighed with fasting and feed intake was calculated to calculate growth performance.Two chickens from each replicate at 21 and 63 days of age were slaughtered.Blood and liver tissue samples were collected and biochemical parameters were determined.At 21 days of age,cecum contents were collected to determine the number of microflora.Breast muscle was taken at 63 days of age to evaluate meat quality.【Result】 Compared with the control group,there were no significant differences in the growth performance of Yellow-feathered broilers in complex enzymes group,Bacillus subtilis group and the combined group (P＞0.05),but the liver index at 21 days was significantly increased (P＜0.05),and the muscle dripping loss at 63 days of age was significantly decreased (P＜0.05) in three groups.Compared with the control group,the supplementation of complex enzymes or Bacillus subtilis alone significantly decreased Enterococcus microflora in cecum of Yellow-feathered broilers at 21 days of age (P＜0.05),significantly decreased plasma urea nitrogen (BUN) level at 63 days of age (P＜0.05),and significantly increased the activities of T-superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) in liver (P＜0.05).Compared with the control group,the plasma BUN level of Yellow-feathered broilers at 21 days of age was significantly decreased by complex enzymes supplementation alone (P＜0.05).And the supplementation of Bacillus subtilis alone significantly increased the activity of choline esterase(CHE) in liver of 21-day-old Yellow-feathered broilers (P＜0.05).Compared with antibiotic group,the combined supplementation of complex enzymes and Bacillus subtilis had no significant effect on liver index of 63-day-old Yellow-feathered broilers (P＞0.05),but significantly increased the meat yellowness value at 24 h (P＜0.05).Compared with the combined group,the supplementation of complex enzymes or Bacillus subtilis alone significantly increased GSH-Px and succinate dehydrogenase (SDH) activities in the liver of 63-day-old Yellow-feathered broilers (P＜0.05),but had no significant effect on the composition of cecal microbiota (P＞0.05).【Conclusion】 Dietary supplementation of complex enzymes or Bacillus subtilis alone could enhance the antioxidant capacity and metabolic function of liver,improve meat quality and reduce the number of harmful bacteria in cecum of Yellow-feathered broilers.However,there was no synergistic effect of the supplementation of complex enzymes and Bacillus subtilis.

【Objective】 The experiment aimed to investigate the optimal process conditions for lignin degradation of reed straw at the dead leaf stage by combined pretreatment with calcium oxide (CaO) and sodium carbonate (Na₂CO₃) and its structural changes.【Method】 The experiment was conducted to investigate the removal of lignin and hemicellulose by selecting four factors:Treatment time,treatment temperature,feed-to-liquid ratio,and CaO addition on the basis of 2% Na₂CO₃ addition in a gradient test.The Box-Behnken response surface optimization test was conducted to investigate the optimal pretreatment process for lignin degradation based on the results. Subsequently,the structural changes of reed under the optimal treatment conditions were analyzed by scanning electron microscopy (SEM),Fourier infrared spectroscopy (FTIR),and X-ray diffraction (XRD).【Result】 The lignin and hemicellulose removal rates of reed straw differed significantly (P＜0.05) at different treatment times,treatment temperatures and CaO additions,and there were no significant differences at different feed-to-liquid ratios (P＞0.05).The response results showed that the regression model was extremely significant (P＜0.01),and all three factors had extremely significant effects on lignin removal rate (P＜0.01),and the magnitude of the effects of the three factors on lignin removal rate were treatment temperature,CaO addition,and treatment time in order.The optimal pretreatment process conditions were 8.44% CaO addition,5.52 h treatment time,and 49.58 ℃ treatment temperature when Na₂CO₃ addition was 2% and material-to-liquid ratio was 1∶3.Under this pretreatment condition,the lignin removal rate of reed straw reached 23.75%,which was close to the model predicted value (24.97%). After optimization, SEM results showed that the surface of reed straw was broken and the pores were enlarged and increased;FTIR results showed that the absorption peak intensities of hemicellulose and lignin corresponding to chemical bonds of reed straw were reduced;XRD results showed that the relative crystallinity index (CrI) of cellulose of reed straw changed from 37.21% to 46.66%.【Conclusion】 The process conditions of combined pretreatment of reed straw with CaO and Na₂CO₃ by response surface methodology were reasonable and feasible.The surface morphological structure,chemical structure of lignocellulose and cellulose crystalline structure of the pretreated reed straw were significantly changed. The results could provide technical support for the high-value development and utilization of non-conventional feed resources.

