Myostatin (MSTN) was a master regulator of skeletal muscle development. In order to obtain the sequence and expression pattern of MSTN gene in New Zealand White rabbits, the MSTN gene sequence was amplified and cloned from leg muscle tissue of New Zealand White rabbit by RT-PCR and then was analyzed by online bioinformatics software. The expression level of MSTN gene in different tissues and different periods of leg muscle was detected by Real-time quantitative PCR. The results showed that the CDS region of MSTN gene in New Zealand White rabbit was 1 128 bp in length, encoding 375 amino acids, and had 95.9%, 94.9%, 94.9%, 91.7%, 91.5%, 91.3% and 84.2% similarity with the nucleotide sequences of Homo sapiens, Sus scrofa, Equus caballus, Mus musculus, Canis lupus familiaris, Bos taurus, Gallus gallus published in GenBank, respectively. MSTN protein was an acidic and hydrophilic secretory protein, without transmembrane structure and contains a signal peptide. It mainly distributed in endoplasmic reticulum and mitochondria. The prediction results of secondary structure and tertiary structure of MSTN protein were consistent, which were mainly composed of random coil, followed by alpha helix and extended chain. It was a mixed protein. Protein interaction prediction found that MSTN protein interacted with PAX7, MYOG, IGF1, FST, Smad3 and Smad2 proteins related to muscle growth and development. Results of expression analysis in different tissues showed that MSTN gene was widely expressed in heart, liver, spleen, lung, kidney, leg muscle, uterus and stomach of New Zealand White rabbits. The expression level in leg muscle was the highest, which was extremely significantly higher than that in other tissues (P<0.01), followed by kidney, and the lowest expression in liver. Results of expression analysis in leg muscle tissue at different stages showed that the expression of MSTN gene in 180-day-old leg muscle tissue of New Zealand White rabbits was significantly higher than that in 90- and 240-day-old (P<0.05). The results laid a foundation for further study on the regulatory mechanism of MSTN gene on muscle growth and development in rabbits.

The research was designed to screen the active region and influence factors of buffalo HSD17B1 gene promoter, predict its transcription binding factor, and provide a theoretical basis for exploring the regulatory mechanism of HSD17B1 gene in buffalo reproductive performance. Using buffalo blood genomic DNA as template, PCR was used to amplify three active region sequences of HSD17B1 gene promoter, and then was directed to the pGL3-promoter vector. Recombinant plasmids were transfected into buffalo follicle granulosa cells, and the activity of relative luciferin enzymes was measured through a dual-luciferase detection system and their relationship to luteinizing hormone (LH) and follicle stimulating hormone (FSH) were explored. Transcription binding factor prediction was made in the HSD17B1 gene promoter region by bio-informational method. The results showed that three different length of HSD17B1 gene promoter fragments were successfully cloned and their dual-luciferase reporting vector were successfully constructed. The active detection of different length promoter fragments found that pGL-pro-HSD17B1-1500 was the most active, and -866/-1 500 bp was the core promoter region of HSD17B1 gene, indicating that this region played an important role in the transcription regulation of HSD17B1 gene. The luciferase activity test results showed that the addition of LH could enhance the activity of HSD17B1 gene promoter. Bio-information analysis of the HSD17B1 gene promoter region sequence found that there were 6 transcription factor binding site (Sp1(-2 327/-2 317 bp), HOXA4(-2 162/-2 146 bp), Sp1(-1 409/-1 395 bp), Sp1(-1 391/-1 380 bp), Sp1(-1 345/-1 319 bp) and GATA1(-812/-801 bp)), but no CpG island, and there were one TATA-box and two CAAT-box. The results indicated that the luciferase reporter gene vector of buffalo HSD17B1 gene promoter was successfully constructed, the core region of HSD17B1 gene promoter was determined, and the activity of the promoter was enhanced by LH adding.

