In order to investigate the expression pattern and biological function of lncRNA TCONS_00143126 in E.coli type mastitis of cows in depth.This study used cDNA from bovine mammary epithelial cells as a template,and confirmed the presence of lncRNA TCONS_00143126 using PCR cloning and sequencing techniques.Subcellular localization analysis of lncRNA was performed,and potential target miRNAs and genes were predicted.The potential mechanism of its action in bovine mastitis was explored through KEGG pathway enrichment analysis.In addition,LPS was used to induce bMECs to construct an in vitro model of bovine mastitis,and the expression of lncRNA TCONS_00143126 in LPS-induced bMECs at 6,12 and 24 h was detected by RT-qPCR.The results showed that lncRNA TCONS_00143126 was real,and its expression was significantly up-regulated in LPS-induced bMECs,and it was mainly distributed in the nucleus.The results of target gene prediction and KEGG enrichment analysis showed that lncRNA TCONS_00143126 might regulate inflammatory signaling pathways such as JAK-STAT,mTOR and MAPK by targeting miRNAs(bta-miR-133a,bta-miR-193a-5p and bta-miR-375,etc.)and target genes(IFNE,SLC2A10,MEX3B),and then play a role in the inflammation of bovine mammary epithelial cells.

Anoctamin 5(ANO5)is a multichannel membrane protein localized in the sarcoplasmic and sarcoplasmic reticulum that primarily plays a role in myosin membrane repair and phospholipid scrambling,mutations in the ANO5 gene can lead to jaw hypoplasia as well as various myopathies.It aimed to investigate the association of SNPs in the ANO5 gene with fat deposition traits in sheep.A population of 1 005 healthy and clearly genealogical Hu sheep male lambs was selected for the study,and PCR amplification and KASPar typing techniques were used to detect the locus polymorphisms of the ANO5 gene in the experimental population and analyze the associations with fat deposition traits.The expression level of ANO5 gene in different tissues was analyzed by qPCR.The results showed that sheep ANO5 gene was widely expressed in a variety of tissues in Hu sheep,and the highest expression of ANO5 gene was found in heart tissue compared with other tissues.Three genotypes of CC,CT and TT with the g.58010 C>T polymorphic locus were detected in the 10th intron of the sheep ANO5 gene.Descriptive statistics showed that the perirenal fat weight was the most different and had the highest degree of variability compared with other fat weights.Correlation analysis showed that fat deposition related traits were positively correlated with growth and feed efficiency traits,and the results of association analyses showed that the polymorphic locus was significantly associated with perirenal fat weight and its related traits in the Hu sheep.Among them,the perirenal fat weight of individuals with CC genotype was significantly lower than that of individuals with TT genotype.In conclusion,the g.58010 C>T mutation locus of the ANO5 gene can be used as a candidate molecular marker for perirenal fat deposition traits in Hu sheep.

This study aimed to compare and analyze the milk composition,serum biochemical indices and expression levels of Leptin and LF genes during lactation between Bamei pigs and Yorkshire pigs.The six purebred Bamei pigs and Yorkshire pigs with healthy body conditions were selected respectively,which also had close parity,mating and delivery periods(within three days).A total of 50 mL milk,2 mL serum and 2 mL whole blood samples were collected from Bamei pigs and Yorkshire pigs on the 1st,7th,14th,21st and 28th day after delivery,respectively.Then,the milk,serum and whole blood samples were used to detect the milk compositions,serum indexes of alkaline phosphatase(ALP),albumin(ALB),globulin(GLB),triglyceride(TG),urea nitrogen(BUN),alanine aminotransferase(ALT)and aspartate aminotransferase(AST),and the expression levels of Leptin and LF genes.The results showed that on the 1st day of lactation in Baimei pigs,in addition to milk fat(Fat),the content of casein(CS),total protein(TP),total solid matter(TS),non-fat milk solids(SNF),lactose(L),free fatty acid(FFA)and acidity in the colostrum of Bamei pigs were all extremely significantly higher than those in the colostrum of Yorkshire pigs,while,the content of CS,TP,Fat,TS,SNF and L of Bamei pigs on the 21st day after delivery were extremely significantly lower than those of Yorkshire pigs.The serum biochemical indexes of two pig breeds showed different trends during the whole lactation.The content of ALT,AST,GLB,BUN and TG of Bamei pigs were significantly higher than those of Yorkshire pigs on the 1st day after delivery,while the content of ALP and ALB were all significantly lower than those of Yorkshire pigs.There were no different levels of these two genes on the 1st day,however,from the 7th day after delivery,the expression levels of LF in the Bamei pigs were significantly different than those in the Yorkshire pigs,while Leptin gene was extremely significantly expressed in Yorkshire pigs than in Bamei pigs.In general,the milk nutrients of Bamei and Yorkshire pigs showed a decreasing trend as the extension of the lactation periods,and the expression levels of Leptin and LF genes were likely related to the milk yield and quality between the two breeds,which may need to be further verified.

The purpose of this study is to screen candidate genes related to the body weight at 8 weeks age of Kangle yellow chicken based on the omics data,and provide the theoretical foundation for molecular marker-assisted breeding of growth traits in Kangle yellow chickens.It also provides key basic data for improving molecular breeding methods for high-quality broilers and accelerating,the progress of breed selection.To detect SNPs significantly associated with body weight traits at 8 weeks age of Kangle yellow chickens,the body weight was measured from 8 to 22 weeks of age of 434 Kangle yellow chickens.Genome-wide association study(GWAS)was performed using the gene chip technology.Genes in the candidate regions with 2 Mb windows surrounding each significant SNP were found for GO and KEGG function analysis.Combined with the data of transcriptomic sequencing and published literature,key candidate genes related to the body weight at 8 weeks age of Kangle yellow chicken were screened.Two potential SNPs significantly associated with target traits were detected,located on chromosome 2(131 485 613 bp)and chromosome 4(60 413 848 bp).A total of 118 candidate genes were screened near SNP sites.Gene function annotation analysis showed the most significant enrichment of biological processes was retinoic acid metabolism.The significant enrichment of 12 KEGG pathways was found,including fatty acid degradation,tyrosine metabolism and drug metabolism-cytochrome P450.OXR1, RSPO2,EIF3E,TRHR,BMPR1B, ADH1C, MTTP, LAMTOR3, PPP3CA and PDLIM3 were preliminarily identified as key candidate genes for body weight traits at 8 weeks age of Kangle yellow chickens.Two SNPs and 10 key candidate genes were preliminarily identified related to the body weight at 8 weeks age of Kangle yellow chicken.

In order to explore the sequence characteristics of yak Akirin1 gene and understand its tissue expression characteristics in detail.Gannan yak muscle tissue cDNA was used as a template to clone the CDS sequence of Gannan yak Akirin1 gene by RT-PCR technology,and bioinformatics analysis was performed using a variety of biological online software and tools.The qPCR technique was used to detect the difference expression of Akirin1 gene in Gannan yak heart,liver,spleen,lung,kidney,muscle,fat and testis tissues.The results showed that the CDS of Akirin1 gene in Gannan yak was 579 bp,encoding 192 amino acids.The protein molecular formula encoded by the gene was C₉₆₄H₁₅₂₅N₂₇₅O₂₉₁S₈,the theoretical isoelectric point of the protein was 8.91,the instability coefficient and the total average hydrophilicity were 84.14 and -0.822,respectively,which belonged to the unstable hydrophilic protein.There were 29 potential phosphorylation sites,including 14 serines,11 threonines and 4 tyrosines.There was no signal peptide and transmembrane helix structure.Subcellular localization showed that it mainly existed in the nucleus and belonged to non-secretory protein.The high-level structure of the protein was mainly composed of 39.06% α-helix and 56.25% random coil structure.Homology analysis showed that the genetic relationship between yak and cattle was the closest,which was in line with the actual situation,indicating that the Akirin1 gene was relatively conservative in the evolution process.Real-time Fluorescence Quantitative results showed that Akirin1 gene was expressed in heart,liver,spleen,lung,kidney,muscle and adipose tissue of adult Gannan yak,and the relative expression in kidney tissue was the highest,which was significantly higher than that in other tissues,and the expression in muscle and liver tissue was relatively low.This experiment provides a theoretical basis for further study on the functional characteristics of yak Akirin1 gene.

The aim of this study was to establish yak muscle and skin fibroblast cell lines,analyze and compare the differences in morphology,growth,marker genes and proteins between two cell lines to provide materials for a better understanding of yak properties.Firstly,the skin and muscle tissue blocks were isolated from the yak fetus at 3 months of gestation,and the primary cells were cultured by tissue block method and enzymatic hydrolysis method respectively,and their growth status and morphological characteristics were observed.Following,the CCK8 method to detect the proliferation viability of skin and muscle fibroblasts,and immunofluorescence staining analysis was performed on the two cells.Finally,RT-qPCR was used to analyze the expression of fibroblast marker genes in the two kinds of cells.It was found that the enzymatic method was easier to obtain fibroblasts than the tissue method,and the viability of skin fibroblasts was significantly higher than that of muscle fibroblasts(P<0.05).The fluorescence intensity of the marker protein VIMENTIN in skin fibroblasts was significantly higher than that of muscle fibroblasts,but the histone-modified H3K27me3 was not significant between the two kinds of cells.Furthermore,VIMENTIN and S100A4 genes were significantly highly expressed in skin fibroblasts,while FAP and SMA genes were significantly high expressed in muscle fibroblasts,consisting with immunofluorescent staining results.In conclusion,it successfully established yak fetal skin and muscle fibroblast cell lines,and compared their physiological properties of two kinds of cells by using CCK8,immunofluorescence staining and RT-qPCR.