【Objective】 This study was aimed at investigating the effect of complex plant essential oils on the intestinal barrier and antioxidant function of broiler chickens challenged with Clostridium perfringens (C. perfringens).【Method】 A total of 288 1-day-old healthy Ross 308 broilers were randomly divided into 4 treatment groups with 6 replicates per treatment and 12 birds each.The birds in the control and infected groups were fed the basal diet.The diet of infection＋CTA and infection＋CAT groups were supplemented with 100 mg/kg of complex plant essential oils,respectively.CTA was composed of cinnamaldehyde,thymol and anisaldehyde with a ratio of 1∶3∶1,and CAT was composed of cinnamaldehyde,anisaldehyde and thymol with a ratio of 1∶3∶1.Continuous oral irrigation 1×10⁸ CFU/mL C.perfringens (1 mL/d) was administered to broilers in the infection,infection＋CTA and infection＋CAT groups from day 14 to day 20 of the trial.The birds in the control group were fed with an equal volume of blister meat medium.The animal trial lasted for 42 days.On days 21 and 28 of the trial,the blood,duodenum,jejunum and ileum of broilers were collected.The serum diamine oxidase (DAO) activity and the plasma levels of antioxidant-related indexes were measured.Additionally,the intestinal morphology was observed,and then the mRNA levels of jejunal barrier-related genes and antioxidant-related indexes in the jejunum were investigated.【Result】 Compared with the control group,C.perfringens infection increased the serum DAO activity on days 21 and 28 (P＜0.05),and downregulated the ratio of villi height to crypt depth (VH/CD) of jejunum on day 21 and of duodenum on day 28 of the trial (P＜0.05).Compared with the infection group,the serum activity of DAO was reduced and VH of ileum was raised with CTA and CAT treated (P＜0.05).In addition,the mRNA levels of occludin,claudin-1 and mucin-5ac in the jejunum on day 21 and ZO-1 on day 28 were upregulated with CTA supplemented.The transcription levels of ZO-1,occludin,claudin-1 and FABP2 on day 28 were elevated with CAT treated (P＜0.05).Additionally,the plasma activity of GSH-Px was increased with CTA and CAT treated (P＜0.05).【Conclusion】 Dietary supplementation of compound plant essential oil could improve the intestinal barrier and antioxidant function of broilers challenged with C.perfringens.

【Objective】 Feeding with Clostridium butyricum(C. butyricum) in broilers can promote growth and improve feed conversion efficiency,but its mechanism remains unclear.Therefore,the purpose of this study was to investigate the effects of dietary supplementation with C. butyricum on the cecal mucosa in broilers using transcriptome sequencing technology and histological staining,and provide experimental basis and theoretical support for the application of C. butyricum.【Method】 120 Arbor Acres (AA) broilers were randomly divided into 2 groups with 6 replicates each,and each replicate included 10 broilers.The broilers in control group were fed the basal diet,and in test group were fed basal diet supplemented with C. butyricum (1×10⁹ CFU/kg).The trial period was 28 days,with free food and water intake and drinking.1 broiler chicken was randomly selected from each replicate and sacrificed at 28 days of age,and the cecal mucosal sample was taken.RNA-Seq was applied to detect the transcriptomic characteristics of cecum mucosa tissue,and tissue staining was applied to analyze the changes of cecum mucosa.【Results】 123 differentially expressed genes were identified,of which 70 were up-regulated and 53 were down-regulated.Gene Ontology (GO) function enrichment analysis revealed that significant or extremely significant enrichment of functional genes were related to substance transport,cell viability and antioxidant (P＜0.05 or P＜0.01).Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the genes involved in pathway related to vitamin and amino acid metabolism,O-glycan biosynthesis and apelin signaling were the most abundant.Compared with control group,the relative expression of intestinal barrier function related proteins such as Claudin 2,Claudin 15,Claudin 19,Claudin 23,TJP 1,TJP 2,TJP 3 and Mucin 1 in cecal mucosa were significantly or extremely significant increased in test group (P＜0.05 or P＜0.01).HE and PAS staining microscopy of cecal mucosa showed that the depth of intestinal gland,thickness of mucosal layer and thickness of muscle layer,as well as the number and density of goblet cells in test group were significantly higher than those in control group.【Conclusion】 Feeding C.butyricum could improve the substance transport of cecum mucosa,enhance the antioxidant capacity and mucosal barrier function in broiler,thus improving intestinal health and promoting growth performance.