The purpose of this study was to clone Smad4 gene and analyze its bioinformatics and tissue expression in chicken. Suqin No. 3 chicken was selected as experimental subjects, the full-length sequence of Smad4 gene in chicken was amplified by Race technology and cloned, the bioinformatics analysis of Smad4 gene was carried out, and the phylogenetic tree was constructed. The expression of Smad4 gene in chicken tissues was detected by Real-time quantitative PCR. The results showed that a full-length of Smad4 gene in chicken was obtained, which contained 17 bp 5'UTR, 1 842 bp open reading frame and 466 bp 3'UTR. There were 11 exons and 10 introns, the length of mRNA was 2 325 bp, which encoded 613 amino acids. The phylogenetic tree showed that Smad4 gene in chicken was clustered with other birds. Bioinformatics analysis showed that the molecular mass of Smad4 protein was 65.43 ku, the theoretical isoelectric point was 10.04, it was a hydrophilic protein. Smad4 protein contained two conserved domains MH1 and MH2. The secondary structure was consist of alpha helix (18.11%), extended chain (17.29%) and random coil (64.60%). Tissue expression analysis showed that Smad4 gene was highly expressed in heart, liver, jejunum and ovary, but there was no expression in breast muscle. In this study, the full-length sequence of Smad4 gene in chicken was successfully obtained, and its tissue expression pattern was preliminarily studied, which provided a theoretical basis for further study on the molecular mechanism of Smad4 gene in reproductive activity of chicken.

The purpose of this study was to modify the exon 1 of sheep (Ovis aries) fibroblast growth factor 5 (FGF5) gene by single base editing technique to introduce the stop codon, to obtain sheep embryos with site-directed editing, and to provide experimental materials for breeding sheep with long wool traits. Firstly, four single guide RNA (sgRNA) oligonucleotide chains (sgRNA-T1 to sgRNA-T4) were designed and synthesized, and four groups of recombinant expression vectors were constructed. The constructed pGL3-U6-sgRNA-PGK-puromycin and pCMV-AncBE4max-P2A-GFP plasmids were transferred into four groups of sheep fibroblasts by co-transfection, and then the activity of the transfected cells was detected by Cruiser^TM enzyme and sequenced at embryos level. The results showed that the PCR products of sgRNA-T1 and sgRNA-T4 groups with targeted effects were screened at cell level and could be cleaved by Cruiser^TM enzyme, and the sequencing results showed that both groups had targeted effects, with editing efficiency of 68.75% and 47.37%, respectively. Different concentrations of AncBE4max mRNA and effective sgRNA were mixed and transforred into sheep parthenogenetic activated embryos by microinjection technique, and the cleavage rate, blastocyst rate and editing efficiency of the embryos were detected. The results showed that at the embryo level, the best injection concentration combination of AncBE4max(ng/μL):sgRNA(ng/μL) was 100:50. The sequencing results of single embryos randomly selected from this concentration group showed that the editing efficiency of introducing stop codons was 80%. However, no editing was detected in the injected embryos with different concentrations of sgRNA-T1. In this study, aiming at the exon 1 of FGF5 gene, two sgRNA (T1, T4) targeting sheep FGF5 gene were successfully screened by transfection expression vector in fibroblasts, and the transformation of the target site C→T of exon 1 of FGF5 gene was successfully realized in embryos by microinjection of sheep parthenogenetic activated embryos, which laid a foundation for the production of sheep with FGF5 gene targeted editing in the later stage.

This study was aimed to obtain the prokaryotic expression protein of embryonic ectoderm development (EED) in chicken and explore its biological characteristics by bioinformatics. EED gene was amplified from spleen in chicken by RT-PCR, and cloned it into prokaryotic expression vector pET-32a(+) to construct the recombinant expression vector pET-32a-EED. Then pET-32a-EED was transformed into E. coli Rosetta to induce the EED fusion protein expression by IPTG. The recombinant protein was purified, and it was detected by Western blotting. Moreover, the bioinformatics analysis of EED protein in chicken was detected by online software. The results showed that the sequence of EED gene was 1 341 bp, which was 99.85% similarity with the reference sequence (GenBank accession No. :NM_001031376.1). The fusion protein with molecular weight of about 70 ku was obtained when the induction temperature was 28 ℃ and the expression of 1 mmol/L IPTG inducer was added for 7 h. The results of online software analysis showed that the relative molecular weight of EED protein in chicken was 50 377.92, and the isoelectric point was 6.54. It was a hydrophilic protein without transmembrane domain and signal peptide sequence. The tertiary structure prediction of EED protein in chicken was similar to human (6c23.1), and it also had the WD40 domain. In conclusion, the EED soluble protein in chicken was successfully expressed and purified, and EED protein had important functional elementsis, which provided materials for further study on the function of EED protein in chicken.