To investigate the infection of BEFV and the genetic diversity of its prevalent strain G gene in a dairy farming community in Dali Prefecture,Yunnan Province.It designed and synthesized two pairs of specific amplified G genes for 10 suspected cases in dairy cattle breeding community of Dali Prefecture,with reference to the G gene sequence of bovine ephemeral fever virus in GenBank,sequenced and completed sequence analysis.At the same time,60 serum samples were collected for antibody detection.The results showed that 6 out of 10 whole blood samples of cattle suspected of BEF were positive for BEFV nucleic acid.The G gene of four bovine ephemeral fever virus Yunnan epidemic strains was amplified and sequenced by removing the repetitive sequence.The total sequence length was 1 872 bp,encoding 623 aa.The nucleotide sequence homology among the four strains was 99.7%-99.9%.Phylogenetic analysis showed that the four strains were grouped together with Asian strains and were a small branch alone.It had the closest relationship with TH-NP0065 strain isolated from Thailand in 2017 and Chinese strain.Compared with the amino acid homology of the vaccine strain JB76H in China,seven amino acid mutation sites were found:K⁵³ → N⁵³,K⁶² → R⁶²,P¹⁸³ → S¹⁸³,G²⁶³ → E²⁶³,K⁴⁶¹ → E⁴⁶¹,K⁴⁵⁷ → R⁴⁵⁷,I⁴⁷⁵ → M⁴⁷⁵.Except the neutralizing antigen site G1,mutation sites were present in G2,G3 and G4.According to the results of antibody test kit,19 samples with antibody concentration≥52.5 ng/L critical value were found,with a positive rate of 31.67%.In conclusion,BEFV infection was present in this dairy farming community and there were multi-locus variants in the G gene of the prevalent strain.

The aim of this study was to explore the mechanism of flavor difference of Angus beef at different months of age based on metabonomics analysis.Angus longissimus dorsi muscle of 17,20 and 24 months old was tested for meat quality traits and metabolomics analysis under the same feeding and management conditions.The metabolome results showed that 69,51 and 53 positive differential metabolites and 53,46 and 58 negative differential metabolites were identified in the B17 vs B20, B17 vs B24 and B20 vs B24 groups,respectively.The expression of 166 differential metabolites was up-regulated and 164 differential metabolites were down-regulated.KEGG enrichment analysis showed that differential metabolites were mainly enriched into multiple metabolic pathways such as lipid metabolism,amino acid metabolism,carbohydrate metabolism and endocrine system,including insulin resistance,cholinergic synapse,linoleic acid metabolism,phosphopentose pathway,carbohydrate digestion and absorption,glucagon signaling and arachidonic acid metabolic pathway.Correlation analysis showed that key metabolites such as proglutathione,betaine,carnitine,choline,hypoxanthine,palmitoleic acid,citric acid and lactose were significantly correlated with phenotypic data and could be used as potential candidate metabolites for Angus beef.Our results indicate that there are significant differences in the quality and flavor of Angus beef at different months of age,and the identified differences in metabolites and metabolic pathways can provide a theoretical basis for future Angus cattle breeding.

bta-miR-6523a is one of the genes related to bovine fat deposition screened by the previous research Shi Yuangang's group through sequencing data.In order to screen the target gene of bta-miR-6523a,identify its expression rule in different tissues,and explore the possible regulatory mechanism of bta-miR-6523a on bovine fat deposition,this study used a variety of database information to predict and screen the target gene of bta-miR-6523a,and enriched and analyzed the predicted functional pathway of the target gene.Finally,the expression of bta-miR-6523a in some tissues of adult cattle and calves was detected by qRT-PCR.The results showed that the predicted target genes of bta-miR-6523a were SCD,CIDEA,LIPE,etc.These predicted target genes were significantly enriched in Ras,Rap1,actin cytoskeleton regulation,Hippo and other signaling pathways related to adipocyte generation and differentiation,cytoskeleton organization and maintenance,and metabolic pathways.qRT-PCR results showed that the expression level of bovine miR-6523a was the highest in lung and the lowest in perirenal fat.The expression of bta-miR-6523a in heart and liver was significantly higher than that in subcutaneous fat,intermuscular fat,longissimus dorsi muscle and perirenal fat,but there was no significant difference among subcutaneous fat,intermuscular fat,longissimus dorsi muscle and perirenal fat.The expression of miR-6523a in subcutaneous fat,intermuscular fat and longissimus dorsi muscle of calves was significantly different,and significantly lower than that in adult bovine tissues.The high expression of bta-miR-6523a in lung tissue and lower expression in adipose tissue suggest that the biological effects of bta-miR-6523a and its target genes may be extensive.

In order to grasp the genetic characteristics of sheep tail length trait,evaluate the genetic effect of c.C334A genotype of TBXT gene,a candidate marker related to sheep tail length variation,and provide a reference for breeding sheep short tail trait.The long-tailed Texel sheep,tailless Kazakh sheep,and hybrid F₁ and F₂ progeny populations were selected as the research objects.The genetic pattern of the number of tail vertebrae was analyzed,and the correlation between the number of tail vertebrae and tail length was analyzed.The genotype effect of TBXT c.C334A on tail length variation was evaluated in the hybrid population of Texel sheep and Kazakh sheep using SPSS 26.0 regression analysis.The results showed that the average number of caudal vertebrae,skewness and peakedness of Texel sheep were 17.87±1.30,1.18±0.58 and 1.37±1.12,respectively.The average number of caudal vertebrae,skewness and peakedness of Kazak sheep were 3.52±1.03,0.54±0.50 and 0.34±0.97,respectively.The average number of caudal vertebrae,skewness and peakedness of the F₁ population were 12.61±1.82,-0.40±0.54 and 1.52±1.08,respectively.The average number of caudal vertebrae,skewness and peakedness of the F₂ population were 11.67±2.63,-0.70±0.23 and-0.12±0.46,respectively.It showed that the inheritance of the number of sheep tail vertebrae in parents and offspring conformed to the genetic pattern of quantitative traits.The coefficient of determination R² of the number of tail vertebrae to the tail length of sheep was 0.726,indicating that the change of the number of tail vertebrae was the main influencing factor of the tail length variation.The additive effect,dominant effect and substitution effect of TBXT c.C334A on tail length variation were 2.62,0.14 and 2.57,respectively.This locus explained 43.96% of the phenotypic variation of tail length,suggesting that TBXT c.C334A is a genetic marker that affects the tail length by affecting the number of tail vertebrae.

The aim of the study was to investigate the stress changes in the intestinal tract of piglets after weaning and the effect on the expression of AA transporter carriers in the intestinal mucosa of Wuzhishan pigs.A total of 24 Wuzhishan piglets were used, which were slaughtered on 0 days of weaning,and 3,7 and 10 days after weaning, and serum, small intestinal segments and mucosa were collected. Their blood immunity indexes,small intestine morphology and intestinal mucosa AA transporter carrier-related gene expression were determined.The results showed that compared with the pre-weaning period,the intestinal villi were sparsely broken,the duodenal and ileal villi heights were reduced in the 3rd day after weaning,and the differences in the changes in the surface area of jejunal and ileal villi were significant.All the values of villi height improved after the 7th day after weaning; the content of serum total protein,albumin,globulin and glucose decreased significantly after weaning,and the differences in aspartate aminotransferase,total cholesterol,lactate dehydrogenase and aspartate aminotransferase content were significant; the changes in serum diamine oxidase,D-lactic acid and lipopolysaccharide were significant after weaning; the relative expression abundance of the small peptide transporter PepT1 increased in the order of duodenum,jejunum and ileum after weaning,the relative expression abundance of the acidic AA transporter EAAT3 was not significant in the intestine,the relative expression of the neutral AA transporters B⁰AT1,ASCT2,and rBAT was higher in the jejunum as well as in the ileum,and the basic AA transporters CAT1,y+LAT1 and 4F2hc,had reduced expression in the duodenum after weaning,and the vectors were relatively more stable in ileal expression.In summary,early weaning stress caused obvious intestinal changes such as shortening of villi and crypt hyperplasia in Wuzhishan piglets,and decreased the content of blood immunoproteins and diamine oxidase,D-lactic acid and lipopolysaccharides,etc.At the same time,the expression of intestinal AA transporter carriers was changed,especially in the 3rd day of the post-weaning period,and all the indexes changed significantly,but with the growth of weaning day old up to the 10th day of the weaning period,it gradually recovered,and the piglets' immunity increased,and the permeability of the intestinal mucosa increased.

In order to investigate the regulatory level of PLA2G4A gene on inosine monophosphate(IMP)in the leg muscle tissue of Bashang Long-tailed chicken,and further explore the biological function of PLA2G4A gene in Bashang Long-tailed chicken.The leg muscle tissue of 270-day-old Bashang Long-tailed chicken was categorized based on the inosine monophosphate content,with five samples allocated to each group.Transcriptome sequencing analysis was conducted on the muscle tissue,followed by KEGG enrichment analysis,GO function annotation and protein interaction network analysis.RT-qPCR was used to analyze the mRNA expression level gene in the leg muscle tissue of Bashang Long-tailed chicken.KEGG enrichment analysis showed that the signal pathways related to IMP synthesis included glycerophospholipid metabolism,etherlipid metabolism and arachidonic acid metabolism.GO functional annotation showed that the PLA2G4A gene was involved in 11 biological processes,including glycerophosphate metabolism,etherlipid metabolism,arachidonic acid metabolism and linoleic acid metabolism.Protein interaction revealed that the PLA2G4A gene shared important regulatory enzyme functions in arachidonic acid metabolism and glycerophosphate metabolism.The Real-time Fluorescence Quantitative PCR results showed that there was a significant difference in the expression level of the PLA2G4A gene in the leg muscle tissue of Bashang Long-tailed chicken in the sample.In summary,the PLA2G4A gene may be the main gene affecting IMP deposition in chicken.

The transcriptional regulation of spleen tissue at different time points after Salmonella infection in Muscovy ducks was analyzed by transcriptome sequencing technology,to investigate the impact of Salmonella infection on Muscovy ducks.Fourteen-day-old Muscovy ducks were infected with Salmonella enteritidis.After 1,3,and 10 days of infection,spleen tissue samples were collected from the experimental and control groups,and RNA was extracted.The Illumina sequencing platform was used for transcriptome sequencing,and changes in transcriptome,signaling pathways,and functions at different time points were identified through bioinformatics analysis.The results showed that 95,53,and 12 differential genes(P<0.05,| log₂(fold change)|>1)were found in Muscovy ducks infected with Salmonella for 1,3 and 10 days,respectively.GO analysis showed that these differential genes were enriched in biological processes,molecular functions,and cellular components at the three time points.KEGG analysis showed that at 1 day of infection,the most differentially expressed genes of CXCR4,CCL3,GDF9 and CNTFR were enriched in the cytokine-cytokine interaction signaling pathway.During the third day of infection,the differentially expressed genes of ADRA1A,ADRA1b,GRIA1,and MC5R were enriched in the neural active ligand receptor interaction signaling pathway;after 10 days of infection,the differentially expressed gene H2-EB1 participates in multiple signaling pathways,indicating that these signaling pathways and differentially expressed genes may play an important role in the infection process of Salmonella in Muscovy ducks.