【Objective】 The experiment was conducted to study the effect of different dietary nutrition levels and feeding modes on growth performance,slaughter performance and economic benefits of Shuxing 1 commercial rabbits.【Method】 360 28-day-old Shuxing 1 commercial rabbits were randomly divided into 4 groups. 5 replicates were set in each group and each replicate had 18 rabbits (half male and half female).Combination of high nutrition level and free feeding were set in group 1,high nutrition level and restricted feeding were set in group 2,low nutrition level and free feeding were set in group 3,low nutrition level and restricted feeding were set in group 4.After the 77-day-old feeding,12 rabbits from each group were randomly selected and the slaughter performance such as pre-slaughter body,all carcass weight,half carcass weight were measured.【Result】 ①The 56 and 77 d body weight,28-56 and 28-77 d daily gain under low nutrient level were significantly higher than those in high nutrient level group (P＜0.05).The diarrhea rate of 28-56 and 28-77 d were significantly lower than those in high nutrient level group (P＜0.05).The 56 and 77 d body weight,28-56 d daily gain and feed gain ratio,56-77 d feed gain ratio and 28-77 d daily gain under free feeding condition were significantly higher than those in restricted feeding group (P＜0.05).The 56-77 d daily gain,28-56 and 28-77 d diarrhea rate,28-77 d mortality rate were significantly lower than those in restricted feeding group (P＜0.05).The interaction effect of nutrition level and feeding methods had significant effects on diarrhea rate at 28-56 and 28-77 d (P＜0.05),but had no significant effects on other growth performance indexes of Shuxing 1 commercial rabbits (P＞0.05).②The pre-slaughter body weight,all carcass weight and half carcass weight of free feeding group were significantly higher than those of restricted feeding group (P＜0.05).The pre-slaughter body weight of high nutrient level was significantly lower than that of low nutrient level,and there was no significant difference in slaughter performance (P＞0.05).Feeding methods significantly affected the slaughter performance of Shuxing 1 commercial rabbits (P＜0.05),and the indexes of free feeding group were higher than those of restricted feeding group.③The different dietary nutrition levels and feeding modes had no significantly affected on the economic benefit of Shuxing 1 commercial rabbits (P＞0.05).【Conclusion】 The body weight and diarrhea rate of Shuxing 1 commercial rabbits were significantly affected by nutrition level and feeding mode.The best benefits could be obtained by free feeding with low nutrition level.

Horsemeat has the characteristics of high protein,low fat and low cholesterol,which can be used as a high-quality source of protein.At present,consumers are not familiar with horsemeat and domestic consumption is not high.Most horsemeat is mainly exported and processed in a single way,mainly smoked horsemeat from Xinjiang region.Therefore,it is of great significance to further clarify the nutritional value and efficacy of horsemeat for the development and utilization of horsemeat products,which is conducive to the transformation and development of the horse industry.The contents of moisture,ash,protein and fat of horsemeat of different breeds are different,and the chemical composition of horsemeat of different ages is different.The tenderness,juiciness,flavor and other meat qualities of horsemeat are different with different dietary nutrient levels.The color,tenderness and nutrient composition of different muscle parts are different.Adding plant additives in diets can change feed digestibility and improve meat quality.Processing techniques such as salting,electrical stimulation or enzyme treatment can effectively improve the tenderness and quality of horsemeat.The author summarized the characteristics of horsemeat quality and nutritional composition, and the effects of genetic factors,individual factors,nutritional level,slaughter,processing and storage on horsemeat quality and nutritional value, in order to provide reference for the improvement of horsemeat quality and product development.