In order to explore the mechanism of myostatin (MSTN) on the growth and development of bovine skeletal muscle, quantitative proteomics and phosphorylated proteomics were used to analyze the differences of protein and phosphorylated modification levels in leg gluteal muscle of wild type Mongolian cattle (MG. WT) and MSTN^+/- Mongolian cattle (MG. MSTN^+/-). The established model of muscle differentiation of bovine skeletal muscle satellite cells in vitro was used to detect the interference effect of the designed and synthesized MSTN siRNA (si-MSTN). At the same time, Real-time quantitative PCR and Western blotting were used to detect the expression of mRNA and protein levels of genes related to actin cytoskeleton regulatory pathway in proliferative (GM) and day 3 of differentiation (DM3) bovine skeletal muscle satellite cells transfected with si-MSTN, and study the effect of knockout MSTN expression on actin cytoskeleton regulatory pathway. The results showed that 16 genes related to actin cytoskeleton regulatory pathway were up-regulated in muscle tissue of MSTN^+/- Mongolian cattle, the expression level of MSTN in si-MSTN transfected cells was extremely significantly decreased (P<0.01). In si-MSTN transfected GM phase bovine skeletal muscle satellite cells, the mRNA levels of ENAH, ACTN4 and Cdc42 genes related to actin cytoskeleton regulatory pathway were significantly increased (P<0.05), and the protein levels of PFN1, RhoA and ACTN4 were significantly or extremely significantly increased (P<0.05;P<0.01). In si-MSTN transfected DM3 bovine skeletal muscle satellite cells, the mRNA levels of ENAH, CFL1, SCIN and Cdc42 genes were significantly increased (P<0.05), the mRNA level of RhoA gene was extremely significantly increased (P<0.01), and the protein levels of PFN1 and ACTN4 were significantly increased (P<0.05). The results of this study indicated that interfering with MSTN could promote the expression of genes related to actin cytoskeleton regulatory pathway, and explore the molecular mechanism that MSTN might mediate actin cytoskeleton regulatory pathway to affect the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells, which provided reference for further study of the regulatory mechanism of MSTN on bovine myogenic differentiation.

Skeletal muscle is the largest tissue in mammals, and the loss of its functional or regenerative properties could lead to dysplasia and muscle skeletal diseases. The animal body has more than 600 muscles, which play a role in support and exercise. Skeletal muscle can be autonomously controlled by the body, which is a feature different from smooth muscle and cardiac muscle. The skeletal muscle of agricultural animals is the main source of meat products, providing humans with high-quality animal protein and nutrients. There are many factors that affect growth and development. The growth and development of muscle determines the yield and quality of pork, which arecrucial economic characters in agricultural animal production. The regulation of growth and development of skeletal muscle involves the activation or silencing of many genes and related pathways, which is a complex multi-level regulatory network. Here, the structure and composition, and growth and development process of skeleta muscle are reviewed. This paper also introduces many regulators on muscle development, including growth factors and cytokines. Base on the available results, it is believed that the future research will focus on the experimental validation of important candidate genes in vitro and in vivo, and then finally applytobreeding practice, which will provide a theoretical foundation for the study on molecular regulation mechanism of growth and development of skeletal muscle and the treatment of muscle genetic diseases.

The purpose of this study was to improve the dispersion and bioavailability of altrenogest (ALT) in water. The solvent method (ethyl cellulose, hydroxypropyl methyl cellulose, lactose) and solvent-melt method (polyethylene glycol 6000, glycerin monostearate, lactose) were used to preparation the ALT solid dispersions and sustained-release silicone suppository. The linearity, stability of the solid dispersion, precision of the instrument and the recovery rate of the standard were investigated. Fourier transform infrared spectroscopy (FTIR) and microscope were used to observe and analyze its apparent morphology. The sustained-release degree of ALT in 14 d was measured by sustained-release test. The results showed that ALT had a good linear relationship (R²=0.9996) in the concentration range of 1~12 μg/mL, and the instrument precision was good (RSD=0.48%). The stability of ALT solid dispersion prepared by solvent method and solvent-melt method was good, with RSD of 0.57% and 0.58%, respectively. The variation between the spiked recovery data of ALT solid dispersion prepared by solvent method and solvent-melt method was small, and the recovery rate was high. Infrared spectra and microscope images of active agent and solid dispersions showed that ALT solid dispersion was formed. The optimal combination of solvent method to prepare ALT solid dispersion was ethyl cellulose:hydroxypropyl methyl cellulose:lactose=1:0.6:1:6. The optimal combination of solvent-melt method to prepare ALT solid dispersion was PEG6000:glycerol monostearate:lactose=1:10:1.5:6. The sustained-release experiments of the sustained-release silicone suppositorials prepared by solvent method and solvent-melt method showed that the concentration of altrenogest increased gradually from 0-48 h, and reached the peak value of 37.73 and 30.46 μg/mL at 144 and 48 h, respectively, and then remained above 21 and 13 μg/mL, respectively. By comparison, the sustained release effect of the solid dispersion of ALT prepared by solvent method was better than that prepared by solvent-melt method, and the preparation method of the solid dispersion of ALT was simple, and the used carrier materials were non-toxic, which provided a reference basis for the further application of the solid dispersion in vivo.