In order to effectively control the nitrogen loss during the livestock manure composting process,a reactor was used for a simulated composting test.Fresh cattle manure as raw material,and maize straw as assist material,to investigate the effects of different clay minerals and chemical substances compound additives on nitrogen loss,nitrogen morphological transformation law and physicochemical properties during the cow manure composting process.The results showed that the compound additives were no impact on the decay process of the material.The index of seed germination rate and pH meets the standard requirements of organic fertilizer(NY525-2002).Adding clay minerals of vermiculite or bentonite,total emission of ammonia decreased by 21.83% and 14.22% than CK,respectively.While,the combination of clay mineral+acid additive could further reduce the ammonia emission.Vermiculite+calcium perphosphate(ZK+CaP)was the best treatment,the second was vermiculite+calcium dihydrogen phosphate combination,total emission of ammonia significantly decreased by 28.47% and 21.18% than single vermiculite treatment.Results of the correlation analysis showed linear correlations were found between pH and total nitrogen content(y=52.97x-351.77,r=0.83),in the pH range of 7.0-8.0.The total ammonia emission was reduced by 5.30 g/kg,because the pH decreased by 0.1 units in a certain range.Total nitrogen content increased by 6-14 percentage points compared with CK.So clay minerals were combined with calcium dihydrogen phosphate,wood acetate and calcium perphosphate,which could reduce the pH effectively,further reduce the ammonia emissions,increase the nitrogen content,and realize the effect of nitrogen preservation and fertilizer increase in the process of cow manure composting,and the combination of vermiculite+calcium perphosphate was the best treatment.

Investigating the predominant genotypes of Bovine viral diarrhea virus(BVDV)infecting cattle in Tongliao,Inner Mongolia,to provide reference for the BVDV epidemiology and prevention and control.In the preliminary phase of the experiment,fecal samples from diarrheic calves were collected at a cattle farm in Tongliao,Inner Mongolia.These samples were tested using PCR to detect BVDV positivity.Positive fecal samples were then inoculated into madin-darby bovine kidney cells(MDBK)for isolation.The isolated strains were identified using RT-PCR and indirect immunofluorescence staining.Subsequently,the full-length genome of the isolates was sequenced,followed by genetic evolution analysis and genotype determination based on sequences of the 5'UTR,N^pro,and E2 genes.The results indicated that this experiment successfully isolated a strain of BVDV,designated as NM-21.Inoculation of NM-21 into MDBK did not induce cytopathic effects,indicating it was a non-cytopathic strain(NCP).Both RT-PCR and indirect immunofluorescence staining confirmed its positivity,with a virus titer of 10^-3 TCID₅₀/mL.Based on the full-length genomic sequence,and homology and genetic evolution analysis of the 5'UTR,N^pro,and E2 gene sequences,the isolate NM-21 showed the highest nucleotide homology with the BVDV-1c subtype strain NM2103(GenBank accession number ON337882.1)from Inner Mongolia,China.

To study the structure and function of phosphotyrosine interaction domain 1 (PID1) gene in yak,and to explore its expression in various tissues.The CDS region of Sangsang yak PID1 gene was cloned using Sangsang yak adipose tissue cDNA as template,and the sequence was analyzed bioinformatically.Meanwhile,the relative expression level of PID1 gene was detected in seven tissues of yak namely heart,liver,spleen,lung,kidney,longissimus dorsi muscle and adipose tissue by Real-time Fluorescence Quantitative PCR(RT-qPCR).The results showed that the PID1 gene in yaks had a coding region length of 654 bp,which encoded 217 amino acids.Homology comparison showed that yak and wild yak were closely related,and the similarity reached 100%.The molecular weight of yak PID1 protein was about 24.84 ku and the theoretical isoelectric point was 6.30.According to the calculation results of instability coefficient,the instability of the protein was high (47.96),and it belonged to an unstable protein.The protein had one N-glycosylation site and 23 phosphorylation sites,with no signal peptide or transmembrane structure.The results of RT-qPCR showed that the expression of PID1 gene could be detected in all tissues,with the highest expression in the lung.The yak PID1 gene was cloned and its protein structure was analyzed.The expression of PID1 gene in yak tissue was also studied.Further study on the role of PID1 gene in yak fat deposition provided preliminary data.

The highly contagious gastrointestinal infectious disease caused by Porcine epidemic diarrhea virus(PEDV)has led to significant economic losses in China's pig industry.It aimed to establish a basis for PEDV antibody detection methods and functional research of the N protein through the screening of specific nanobodies(Nbs)using phage display technology.Peripheral blood lymphocytes were isolated from a camel immunized with the N protein,and total RNA was extracted and reverse transcribed into cDNA.The variable domain of the heavy chain of heavy chain antibodies(VHH)was amplified by PCR,subcloned into the pCANTAB5E-ccdb vector,and electroporated into ER2738 competent cells to construct the VHH phage antibody display library.Subsequently,the library was subjected to four rounds of panning against the PEDV N protein,and positive phage clones were cloned into the pET-30a vector.The binding affinity and specificity of the Nbs were determined by indirect ELISA and Western Blot.The results showed that after the fifth immunization,the antibody titer reached 1∶25 600.The constructed phage display library had a capacity of 4.72×10⁸ and an abundance of 4.3×10¹⁰ cfu/mL,with a 93.75% positive rate.After four rounds of screening,16 Nb clones with different amino acid sequences were obtained,and Nb45 was validated to possess excellent specificity and binding ability to the PEDV N protein.This study successfully screened and obtained specific N protein-targeting Nbs,providing biological materials for the establishment of PEDV detection methods and foundational research.

The aim of this study is to analyze the structure and biological function of ORF2 protein of Avian hepatitis E virus (aHEV)CaHEV-GDSZ01 strain,and the recombinant ORF2 protein was expressed in E.coli prokaryotic expression system and polyclonal antibodies against ORF2 protein were prepared to verify the immunogenicity and reactivity of the recombinant protein.The physicochemical properties,structure and function of ORF2 proteins of CaHEV-GDSZ01 strain were analyzed by bioinformatics software.The prokaryotic expression plasmids of pET-32a-ORF2-His and pET-32a-ORF2 were constructed by digesting and ligating pET-32a(+)vector with ORF2 gene of CaHEV GDSZ01 strain,and then transformed into DE3 competent cells for induction and expression,and the expression form was analyzed.The recombinant ORF2 protein was purified by gel cutting,its reactivity was verified by Western Blot test,and then rabbits were immunized to obtain rabbit anti-ORF2 protein polyclonal antibody.The polyclonal antibody was identified and analyzed by Western Blot,and the antibody titer was detected by indirect ELISA.The results of bioinformatics analysis showed that the ORF2 protein of CaHEV-GDSZ01 strain was hydrophilic and unstable,and the probability of the existence of signal peptide was as high as 93.59%.The secondary structure of the protein was mainly irregular curl and had the structural conditions for binding antibodies.And the overall prediction score of protective antigen of ORF2 protein showed that it had good immunogenicity.ORF2 gene prokaryotic expression plasmids was successfully constructed,and the target protein was successfully expressed.SDS-PAGE showed that the protein was expressed as insoluble protein,and the protein was proved to have good reactivity by Western Blot.The titer of the prepared polyclonal antibody was 1∶102 400,and it could specifically recognize immunogen ORF2 protein,non-immunogen ORF2-His protein and truncated expressed sORF2 protein.The physicochemical properties and some biological functions of ORF2 protein was compared and analyzed,the related proteins were expressed successfully,and the rabbit polyclonal antibody against ORF2 protein was prepared.

In order to analyze the possible role of dTLR2 in duck innate immunity,we study the expression of dTLR2 mRNA in immune organs during 1 to 10 weeks,as well as the expression in blood after challenging with Escherichia coli and Salmonella enteritis by Fluorescence Quantitative PCR.We also prepared polyclonal antibodies of the extracellular domain of dTLR2 by recombinant protein expression for further studying the biological function of dTLR2.The results showed that dTLR2 mRNA was expressed in spleen,thymus and bursa at different weeks.The expression of dTLR2 mRNA was significantly different among different weeks in the spleen and thymus tissues,and there was no significant difference in bursa.After infection with E.coli and S.enteritis,the expression of dTLR2 mRNA was significantly higher than that of the control group in the first and second days,but there was no significant difference among groups in the third day.Analyzing the sequence of dTLR2 gene,we predicted the extracellular domain,designed primers for PCR assay,and built pET28a-TLR2 recombinant plasmid.The recombinant plasmid was translated into E.coli BL21 (DE3) for expression of dTLR2-His protein.The SDS-PAGE results showed that the fusion recombinant protein efficiently expressed in the supernatant,molecular mass was around 29 ku.The results of Western Blot showed that the antibody which harvested from immunizing rabbits could detect recombinant dTLR2 and endogenous dTLR2.The analysis of the expression of dTLR2 and successful preparation of polyclonal antibody provide favorable support for further studying of dTLR2 biology function.