【Objective】 To study the effects of supplemental feeding of Chinese herbal medicine feed additives and active dry yeast on the growth performance and blood index of suckling calves.【Method】 Twenty-four suckling Yanbian Yellow calves of similar weight (93.06 kg±11.20 kg) and age (3.5 months) and in good condition were selected and randomly divided into three groups of eight calves each (five male calves and three female calves) using a completely randomised trial design. The control group was fed with basic diet,the Chinese herbal group was supplemented with 0.5% Chinese herbal feed additive on top of the control group and the yeast group was supplemented with Raman active dry yeast at 1 g/d on top of the control group.The trial period was 31 days,with a pre trial period of 10 days and a formal trial period of 21 days.During the experiment,feed and fecal samples were collected,and after the experiment,blood samples were collected to measure the feed intake,digestion rate,and serum indicators of the calves.【Result】 Compared with control group,① The average daily gain (ADG) was significantly higher in both the herbal and yeast groups (P＜0.05);② The digestibility of dry matter (DM),crude protein (CP),neutral detergent fibre (NDF),acid detergent fibre (ADF) and crude fat (EE) were significantly higher in the herbal and yeast groups of calves (P＜0.05);③ Serum growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels were significantly higher in calves in the herbal and yeast groups (P＜0.05),while triiodothyronine (T₃) and thyroxine (T₄) levels were slightly higher but did not show significant differences (P＞0.05);④The serum immunoglobulin G (IgG) content was significantly higher in the herbal group (P＜0.05) and the serum immunoglobulin A (IgA) content were significantly higher in both the herbal and yeast groups (P＜0.05);⑤The serum interleukin-1β (IL-1β) and gamma interferon (IFN-γ) levels were significantly lower in the herbal and yeast groups (P＜0.05);⑥ The activity of serum catalase (CAT) were significantly higher in the herbal group (P＜0.05), and there was no significant difference in the yeast group(P＞0.05).【Conclusion】 Supplementation with 0.5% herbal feed additives or 1 g/d active dry yeast preparations could improve the growth performance and development of suckling calves to varying degrees,and could also promote calf immune and antioxidant capacity, and antibacterial and anti-inflammatory properties.

【Objective】 This experiment was conducted to study the effects of different concentrate to forage ratio of Caragana diet on growth performance,meat quality and intestinal microflora of Hu sheep.【Method】 60 Hu sheep about 2 months of age with uniform weight (15.36 kg±3.36 kg) were divided equally into three groups,there were 5 replicates per group,4 sheep per replicate,with 60％ caragana hay,20％ peanut hay and 20％ alfalfa hay as the roughage diets,and the ratios of roughage to concentrate in the three groups were 60∶40,50∶50 and 40∶60,respectively.The adaption period was 10 days,and the experiment period was 60 days. The body weight of each group was recorded every 15 days during the test period,and the slaughter test was conducted on the second day after the end of the test period.Longissimus dorsal muscle and triceps femoris samples, and jejunum contents of Hu sheep were collected for meat quality analysis and intestinal flora testing.【Result】 The results showed that there was no significant difference in body weight among the three groups at 15,30 and 45 days (P＞0.05).On the 60th day of the experiment,the average body weight and average daily gain of the 60∶40 group were significantly higher than those of the 40∶60 group (P＜0.05).The total amino acid content of triceps femoris in the 50∶50 group was the highest (P＜0.05), and flavor amino acids contents in which were higher too. There was no significant change in the content and proportion of fatty acids in the muscle of Hu sheep among the three groups (P＞0.05).The main bacterial composition of the gut microbiota phylum level in three group of Hu sheep was Firmicutes,Actinobacteria and Patescibacteria.The abundance of Firmicutes,Proteobacteia and Tenericutes decreased with the decrease of roughage (P＜0.05).At the genus level,Aeriscardovia abundance tended to increase with roughage reduction (0.05<P＜0.1).【Conclusion】 When the ratio of roughage to concentrate was 60∶40,the growth performance of Hu sheep was the highest,and the mutton quality was better at 50∶50.Feeding different ratio of roughage to concentrate affected the intestinal microbial structure to some extent.