This study aimed to detect the SNPs of CDKN2A gene in Wenshang Barred chicken,and perform the analysis of its bioinformatics and expressions in skin tissues.Based on the high quality chromosome-level genome of Wenshang Barred chicken assembled by the research group of Resource Conservation and Evaluation in Jiangsu Institute of Poultry Science,the sequences were blasted to chicken reference genome (GRCg7b) and the SNPs were acquired.These SNPs were detected and genotyped by MassARRAY nucleic acid mass spectrometry in the chicken population.Bioinformatics software was used to predict the regulatory sequence of CDKN2A gene.The physicochemical properties and structural characteristics of the encoded protein were analyzed.The expressions of this gene in the skin tissues of Wenshang Barred chicken at different periods were detected by transcriptome and Real-Time Fluorescence Quantitative PCR.The results showed that the total length of CDKN2A and its CDS were respectively 9.854 kb and 183 bp,encoding 60 amino acids.Forty-one SNPs were found in this gene and its upstream and downstream 1 kb,of which four SNPs were newly discovered loci.These four new SNPs were located in the downstream and the intron,respectively.The first exon had two SNPs,both of which were missense mutations:V9D and C10R.The detection rates of 34 SNPs among the 41 SNPs were all over 91% in the Wenshang Barred chicken population.These SNPs were almost homozygous mutants without polymorphism.Eight promoters and five CpG islands were predicted in the 2 kb upstream of CDKN2A in Wenshang Barred chicken.The promoter region contained a total of 12 transcription factor binding sites,including Sp1,MEB-1,YY1,C/EBPα,AP-2α.The protein encoded by this gene was a positively charged,unstable hydrophilic protein with a molecular mass of 7.30 ku and a theoretical isoelectric point of 12.82.The predictive subcellular localization of the protein was mainly in mitochondria.The protein had no signal peptide regions and signal peptide shear sites.It had eight potential phosphorylation sites and no glycosylation sites.The protein contained a typical TRP_2 (transient receptor potential ion channelⅡ) conserved domain and its secondary and tertiary structures were mainly α-helices and random coil.In addition,it had interactions with MDM2,NPM1,USP36 proteins.The expressions of CDKN2A gene in the back skins increased from 1 to 35 days old in Wenshang Barred chicken.

The aim of this study was to investigate the correlation of single nucleotide polymorphisms of the carboxyl ester lipase gene (CEL) with eye muscle area in Hu sheep.The 1 049 Hu sheep with accurate phenotypic records and in good health were selected,their eye muscle area was determined after slaughter,and blood was collected for genomic DNA extraction.Amplification of target fragments and genotyping of CEL gene by PCR and KASPar SNP typing,and association analysis with eye muscle area of Hu sheep,RT-qPCR was used to detect the expression of CEL gene in 10 tissues of Hu sheep.The results showed that the Hu sheep CEL gene was expressed in different tissues and was highest in the duodenum.There was a synonymous mutation of g.4039718 C>T in this gene,which was moderately polymorphic.Trait association analysis showed that this polymorphic locus was significantly associated with eye muscle area in Hu sheep,with TT-type individuals having significantly higher eye muscle area than CC-type individuals.Descriptive statistics showed that the coefficient of variation for eye muscle area was 12.29%,which had high selection potential.Correlation analysis showed that eye muscle area was significantly and positively correlated with growth and slaughter traits such as body weight,body height,breast circumference,slaughter rate,carcass weight and live weight before slaughter.The results indicated that the g.4039718 C>T polymorphic locus could be used as a candidate molecular marker for genetic improvement of eye muscle area traits in Hu sheep.

To investigate the regulatory effect of DEAD-box helicase 21(DDX21)on the replication of Transmissible gastroenteritis virus(TGEV).Firstly,Western Blot was utilized to analyze the effect of TGEV infection on DDX21 expression.Furthermore,we constructed eukaryotic expression plasmids of porcine DDX21 and established knockdown stable cell lines.RT-qPCR,Western Blot,Indirect immunofluorescence(IFA)and TCID₅₀ assays were used to investigate the regulatory effect of DDX21 on TGEV replication in vitro.Western Blot analysis showed that the protein levels of DDX21 were significantly up-regulated in PK-15 cells at the early stage of TGEV infection.RT-qPCR,Western Blot,IFA and TCID₅₀ experiments showed that over-expression of DDX21 significantly increased the mRNA level and protein of TGEV N in a dose-dependent manner.And the amino acids 601—784 aa of DDX21 were critical for promoting TGEV replication.Otherwise,the titer of TGEV was significantly down-regulated in DDX21 knockdown cell lines,whereas the titer of TGEV in DDX21 knockdown cell lines was reversed under the rescue experiment.This study revealed for the first time that DDX21 promotes the proliferation of TGEV and identified the key domain of DDX21 in regulating TGEV replication,which provided a theoretical a basis for future research on the function of DDX21 protein and the pathogenesis of TGEV.

Epidermal growth factor receptor(EGFR)is excessively expressed in numerous solid tumours,establishing a stable over-expressing canine EGFR protein MDCK cell line will prove beneficial in providing an exceptional cellular model and research foundation for the study of canine or human tumour diseases.Firstly,the target fragment was prepared for construction of an overexpression vector of the EGFR gene.It was co-transfected with a lentivirus packaging vector into 293T cells.After 48 hours of transfection,the supernatant was extracted and purified to obtain a lentivirus containing the overexpressed EGFR gene.This virus was then transduced into MDCK cells,following puromycin selection and single-cell cloning.The infectivity efficiency was observed,expressing gene and protein levels were detected through RT-qPCR,Western Blot,and indirect immunofluorescence assays,and an EGFR protein overexpression MDCK cell line was established.Using flow cytometry,apoptosis and cell cycle of overexpression and control cell lines were separately determined.The canine EGFR gene was located on chromosome 18 and consisted of 30 exons encoding a transmembrane glycoprotein.Through the reaction system,PCR products were ligated into linearized expression vectors,generating 8 positive transformants with a size of 738 bp.The obtained lentivirus titer of EGFR gene overexpression was 3.5×10⁸ TU/mL.Transfected MDCK cells exhibited superior fluorescent effect was remarkable under fluorescent microscopy,resulting in a significant increase in the expression level of the EGFR gene and its protein within the cells.Indirect immunofluorescence assay revealed a substantial enhancement in the expression of EGFR in cells.In contrast,early apoptosis and late apoptosis rates among the transfected cells increased notably,with a clear arrest occurring at the G2/M phase.This research successfully developed a stable canine EGFR overexpressing MDCK cell line.Overexpression of canine EGFR stimulated cell division,elevated DNA replication,and concurrently triggered programmed cell death.

The aim of this study was to investigate the effect of Ezrin on the maturation of mouse oocytes,fertilization,and early embryo development,as well as to reveal its mechanisms.Mouse germinal vesicle(GV)stage oocytes and pronuclear-stage embryos were injected with siRNA targeting Ezrin(si-Ez)or a negative control siRNA(si-NC),then the rates of oocyte maturation,fertilization,and early embryo development were calculated,the length and density of microvilli on the surface of the MⅡ stage oocytes were detected with scanning electron microscopy,the spindle localization and cortical granule migration were observed using immunofluorescence staining.The results showed that knocking down Ezrin during the GV stage significantly reduced the maturation rate of mouse oocytes and extremely significantly lowered the developmental rate of early-stage embryos after fertilization.While it decreased the fertilization rate of mature oocytes,the difference was not significant.Knocking down Ezrin during the pronuclear stage extremely significantly reduced the total cell number of mouse blastocysts.Although there was a downward trend in the rates of morula and blastocyst survival,there was no significant difference compared to the control group.Scanning electron microscopy revealed that knocking down Ezrin in GV stage mouse oocytes significantly decreased the length and density of microvilli on the surface of MⅡ stage oocytes.Immunofluorescence results showed that knocking down Ezrin in GV stage mouse oocytes affected the migration of the spindle and cortical granules.These results suggest that Ezrin,through its influence on microvilli formation,spindle localization,and cortical granule migration,blocks the maturation of mouse oocytes,consequently affecting fertilization and early embryo development.

In order to explore the expression of uncoupling protein 3(UCP3)in different tissues of Min pig and its effect on the expression of mitochondrial-related genes and ATP synthase-related genes in Min pig preadipocytes,the back adipose tissue of 1-month-old Min pig was collected as the research material.The preadipocytes were isolated by enzymatic digestion and cultured in vitro.At the same time,axillary fat,chest fat,back fat,inguinal fat,perirenal fat and intramuscular fat were collected to construct tissue expression profiles.Real-time Quantitative PCR was used to detect the expression of MCU family genes(MCU,MICU1,MICU2),ATP synthase(ATP5B,ATP5E)and 1,4,5-trisphosphate inositol receptor type 1(ITPR1)genes and the expression of UCP3 gene in different adipose tissues by in vitro transfection of UCP3 gene overexpression vector or interference fragment.The results showed that the enzymatic digestion method could quickly obtain sufficient preadipocytes in the back adipose tissue.The cell edge had good refractive index,clear boundary,and similar morphology to fibroblasts.UCP3 gene was expressed in different adipose tissues,with the highest expression in dorsal adipose tissue and the lowest expression in abdominal fat.After overexpression of UCP3 gene,the expression levels of MCU family genes and ITPR1 genes were significantly increased,and the expression level of ATP5B gene was significantly decreased.After UCP3 gene interference,the expression levels of MCU family genes and ITPR1 genes were significantly decreased,and the expression levels of ATP synthase genes were significantly increased.The results showed that UCP3 gene was differentially expressed in different adipose tissues,which could promote the expression of MCU family genes and ITPR1 genes and inhibit the expression of ATP5B gene.

In order to investigate the sequence characteristics of bovine Bta-miR-494 and its tissue expression,bioinformatics methods were used to analyze the characteristics of CpG islands,promoters and transcription factors of its upstream sequence and their conservatism among different species;then predicted and functionally enriched the target genes of bovine Bta-miR-494;and finally detected its spatial and temporal expression characteristics among different tissues and the same tissue at different periods of time of the bovine Bta-miR-494 by qRT-PCR method.The results showed that Bta-miR-494 was highly conserved among different species and had the closest affinity to goat,with no CpG island upstream and two transcriptional start sites;the target gene enrichment analysis showed that the target genes of Bta-miR-494 were significantly enriched in the signaling pathways related to muscle growth and metabolism,such as PI3K-Akt,p53 and MAPK; the predictive target gene enrichment analysis showed that the target genes of Bta-miR-494 were significantly enriched in the signaling pathways related to muscle growth and metabolism.qRT-PCR results showed that the expression of Bta-miR-494 was highest in the liver and lowest in the testis of adult cows;and the expression of Bta-miR-494 was significantly higher in the biceps femoris of adult cows than that of newborn calves,whereas the expression of Bta-miR-494 in the cardiac muscle and longitudinal muscle of dorsal cows was significantly lower than that of newborn calves,while the expression of Bta-miR-494 in the cardiac muscle and the longissimus dorsi muscle was significantly lower than that of newborn calves.This inter-tissue differential expression suggests that Bta-miR-494 may dominate different myogenic expression patterns during the development of muscle tissues at different growth stages in cattle.

The aim of this study was to express the ORF3 protein of Avian hepatitis E virus (aHEV)and prepare its polyclonal antibodies,which can provide biomaterials for further study on the biological function of new vaccine of aHEV ORF3 protein.The ORF3 gene of CaHEV-GDSZ01 strain was amplified by RT-PCR,ligated with pET-32a(+)expression vector to construct recombinant prokaryotic expression plasmid,and transformed into BL21(DE3)competent cells to induce expression.The expression of ORF3 protein was analyzed by SDS-PAGE gel electrophoresis and immunization of rabbits to obtain rabbit anti-ORF3 polyclonal antibody.The titer of polyclonal antibody was determined by indirect ELISA,and its specificity was detected by Western Blot and IFA.The pET-32a-ORF3 recombinant prokaryotic expression plasmid was successfully constructed,and the 27 ku insoluble recombinant protein of aHEV ORF3 was successfully expressed by prokaryotic expression system.The results of Western Blot and IFA showed that the prepared polyclonal antibody could specifically recognize ORF3 protein,and the ELISA results showed that the titer of polyclonal antibody was 1∶51 200.In this study,the ORF3 protein of CaHEV-GDSZ01 strain was successfully expressed and its polyclonal antibody was prepared.

Luchuan pig myoblast determining gene 1(MyoD1)was cloned,bioinformatics analysis was performed,and its expression was analyzed in different tissues of Luchuan pigs,to explore the role of MyoD1 gene in the growth and development of skeletal muscle.Published MyoD1 gene of wild boar sequence on NCBI as the template,designed specific primer,cloned the CDS region of MyoD1,and compared the similarity with wild boar,cattle,sheep,human,mouse and rat gene sequence,constructed system tree for bioinformatics analysis by online software.Then,the relative expression of MyoD1 gene in different tissues of Luchuan pigs by RT-PCR.The CDS of MyoD1 gene in Luchuan pig was 960 bp,encoding 319 amino acids,five base mutations.The similarities of MyoD1 genes between Luchuan pig and wild boar,cattle,sheep,human,mouse and rats were 99.2%,93.0%,92.3%,90.0%,84.3% and 84.0%,respectively.Total number of MyoD1 gene atoms in Luchuan pigs was 4 680,molecular mass of the protein was 33.99 ku,molecular formula was C₁₄₅₇H₂₂₉₆N₄₄₂O₄₇₁S₁₄;the instability coefficient was 63.87,indicating a lack of stability;isoelectric point was 5.63,which belongs to the acidic protein;no transmembrane structure existed,found 35 phosphorylation sites and 1 glycosylation site;protein secondary structure was dominated by an irregular coil,which accounting for 60.69%.The MyoD1 gene in Luchuan pigs had the highest expression in the longest dorsal muscle,which was significantly higher than in other tissues,while kidney had the lowest expression.MyoD1 gene was expressed in all tissues of Luchuan pigs and mainly in the longest dorsal muscle,we speculated that MyoD1 gene might play a crucial role in the muscle growth and development of Luchuan pigs.

This study aims to obtain the MFSD4A gene sequence in Maiwa yak and elucidate its expression patterns in different tissues,analyze the mRNA expression levels of the interacting proteins of MFSD4A,and determine the subcellular localization of the MFSD4A protein,and provide a theoretical basis for further research on regulating MFSD4A in the growth and development of yak testis.RT-PCR was used to obtain the MFSD4A gene CDS sequence in Maiwa yak and perform bioinformatics analysis.RT-qPCR was used to detect the expression differences of the MFSD4A gene in 10 tissues,including heart,liver,spleen,lung,kidney,gluteal muscle,back muscle,fat,testis,and lymph,and the correlation between the interacting proteins and MFSD4A.pEGFP-MFSD4A fusion plasmid was transfected into Madin-Darby bovine kidney cells(MDBK)to investigate the subcellular localization of the MFSD4A protein.The results indicated that the entire length of the MFSD4A gene CDS region in the macaque yak was 1 530 bp,encoding 509 amino acids with a theoretical isoelectric point(PI)of 8.66.It had 12 transmembrane domains and belonged to the alkaline transmembrane protein.RT-qPCR results showed differential expression of MFSD4A mRNA in various tissues of the macaque yak,with the highest expression in the testis.It was also highly positively correlated with the expression levels of MFSD9,CBY1,INHBA,UNC93A,SVOP,and ID1 in the testis,suggesting that the MFSD4A gene might be related to the regulation of growth and development of yak testis.Fluorescence localization of the pEGFP-MFSD4A fusion vector transfected into Madin-Darby bovine kidney cells(MDBK)showed that the protein was mainly located in the nucleus.

In order to explore the expression and function of interferon-stimulated exonuclease gene 20(ISG20)in the formation of receptivity in beef cattle endometrial epithelial cells.The CDS region of the beef cattle ISG20 was obtained by RT-PCR combined with sequencing.The sequence analysis of the ISG20 and its encoded proteins was performed using bioinformatics online tools.The expression of the ISG20 and its related genes was detected by qRT-PCR in the formation of receptivity in beef cattle endometrial epithelial cells.The results showed that the length of CDS region of the ISG20 was 516 bp,which could encode a single protein consisting of 176 amino acids with no transmembrane domain and unstable basic hydrophilicity.The protein contained the DEDD(or DnaQ-like)exonuclease superfamily domain,interacted with 10 proteins,including ISG15 and MX2,and functioned mainly in the cytoplasm.In addition,the qRT-PCR results showed that compared with the control group,the expression of the ISG20 and its related ISG15 and MX2 was both significantly up-regulated in the formation of receptivity in beef cattle endometrial epithelial cells induced by progesterone(P4)-co-interferon-tau(IFN-τ).ISG20 may be involved in the formation of receptivity in beef cattle endometrial epithelial cells and play a role in biological processes such as early pregnancy.

In order to study the structure and function of DJ-1 gene in yaks,this experiment used Meiren yak adipose tissue cDNA as a template,cloned the coding sequence (CDS) of yak DJ-1 gene using RT-PCR,and performed bioinformatics analysis,including domain prediction and protein physicochemical properties prediction of the gene sequence.Then,tissue expression analysis was performed using RT-qPCR.The results showed that the CDS region of the DJ-1 gene in Meiren yak had a total length of 570 bp and encoded 189 amino acids;the prediction of the DJ-1 domain in yaks showed that there was one PfpI domain containing the Pfam in positions 29—168 of the DJ-1 amino acid sequence.By predicting structure and function of the DJ-1 protein,the results showed no transmembrane structure and no signal peptide region;molecular weight was 20.035 31 ku,total number of atoms was 2 870,theoretical isoelectric point was 6.84,instability coefficient was 28.37,and expressed as a stable protein;the fat coefficient was 101.11,with a total average hydrophilicity coefficient of -0.004.It was a hydrophilic protein with a total of 11 phosphorylation sites.Meanwhile,the results of subcellular localization prediction indicated that the DJ-1 protein of Meiren yak was mainly distributed in the cytoplasm and mitochondria.The phylogenetic tree showed that Meiren yak had the closest genetic relationship with wild yaks and European cattle,and the farthest genetic relationship with Arctic foxes and wide snouted manatees;the RT-qPCR results showed that it was expressed in the heart,liver,spleen,lungs,muscle,fat,and testicular tissues of adult Meiren yaks,with the highest expression level in the heart and muscle tissues,and the lowest expression level in the liver and testicular tissues.

The aim of this study was to analyze the regulatory effect of RXFP2 gene on horned phenotype and related SNP markers in horned and hornless Oula sheep populations,in order to provide technical support for genetic improvement and breed breeding of Oula sheep.DNA was extracted from 100 ewe blood samples of healthy adult Oula sheep in Henan County,Qinghai Province,including 50 blood samples with horns and 50 blood samples without horns,respectively,15 samples with and without horns were randomly selected for whole genome sequencing,and then all blood samples were amplified by multiple PCR to verify the SNPs of RXFP2 gene.Genome-wide detection results showed that there were strong selection signals in chromosome 10 of Oula sheep,including RXFP2 gene,and the strongest selection signals appeared in the RXFP2 gene segment.Multiple PCR tests verified the accuracy of the 4 coding SNPs screened by whole genome resequencing.Seven high frequency SNPs in the intron region of RXFP2 gene were detected in Oula sheep(29508704G>A,29509428A>C,29509766G>A,29512170G>A,2951276G>A,29514968T>A,29521377C>A).The genotypes of the 7 SNP loci were separated in hornless phenotypes.100% of homozygous genotypes appeared horned or hornless trait,89.66% of heterozygous genotypes were hornless trait and 10.34% were horned trait.The frequency of mutant alleles was less than that of wild alleles,and the population showed an unbalanced state of Hardy-Weinberg,which meant subject to high artificial selection intensity,but the content of polymorphic information was moderate,and the potential of breeding and improvement was still large.In conclusion,the present study confirmed the strong regulatory effect of RXFP2 gene on horn trait in Oula sheep,and filtered out 7 molecular markers of RXFP2 gene on horn trait in Oula sheep population.The relevant results can be used as molecular markers for genetic improvement of the hornless population of Oula sheep.