【Objective】 The purpose of this study was to explore the association between insertion/deletion (InDel) mutation of Notch2 gene with cashmere and growth traits in Shaanbei White cashmere goats,in order to provide a theoretical basis for molecular breeding of goats.【Method】 A total of 1 469 adult female Shaanbei White cashmere goats were selected as the research objects,the ear tissue were collected to extract DNA,and their cashmere and growth traits,such as cashmere length,wool length,body length,body height were measured.The primers were designed in reference to the InDel genetic variation information of Notch2 gene in goats found in GenBank and Ensembl databases,and the 17 InDel mutations of Notch2 gene were detected by PCR amplification and direct sequencing,and the association between mutation and cashmere and growth traits in Shaanbei White cashmere goats was analyzed.【Result】 There were two polymorphism loci in 12 bp InDel(rs665021370) of intron 15 and 21 bp InDel(rs653705114) of intron 25 in Shaanbei White cashmere goats.Three genotypes of insertion/insertion type (II),heterozygous type (ID) and deletion/deletion type (DD) were present at the two InDels,which were all in low polymorphism (P＜0.25) and in Hardy-Weinberg equilibrium (P＞0.05).The association analysis results showed that rs665021370 mutation of Notch2 gene was significantly or extremely significantly correlated with body height and body length in Shaanbei White cashmere goats (P＜0.05 or P＜0.01);rs653705114 mutation was significantly or extremely significantly correlated with wool length,chest width and body height in Shaanbei White cashmere goats (P＜0.05 or P＜0.01).There was no strong linkage relationship between two InDels.【Conclusion】 rs665021370 and rs653705114 mutations of Notch2 gene had significant effect on cashmere and growth traits in Shaanbei White cashmere goats,which could be used as molecular markers for excellent trait breeding in Shaanbei White cashmere goats.

【Objective】 The purpose of this study was to investigate the effect of zinc finger BED domain containing protein 6 (ZBED6) gene knockout on skeletal muscle growth and development in adolescent mice and its molecular mechanism.【Method】 Wild type (WT) and Zbed6 gene knockout (Zbed6^-/-) C57BL/6 mice at 8 weeks old were used as experimental subjects.Blood was collected and serum testosterone content was determined by ELISA method.Skeletal muscle tissue was isolated and muscle weight and muscle ratio were recorded.Skeletal muscle fiber area and IGF2 protein expression levels were determined by histological analysis and immunohistochemical analysis.Transcriptome sequencing was performed on WT and Zbed6^-/- male and female mice by Illumina Novaseq^TM 6000 high-throughput sequencing.Combined with ZBED6 target gene data of mice,the downstream target genes that played a role in Zbed6 gene knockout mouse muscle tissue were screened.Real-time quantitative PCR was used to verify the reliability of differentially expressed genes in transcriptome sequencing.【Result】 Compared with WT mice,muscle weight and muscle ratio of Zbed6^-/- male and female mice were extremely significantly increased (P<0.01).After Zbed6 gene knockout,the mRNA expression of Zbed6 gene in skeletal muscle of female and male mice was significantly decreased (P<0.05),and the mRNA and protein expression of IGF2 were significantly or extremely significantly increased (P<0.05 or P<0.01),serum testosterone content was extremely significantly increased (P<0.01).Transcriptome sequencing showed that 38 differentially expressed genes were detected in the skeletal muscle of male mice in Zbed6^-/- group,of which 22 genes were up-regulated and 16 genes were down-regulated.49 differentially expressed genes were detected in skeletal muscle of female mice,among which 19 genes were up-regulated and 30 genes were down-regulated.A total of 10 differentially expressed genes were co-expressed in female and male mice,among which Dock3,Lhx2,Brsk2,Caskin1 and Gbe1 were ZBED6 target genes.Real-time quantitative PCR showed that the expression of the 4 differentially expressed genes was consistent with the transcriptome sequencing analysis,which confirmed the accuracy and reliability of the sequencing results.【Conclusion】 Zbed6^-/- promoted skeletal muscle growth and increased serum testosterone content in adolescent mice.Five ZBED6 target genes including Dock3, Lhx2,Brsk2,Caskin1 and Gbe1 were screened.The results provided new gene targets and research directions for further exploring the function of ZBED6,contribute to improving the functional map of ZBED6,and provided an important theoretical basis for the improvement of large animal varieties and the genetic breeding of lean meat traits of livestock and poultry.