The study aims to investigate the effect and mechanism of PPARA on intramuscular fat (IMF) deposition,and to search for molecular markers related to IMF deposition in sheep.The experiment was conducted using Small-tail Han sheep (STH) and a hybrid F₁ population between Suffolk sheep and Small-tail Han sheep(STH×SFK)as the research object.By measuring their meat quality,the histological structure and fat drop distribution of longissimus dorsi muscle of two sheep populations were compared by H&E staining and Oil Red O staining;qRT-PCR and WB techniques were used to detect the expression differences of PPARA mRNA and protein levels among different populations,and analyze the correlation between PPARA gene and IMF;Sanger sequencing technology was used to detect the PPARA gene SNP site to evaluate the possibility of using it as a genetic marker related to IMF deposition in sheep.The results indicated that the shear force and water loss rate of STH were significantly higher than those of STH×SFK,but pH was extremely significantly lower than that of STH×SFK,marble score of STH was lower than that of STH×SFK population;histological staining showed that STH muscle fibers were thick and tightly arranged,with IMF concentrated in the muscle fiber gaps,and muscle fibers were thin and loose in structure in STH×SFK,and IMF was evenly and widely distributed among muscle cell and muscle fiber spaces,with a higher content.The expression levels of PPARA mRNA in the STH were significantly higher than those in the STH×SFK population,and PPARA protein expressions of the STH were extremely significantly higher than those of the STH×SFK population.Correlation analysis showed that the expression level of PPARA gene was extremely significantly negatively correlated with sheep fat droplet area ratio and pH,extremely significantly positively correlated with shear force and water loss rate,and weakly correlated with marble score.Sanger sequencing results showed that base mutations T49885C,T50007C,G50013A,G50835A,G50942A and G51154C occurred in the second intron of PPARA gene in two sheep populations,but no C49885T mutation was detected in the STH population.The analysis of SNP genetic diversity showed that the homozygosity of SNP loci in the population was greater than the heterozygosity,and they were in a medium to low degree of polymorphism in the population;except for the G50835A mutation,all other SNPs were in line with the Hardy-Weinberg equilibrium,and with a stable genetic basis.It was believed that PPARA gene had an important impact on IMF deposition in sheep,and could be used as a potential molecular genetic marker for IMF trait selection in sheep.

The aim of this study was to investigate the effect of bisphosphoglycerate mutase(BPGM)on the growth and development of chicken myogenic cells.The transcriptomics and proteomics data was jointly analyzed in the preliminary stage of the study to identify the differentially expressed genes that might affect the growth and development,and investigated their effects on the proliferation,apoptosis and expression levels of related marker genes of myogenic cells.Inosinic acid(IMP)was detected by ELISA and adenosine triphosphate(ATP)was detected by colorimetric assay,which revealed the important roles of the key candidate genes on muscle development and meat flavor of Jingyuan chickens.The results showed that a total of 139 differentially expressed genes/proteins were identified between the breast muscle and leg muscle of Jingyuan chickens,including 75 differentially expressed genes/proteins that were up-regulated in both the transcriptome and proteome,45 differentially expressed genes/proteins that were down-regulated in both the transcriptome and proteome,13 differentially expressed genes/proteins that were down-regulated in the transcriptome,and 6 differentially expressed genes/proteins that were up-regulated in the proteome and down-regulated in the transcriptome.7 differentially expressed genes were screened from the glycolysis/glycolysis pathway,which were BPGM, phosphoglycerate kinase 1 gene(PGK1),glucose-6-phosphate isomerase gene(GPI),phosphoglycerate mutase 1 gene(PGAM1),lactate dehydrogenase A gene(LDHA),lactate dehydrogenase B gene(LDHB)and phosphoglycerate isomerase 1 gene(TPI1),and screened for the differentially expressed gene BPGM in combination with KEGG pathway enrichment analysis and protein network interactions analysis.BPGM was expressed in all tissues of Jingyuan hens,the highest expression was in pectoral muscle,and it showed an up-regulation trend during the differentiation of myoblasts.Primary myogenesis cells were isolated and cultured for cell transfection.Interfering with BPGM inhibited myoblast proliferation and promoted apoptosis;it also affected genes upstream and downstream of the glycolytic pathway,and inhibited the biosynthesis of IMP and ATP at the same time.In conclusion,down-regulation of BPGM inhibited the growth and development of myoblasts,as well as the biosynthesis of IMP and ATP.

To investigate the effect of lncRNA2099 on the proliferation and differentiation of porcine preadipocytes,the qRT-PCR was used to detect the expression level of lncRNA2099 in heart,liver,spleen,lung,kidney,back muscle,back fat and proliferation and differentiation stages of porcine preadipocyte.The nucleoplasmic separation technique was used to determine the expression location of lncRNA2099 in adipocyte.The eukaryotic expression vector pcDNA3.1-lncRNA2099 was constructed and qRT-PCR was used to detect its impact on the expression of marker genes related to adipocyte proliferation and differentiation after over expression in adipocyte.The results showed that lncRNA2099 was highly expressed in kidney and back fat,but not in back muscle.The nucleoplasmic localization results showed that lncRNA2099 was expressed in both nucleus and cytoplasm;the expression levels of proliferation marker gene P21 and lipogenic differentiation marker gene LPL significantly increased after overexpression of lncRNA2099 in adipocytes.lncRNA2099 may act as a transcriptional regulator to inhibit proliferation and promote lipogenic differentiation in porcine preadipocytes.

The aim of this experiment was to analyze the transcriptome data of testicle in different size of Shandong black cattle,and discover the key candidate genes affecting the reproductive function of testicles,in order to explore the molecular mechanism of maintaining the reproductive function in cattle testicle.Six bulls were divided into two groups according to different size testicle for transcriptome sequencing.The differential expression multiplier values |log₂(Fold Change)|≥1 and P-value≤0.01 were used as the conditions for selecting the differential expression genes (DEGs),and GO and KEGG analysis were performed to select the candidate gene of reproductive function of testicle.The CDS region of cattle candidate gene was cloned,and qRT-PCR,Western Blot,IHC were used for further confirmation.A total of 353 DEGs were obtained from the sequencing results,including 114 up-regulated genes and 239 down-regulated genes in small testicle.The results of GO functional annotation found that the items related to metabolism,development,reproduction and other processes.The results of KEGG pathway enrichment analysis showed that DEGs were mainly enriched in Wnt signal transduction,PI3K-Akt signaling pathway about reproductive function of testicle,further screening for CCN4 related to testicle reproductive function as candidate gene to validate.The qRT-PCR and Western Blot results showed that the relative expression of CCN4 gene and protein in big testicles was significantly lower than that in small testicle.The IHC results showed that CCN4 protein was mainly positive in the areas outside spermatids (especially sertoli cells) and the relative expression in big testicle was significantly lower than that in small testicle.It is speculated that the CCN4 gene may inhibit sertoli cells to affect cattle testicle function.This study provides a basic data for further study of reproductive function testicle in different size of cattle.

This study aims to explore the effects of different concentrations of zearalenone (ZEN) on the growth of bovine mammary alveolar cell-T (MAC-T) and the expression of genes related to milk fat synthesis.Firstly,MAC-T cells were treated with different doses of ZEN for 36 hours,and the number of cells was counted using a hemocytometer.Then,cells were stained and analyzed for apoptosis and necrosis to determine the appropriate dose of ZEN.Next,acommercial detection kit was used to examine the impact of ZEN on reactive oxygen species (ROS) and mitochondria number in MAC-T cells.Finally,using Real-time Quantitative PCR (RT-qPCR) technology,the influence of ZEN on proliferation,apoptosis,oxidative stress,and milk fat synthesis-related gene expression in MAC-T cells was analyzed.The results showed that low doses of ZEN (0.01—1.00 μmol/L) tended to promote MAC-T growth,whereas high doses of ZEN (5.00—10.00 μmol/L) significantly reduced the number of cells,0.10 μmol/L ZEN had no obvious effect on ROS and mitochondria number in MAC-T,but 10.00 μmol/L ZEN notably elevated ROS levels and decreased mitochondria number.RT-qPCR results indicated that 0.10 μmol/L ZEN significantly promoted the expression of proliferation genes (CDK1,CCND2),antioxidant genes (DHODH,GPX4),and anti-apoptotic genes (BCL-2),but also increased the expression of apoptotic genes (CAS-3,BAX).Whereas,10.00 μmol/L ZEN significantly inhibited the expression of proliferation genes (PCNA,CDK1,CCND2),antioxidant genes (DHODH,GPX4,AIFM2),and anti-apoptotic genes (BCL-2),while significantly promoting the expression of apoptotic genes (CAS-3,BAX).Notably,0.10 μmol/L ZEN obviously promoted the expression of milk fat synthesis-related genes (PPARγ,FASN,JAK-2),but 10.00 μmol/L ZEN significantly suppressed these genes.The aforementioned results suggested different effects of ZEN concentrations on MAC-T cells:0.10 μmol/L ZEN could promote MAC-T cell growth and milk fat synthesis-related gene expression while also inducing the expression of apoptotic genes.In contrast,10.00 μmol/L ZEN induces oxidative stress in MAC-T cells,reduces mitochondria number,inhibits the expression of genes associated with proliferation,antioxidant properties,anti-apoptotic properties,and milk fat synthesis,while promoting the expression of apoptotic genes,leading to cell apoptosis.

The hair follicles of Dorper sheep are characterised by cyclic growth and development,which is generally divided into three periods:anagen,catagen,and telogen.This study aims to explore the regulatory mechanism of key lncRNAs as ceRNA associated with hair follicles anagen and telogen as screened by previous studies.Ten Dorper ewes (shedding group,S group) with extreme shedding phenotypes and 10 non-shedding ewes (non-shedding group,N group) were selected under the same feeding conditions,with similar body weights and sizes,and about 2 years old,and the best 5 shedding ewes and 3 non-shedding ewes were selected for transcriptome sequencing after phenotypic observation.The ceRNA regulatory network of key lncRNAs was constructed by bioinformatics analysis methods such as target prediction,expression pattern analysis,and GO and KEGG enrichment analysis,to further investigate the regulatory mechanisms of key lncRNAs.The results showed a total of 262 mRNAs were screened by lncRNA-miRNA and miRNA-mRNA target prediction analysis,of which 135 mRNAs were consistent with the expression patterns of the corresponding lncRNAs (19 A pattern (Anagen,highly expressed in anagen) and 116 T pattern (Telogen,highly expressed in telogen)).GO and KEGG enrichment analysis of these 135 mRNAs revealed that some genes associated with development of hair follicles,such as CTNNB1,FGF22,FGF5,FZD3,JAK3,Lpar6,NGFR,PAK1,EIF4E,and Wnt10b,were enriched in Rap1,MAPK,Wnt,PI3K-Akt,Ras,mTOR,Jak-STAT,Hippo and other signaling pathways,which were associated with development of hair follicles.A ceRNA regulatory network consisting of 10 lncRNAs,11 miRNAs and 10 mRNAs was finally constructed.The qRT-PCR of several randomly selected differentially expressed lncRNAs and differentially expressed genes showed that the expression trend was basically consistent with that of the transcriptome sequencing results,indicating that the sequencing result was reliable.These 10 lncRNAs obtained from the study together with their target-regulated miRNAs and mRNAs can be the focus of research on the development of hair follicles in sheep.

This study aims to analyze the effects of LncRNA on muscle development of male and female Tan sheep from the gene level,because muscle growth and development is an important indicator of economic benefit in breeding industry,and skeletal muscle development is significantly correlated with meat production performance.In order to preliminarily reveal the molecular mechanism affecting the differences in muscle development,transcriptome sequencing analysis was conducted to screen the LncRNA related to sheep muscle development.Full transcriptome sequencing (RNA-seq) and analysis were performed on longissimus dorsi muscle tissues of 3 male and female sheeps (8 months old),so as to screen out LncRNAs that may be related to muscle development of Tan sheep.The results showed that among the 8 513 LncRNAs were identified,45 intergroup differentially expressed LncRNAs were selected,of which 18 were up-regulated and 27 down-regulated.Prediction of co_location target gene of DELncRNA and GO functional enrichment analysis showed that target genes were significantly enriched in 268 GO terms,including somato-secreting cell differentiation,typical Wnt receptor signaling pathway involved in negative regulation of apoptosis,positive regulation of growth hormone secretion,and cAMP-mediated signaling.KEGG pathway enrichment analysis showed that DEGs was enriched in AMPK,NF-KB,PI3K-AKT,MAPK,cAMP,FOXO and other signaling pathways.Five LncRNAs,namely,TCONS_00017263,TCONS_00147966,TCONS_00163484,TCONS_00079802,and TCONS_00079803,which may be involved in muscle development,were further screened.Real-time Fluorescence Quantitative PCR was performed on 6 randomly selected DELs,and the expression trend was consistent with the trend of transcriptome sequencing,which further verified the accuracy of the sequencing results.These DELncRNAs obtained in the study may cause the differences in muscle growth and development of Tan sheep between males and females,and these related LncRNAs may provide more information for regulating muscle development and provide scientific basis for molecular breeding of meat performance of Tan sheep of different genders in Ningxia in the future.

Beef marbling is an important meat quality trait regulated by multiple genes and is one of the conditions to measure the quality of high grade beef.LncRNA is an important non-coding RNA that plays an important role in regulating the biological processes related to fat deposition.To understand the potential biological role of lncRNA in different marbling phenotypes in beef cattle,this study collected crossbred and the longest dorsal muscle tissues from two extreme grades of high(A5)and low(A1)marbling,and used high-throughput sequencing to detect differential lncRNA associated with meat marbling,predict the trans-acting target genes of differential lncRNA,and perform functional enrichment for the analysis.The results showed that a total of 45 significantly different lncRNA were screened in two grades of marbled beef tissues,including 21 and 24 significantly up- and down-regulated lncRNA in high-grade marbled tissues,respectively,including MSTRG.9509.3,ENSBTAT00000077612,ENSBTAT00000072841 and other lncRNA associated with lipid deposition.Functional enrichment analysis of trans-acting target genes showed that predicted target genes were significantly enriched to lipopolysaccharide binding,lipolysis and other biological processes associated with lipid deposition.Differential lncRNA may be involved in regulating beef marbling phenotypic traits by targeting fat deposition-related genes(FABP4,PLIN1,LIPE,etc.)through trans-activation.The detection of beef marbling-related differential lncRNA,predicted target genes and their enrichment pathways would provide a basis for expanding the understanding of the biological functions of non-coding RNAs and their relationship with beef marbling characteristics,as well as a reference for the analysis of quality traits and breeding of new breeds in agricultural livestock.

To analyze the same protein of whey and milk fat globule membrane (MFGM)of jersey yak crossbreeds milk and yak milk by proteomics method. The milk yield of jersey yak crossbreeds was higher than that of yak. If the function of jersey yak crossbreeds milk is like that of yak milk,it will improve the economic level of local herdsmen. Whey and milk fat were separated by centrifugation,and proteins were extracted respectively. The same protein was obtained by qualitative and quantitative analysis of protein by tandem mass tag (TMT)technique. GO functional annotation,KEGG metabolic pathway,protein interaction and other bioinformatics analysis were performed on the same protein. Result analysis indicated that 651 whey proteins and 990 MFGM proteins were identified in the peak lactation period of jersey yak crossbreeds milk and yak milk by label quantitative proteomics,and 298 whey proteins and 283 MFGM proteins were screened. GO annotation analysis showed that the same proteins of jersey yak crossbreeds milk and yak milk whey were mainly involved in the negative regulation of endopeptidase activity,immune response and immunoglobulin production. The same proteins of MFGM were mainly involved in the negative regulation of endopeptidase activity and complement activation. The cell components of the same proteins of whey and MFGM in the classical pathway were mainly extracellular space and extracellular region. The molecular functions involved were mainly guanosine 5'-triphosphate (GTP)enzyme activity and GTP binding. KEGG enrichment analysis showed that the main enrichment pathways of the same proteins in jersey yak crossbreeds milk and yak milk whey and MFGM included complement and coagulation cascades,phage and protein processing in endoplasmic reticulum. During the peak lactation period,the proteins with higher abundance in the milk of jersey yak crossbreeds and yak showed excellent performance in immunity and promoting nutrient absorption. However,the milk yield of jersey yak crossbreeds was higher than that of yak,which would increase the economic income of local herdsmen and provide more valuable plateau dairy products for people.

With the development of dairy products,milk protein is an important index to evaluate the nutritional value of milk.Optimizing feed formulation is one of the key tasks to improve the quality and efficiency of dairy cow production.At present,although feeding rumen-protected amino acids is the most direct and efficient way to improve milk protein synthesis of dairy cows,most studies only focus on a single limiting amino acid,ignoring the understanding of amino acid nutritional balance.Amino acid nutrition,as the source and signal substance of milk protein synthesis,affects the absorption and utilization of blood amino acids by cows,but its molecular mechanisms is still unclear.Therefore,this article mainly analyzed the effects and regulation of amino acids on protein synthesis in dairy cows.Firstly,it described the basic pathway of milk protein synthesis and the influence of essential amino acids,non-essential amino acids and combined essential amino acids on milk protein yield and concentration.Secondly,it introduced the impact of the supply of essential amino acids and combined essential amino acids on feed intake,blood amino acid concentration,blood flow and mammary gland cells of dairy cows,while emphasizing the optimal supplemental dose of amino acids required for mammary gland cell culture and the specific regulation of essential amino acids on amino acid transporters and signaling pathways in cellular molecular angle.In summary,through the discussion of the factors and molecular regulation mechanism of mammary gland protein synthesis,it can not only help to optimize the ideal amino acid ratio in feed and scientific production mode,but also provide theoretical reference for improving the synthesis of milk protein by amino acid nutrition in related filed.

The purpose of this study was to preliminarily analyze the promoter activity and transcription regulatory elements of porcine CRYBB2. Using bioinformatics analysis,PCR amplification,gene cloning,cell transfection,double luciferase activity analysis and other methods,we obtained the sequence characteristics of CRYBB2 promoter region,constructed double luciferase reporter gene vectors of CRYBB2 promoter region with different fragment lengths,analyzed its luciferase activity,and then determined the core promoter region and key regulatory region of CRYBB2.Finally,the transcription factors and their binding sites in key regulatory regions were predicted. The results showed that the candidate promoter region of CRYBB2 might contain four core promoters and one CpG island.-52—-3 bp might be the core promoter region of CRYBB2 gene,and-505—-19 bp might be the key regulatory region of CRYBB2 gene promoter,which played a positive regulatory role,while-2 060—-505 bp didn't have any regulatory elements that affected the activity of CRYBB2 gene promoter.The key regulatory region of CRYBB2 promoter contained multiple potential transcription factor binding sites,such as TBP,NFIA,FOXP1,NKX2-8,KLF4,Tcf3,Crx,SNAI3,Rfx1 and CREB1. This study laid a foundation for further study on the expression mechanism of porcine CRYBB2.

It aimed to explore the kinship between three black sheep populations in Sichuan by determining the sequence of the zinc finger domain of Prdm9.Anticoagulated whole blood was collected from the black sheep population,genomic DNA was extracted,and the zinc finger domain of the Prdm9 was specifically amplified by PCR,and further sequenced by cloning to analyze genetic diversity.The results showed that nine types of zinc fingers were identified in black sheep,with 1—5 amino acid differences between zinc fingers,constituting five Prdm9 zinc finger domains,defined as different alleles(A,B,C,D and E),of which A to D contained eight zinc fingers,which were the main types of Prdm9 zinc finger domains in the tested black sheep,and E contained nine zinc fingers and was only found in Yanyuan black sheep,with an allele frequency of 0.050.These alleles formed six genotypes,including AA,AC,BB,BD,CC and EE,and four genotypes were detected in both Butuo and Puge black sheep,while six were found in Yanyuan black sheep,and BB and EE were only observed in this population.The Prdm9 allele frequencies and PIC values varied among the three black sheep populations,with richer genetic polymorphisms,of which the Yanyuan black sheep were relatively the most abundant.The allele frequency distribution of the Prdm9 zinc finger domain in all three black sheep populations deviated significantly from Hardy-Weinberg equilibrium(P<0.01),suggesting that they were influenced by artificial selection in breeding.In conclusion,Prdm9 has rich genetic diversity in the black sheep population,and all three populations are closely related to each other.