Endometrial hyperplasia is a common uterine disease in females,which can cause various degrees of functional uterine bleeding,embryo implantation failure and postmenopausal vaginal bleeding.It has a negative impact on reproduction and has the risk to develop endometrial cancer.Steroid hormones,including estrogen,progestogen,and androgens,play important roles in endometrial hyperplasia.High concentrations of estrogen or long-term low concentration of estrogen stimulation without progesterone resistance is the main cause of endometrial hyperplasia.Androgens,similar with progesterone,has an antiestrogen effects.Pregnant mare serum gonadotropin,a reproductive hormone commonly used in fix-timed insemination,can significantly increase the number of ovulations,while cause excessive endometrial hyperplasia by promoting ovarian estradiol secretion.The role and mechanism of steroid hormones in endometrial hyperplasia remains unknown.The review introduces the types of endometrial hyperplasia,the research progress of endometrial hyperplasia in different animals,and the role and mechanism of steroid hormones on endometrial hyperplasia,the influence of other factors and genes on endometrial hyperplasia and the prevention and treatment of endometrial hyperplasia,in order to provide theoretical references for the prevention and treatment of endometrial hyperplasia in animals.

【Objective】 The purpose of this study was to explore the influence of stem cell factor (SCF) on the pigment synthesis process of sika deer.【Method】 Wild type (SCF1-9) and mutant (SCF789) expression vectors of sika deer SCF gene were constructed and transfected into HEK293 cells.Mouse melanoma cells (B16) were cultured with HEK293 cell culture medium to simulate the transport process of SCF in the body,and the melanin content and tyrosinase activity of B16 cells were detected.RT-PCR was used to detect the mRNA expression of microeye transcription factor M type (MITF-M),tyrosinase (TYR),tyrosinase-associated protein 1 (TYRP1),TYRP2,solute carry family 45 member 2 (SLC45A2),SRY box transcription factor 10 (SOX10) and cAMP response element-binding protein (CREB).The protein expression levels of MITF-M and TYR were detected by Western boltting.【Result】 Compared with control and SCF789 groups,the melanin content,tyrosinase activity,mRNA expression of MITF-M, TYR and SOX10 genes,and protein expression of TYR and MITF-M in SCF1-9 group were extremely significantly or significantly increased (P<0.01 or P<0.05).The mRNA expression of SCL45A2 gene in SCF1-9 group was significantly higher than that in SCF789 group (P<0.05),had no significant difference with control group (P>0.05).【Conclusion】 The SCF gene of sika deer regulated expression of pigment genes MITF-M,SOX10,TYR and SLC45A2 through SCF/c-kit signaling pathway to promote pigment synthesis.The results provided theoretical basis for further study on the expression regulation of pigment synthesis related genes.

【Objective】 This study was amied to evalute the genetic diversity and genetic relationship of Liangshan black sheep population in Sichuan province,in order to provide scientific basis for the protection and rational utilization of local black sheep germplasm resources.【Method】 Mitochondrial cytochrome b (Cytb) gene of Liangshan black sheep was amplified by PCR and sequenced.The sequence length and mutation site information were confirmed by BLAST comparison.Combined with 153 sequences from 22 sheep breeds at home and abroad downloaded from GenBank,mitochondrial Cytb gene sequences of sheep were analyzed using Mega 7.0 software for base composition and genetic distance analysis,and DnaSP 5.0 software was used for genetic diversity analysis.【Result】 The Cytb gene size of Liangshan black sheep population was 1 140 bp,no insertion or deletion was found,and the AT content was higher than GC content.A total of 13 variation sites were found in the Cytb gene of 60 black sheep,with 13 haplotypes,of which H2 and H3 were the dominant haplotypes.The haplotype diversity (Hd),nucleotide diversity (Pi) and mean nucleotide variance (K) of Liangshan black sheep were 0.674,0.00120 and 1.371, respectively.Phylogenetic tree analysis showed that the genetic distance between Liangshan black sheep and Wula Black sheep was the smallest,and the genetic distance between Liangshan black sheep and Ovis ammon was the largest.【Conclusion】 The genetic diversity of Cytb gene in Liangshan local black sheep was low,and the genetic diversity of Cytb gene in Yanyuan black sheep was much lower than that in Butuo and Puge black sheeps.The three black sheep groups were closely related,and it was speculated that they had two matrilineal origins.Liangshan local black sheep had the closest relationship with Wula black sheep,but had a distant relationship with Ovis ammon.