In order to study the effects of different combinations of bacteriocin and probiotics on growth performance,slaughter performance,intestinal tissue morphology and intestinal flora of broilers.A total of 300 one-day-old male chicks of Aibayijia(AA)were selected and randomly divided into 5 groups,with 6 replicates in each group,10 in each replicate,and group A(control group)was fed the basal diet,group B(bacteriocin 50+probiotics 50)added bacteriocin 2.57 mL/kg and probiotics 0.84 g/kg to the basal diet,and group C(bacteriocin 100+probiotics 100)added bacteria to the basal diet bacteriocin 5.13 mL/kg,probiotics 1.68 g/kg,group D(bacteriocin 150+probiotics 150)added bacteriocin 7.7 mL/kg,probiotics 2.52 g/kg in the basal diet,group E(antibiotics 50)chloromycin 50 mg/kg was added to the basal diet.The test was divided into the first stage(1—21 days old)and the second stage(21—42 days old),and the test period was 42 days.The results showed that,at 22—42 days and 1-42 days,the average daily gain in group D was significantly higher than that in group A(P<0.05),and the feed-to-weight ratio was significantly lower(P<0.05).The heart weight of group D was significantly higher than that of group A(P<0.05),and the weight of liver of group C was significantly higher than that of group A(P<0.05).Compared with group A,the duodenal villus height in groups C,D,and E were significantly increased(P<0.05).Compared with group A,the relative abundance of Firmicutes in group D increased,while the relative abundance of Bacteroidetes decreased,the relative abundance of Proteobacteria in groups C, D, and E was higher than that in group B. At the genus level,the relative abundance of Ruminococcus in group D was significantly higher than that in other groups(P<0.05).The addition of bacteriocin in the diet improves the overall performance of white feather broilers,improves the structure of intestinal flora of broilers,promotes healthy intestinal development,and increases the weight of heart and liver in broilers.The supplemental level of group D(bacteriocin 150+ probiotics 150)was appropriate.

The aim of this study was to understand the structure and function of ferritin light chain(FTL)gene in yak and its expression pattern in eight tissues of different age groups.The coding sequence(CDS)of FTL gene was cloned from the cDNA of liver tissue of adult(36 months old)Meiren yak,and bioinformatics analysis was carried out.The relative expression of FTL gene in heart,liver,spleen,lung,kidney,longissimus dorsi,fat and testis was detected by Real-time fluorescence quantitative(qRT-PCR).The results showed that the CDS region of FTL gene of Meiren yak was 528 bp,encoding 175 amino acids,and had the closest relationship with wild yak and cattle(99.8%).FTL protein was a stable hydrophilic secretory protein without signal peptide and transmembrane structure.It was mainly distributed in the cytoplasm.The secondary structure of the protein was mainly α-helix,which mainly interacted with FTH1,SCARA5,NCOA4 and other proteins.The results of qRT-PCR showed that the expression levels of FTL gene in adult cattle were liver,kidney,testis,spleen,lung,fat,longissimus dorsi and heart,respectively.The expression levels of FTL gene in fat and longissimus dorsi tissues of 7-day-old calves were the highest,and the expression level of FTL gene in liver tissue of adult cattle was the highest.By comparing the two stages,it was found that the expression levels of fat,longissimus dorsi and heart tissues decreased with the increase of age,while the expression levels of lung,spleen,testis,kidney and liver tissues increased with age.

The aims of this study were to explore the genetic diversity,phylogenetic evolution and genetic relationship from six yak groups of Tibetan yaks by mtDNA ND1.To provide a theoretical basis for the genetic diversity,historical evolution,the protection and utilization of genetic resources of Tibetan yaks.The sequences of ND1 gene protein coding region(CDS)in 95 individuals of Pali yak,Jiali yak,Gongbujiangda yak,Sibu yak,Jiangda yak and Leiwuqi yak were analyzed by PCR and direct sequencing.DNAMAN,DNASP 5.1,Mega 7.0,Arlequin 3.5.2 and other software were used to analyze the sequence polymorphism,haplotype diversity,genetic distance and constructed the haplotype network map and phylogenetic tree.The result showed that the sequence length of CDS region of ND1 gene of Tibetan yaks population were 956 bp,and a total of 78 mutation loci and 16 haplotypes were detected and the average haplotype diversity(Hd)and nucleotide diversity(Pi)were 0.670 and 0.004 21,Tajima's D was all negative.According to the molecular variance analysis of population genetic differences,the degree of variation within Tibetan yaks was greater than among populations;the degree of differentiation between Sibu yak and Jiali yak was large,and the degree of differentiation among most other groups was medium or weak.In addition,cluster analysis found that Leiwuqi yak was grouped into a single group,Jiali yak,Gongbujiangda yak,Sibu yak and Jiangda yak were first grouped into one group,and then grouped with Pali yak;the 16 haplotypes were divided into two clusters,suggesting that there may be two maternal origins in Tibetan yaks.The cluster analysis with other cattle breeds showed that Bos grunniens,Bos taurus and Bos javanicus may be the mixed maternal origin of Tibetan yaks.The genetic diversity of Tibetan yaks was very rich,among which the genetic diversity of Leiwuqi yaks was the most abundant,six yak populations had two maternal origins.

The aim of this trial was to investigate the effect of different levels of NDF on the expression of genes related to growth axis and duodenal growth in lambs,in order to provide a reference for the appropriate NDF level for lamb starter.Thirty-six male Hu lambs were selected for the trial and randomly divided into 15% and 25% NDF level groups.At 56 days of age,six lambs from each group were selected for slaughter,weighed for tissue and organ,and the expression of relevant genes was measured.The results showed that there was no significant effect(P>0.05)of NDF levels in the starter diet on tissue and organ mass and index;in the 15% group of lambs,hypothalamic growth hormone releasing hormone(GHRH),hepatic insulin-like growth factor-1(IGF-1),duodenal IGF-1 and IGF-1 receptor(IGF-1R)expressions were significantly higher in the 25% group(P<0.05);the expression of pituitary growth hormone(GH)and growth hormone receptor(GHR) in the duodenum were significantly higher in the 25% group than in the 15% group(P<0.01).The average daily gain of lambs was strongly negatively correlated with hypothalamic GHRH expression(P<0.01),significantly negatively correlated with hepatic GHR and duodenal IGF-1 expression(P<0.05),and significantly positively correlated with pituitary GH and duodenal GHR expression(P<0.05);pituitary GH was significantly or highly significantly negatively correlated with hepatic GHR,IGF-1,duodenal IGF-1 and IGF-1R expression(P<0.05 or P<0.01),and strongly significantly positively correlated with duodenal GHR expression(P<0.01);hepatic GHR was strongly significantly positively correlated with hepatic IGF-1,duodenal IGF-1 expression(P<0.01),and strongly significantly negatively correlated with duodenal GHR expression(P<0.01).In conclusion,the expression of genes related to GH-GHR levels in the growth axis of lambs is enhanced by feeding 25% NDF level open feed compared to the 15% level group.

The aim was to screen key differentially expressed genes and signaling pathways and explore the molecular regulatory mechanisms of chicken skin follicle traits through transcriptomic analysis of the dorsal skin follicle tissues of Chongren partridge chicken.Total RNA of skin hair follicle tissue was extracted by TRIzol method,and transcriptome high-throughput sequencing(RNA-Seq)was performed by Illumina platform.Finally,the transcriptome sequencing was validated by Real-time fluorescence quantitative PCR.The results were as follows:A total of 3 856 differentially expressed genes were screened,and 971 differentially expressed genes were screened in the cockerel(BGB vs YGB)comparative combinations,of these,274 up-regulated and 697 down-regulated.There were 3 529 differentially expressed genes in comparative combinations of hen(BMB vs YMB),of these,1 477 up-regulated and 2 052 down-regulated.Three highly expressed genes,KRT75,KRT6A,and KRT14,associated with skin follicle traits,were screened among the common differentially expressed genes in roosters and hens.In the significant enrichment of GO functions,516 GO terms were enriched in the BGB vs YGB and 1 020 GO terms were enriched in the BMB vs YMB.Genes were mainly focused on activities involved in cell adhesion and cell activation.Significantly enriched multiple KEGG pathways,with 8 significant KEGG pathways enriched in the BGB vs YGB and 20 significant KEGG pathways enriched in the BMB vs YMB.Three key pathways,Wnt signaling pathway,neuroactive ligand-receptor regulatory pathway and TGF-β regulatory pathway,were screened.Fluorescence quantification showed that the expression of five randomly selected differential genes in skin hair follicles was consistent with the trend towards RNA-Seq.In summary,the screened KRT75,KRT6A,and KRT14 genes may affect chicken skin follicle traits;the neuroactive ligand-receptor regulatory pathway,the Wnt signaling pathway,and the TGF-β signaling pathway are important pathways that regulate follicular traits in chicken skin.

The aim of this study was to obtain the serum inflammatory factor content,spleen pathological changes and transcriptome expression profile characteristics of diarrheic piglets with Clostridium perfringens type C,and to provide a reference for the diarrhea prevention and treatment.Six 7-day-old binary piglets with good health and similar body weight were randomly selected and divided into two groups,namely control group (CS) and treatment group (TS).Each piglet in the TS group was fed with 1 mL bacterial solution (1×10⁹ cfu/mL) every day for 5 consecutive days.Then,piglets in the CS group were fed with culture medium without the bacterial solution.Blood samples were collected and serum was separated on 0,1,3 and 5 days after infection.The serum IL-8,IFN-α and IFN-γ concentration were detected by ELISA.After the experiment,piglets were slaughtered to collect the spleen.The partial spleen was collected for HE staining to observe the pathological changes.Additionally,differentially expressed (DE) genes in the spleen were obtained using RNA-Seq.GO terms and KEGG signaling pathways enrichment analysis were performed.The key spleen genes during the infection of piglets with C.perfringens type C were verified by RT-qPCR.The spleen of piglets in the TS group displayed obvious inflammation,neutrophil infiltration,accompanied by degeneration and necrosis of parenchymal cells;on the 3rd and 5th day,the levels of IL-8,IFN-α,and IFN-γ in the CS group serum were extremely significantly lower than those in the TS group (P<0.01);a total of 1 079 DG genes was screened between TS group and CS group,including 583 up-regulated genes and 496 down-regulated genes.DG genes were mainly enriched in GO terms such as cell response to interferon-β,cell response to DNA damage stimulation,and negative regulation of inflammatory response.Differentially expressed gene were mainly enriched in KEGG signaling pathways such as tuberculosis,hepatitis B,and B cell receptors.These GO terms and KEGG signaling pathways were involved in piglet resistance to C.perfringens type C infection;the RNA-Seq results of the selected candidate genes (such as JAK3,TLR3,ILF2,CXXC1) resistant to C.perfringens type C infection were consistent with the RT-qPCR results.The above results indicated that JAK3,TLR3 and other genes played important roles in the process of piglet spleen resistance to C.perfringens type C infection through key signaling pathways.