The purpose of the experiment was to comprehend the metabolic differences of sex and the sex effects of meat quality and flavor caused by these metabolic differences of different tissues in Dulu pig, and offer basal metabolic information for meat quality and flavor research in pigs. Sixteen (half male and half female) 8-month-old healthy Dulu pigs with similar body weight were selected, and Non-targeted metabolites detection was conducted in serum, liver, longissimus dorsi muscle by gas chromatography/mass spectrometry (GC/MS). The different metabolites selection and metabolism pathway enrich analysis were conducted to analysis the sex effects of meat quality and flavor by metabonomics. The results showed that sexes had some influence on metabolic profiling of serum, liver, and longissimus dorsi muscle. The numbers of different metabolites caused by sex were bigger in serum and liver, and it was the least in longissimus dorsi muscle. The contents of most lipid differential metabolites in serum of male were higher than female (P<0.05), while it was the opposite in the liver (P<0.05). The contents of most carbohydrate differential metabolites in both serum and liver of male were higher than female (P<0.05). The levels of 2-monoolein, behenic acid, cholesterone and dihydrocholesterol in serum of male were more than 1.5 times as much as female. The levels of beta-gentiobiose, L-kynurenine and L-tyrosine in liver of male were 29, 11 and 3 times as much as female, respectively. Lipoic acid of male was significantly lower than female (P<0.05), which was 50% of female. The pathways of hormone, lipid, carbohydrate and animo acid were significantly different between the males and females (P<0.05). In conclusion, the sex effects were different in serum, liver and longissimus dorsi muscle. There were sex effects on hormone, lipid, carbohydrate and animo acid metabolism in serum and liver, however, these different sex effects were not too strong to reflect meat quality and flavor. The higher levels of lipid and carbohydrate metabolites made the meat quality and flavors of male a little better than female.

Skeletal muscle development is closely related to pork yield and quality, which is affected by various factors. In recent years, the influence of non-coding RNA (ncRNA) on skeletal muscle development has become one of the new research hotspot. ncRNA mainly include microRNA (miRNA), long non-coding RNA (lncRNA) and circular RNA (circRNA), which were a class of RNAs that do not have the function of encoding a protein. Initially, they were thought to only regulate gene expression at the transcriptional and post-transcriptional level. However, with the deepening of research, more and more non-coding RNAs have been confirmed to be involved in various biological processes, such as proliferation, differentiation and apoptosis of skeletal muscle cells. In details, miRNAs affect the function of the targeted mRNAs by binding to the complementary sequences of their target mRNA. lncRNA and circRNA can act as molecular sponges of miRNA to weaken the inhibitory effect of miRNA on targets. Therefore, the author mainly reviews ncRNA introduction, and the effects of miRNA, lncRNA and circRNA on the development of porcine skeletal muscle, and put forward the prospect of ncRNA on the growth and development of porcine skeletal muscle.

The competitive endogenous RNA (ceRNA) hypothesis suggests that transcripts with the same microRNA (miRNA) response element (MRE) bind to the miRNA in a competitive manner, thereby affecting the expression levels of transcripts. The ceRNA hypothesis subverts the traditional concept of one-way regulation between miRNA and target genes, and has great biological significance in RNA regulatory networks. Among many transcripts, long non-coding RNA (lncRNA) is a class of non-coding RNA with a sequence length of more than 200 nucleotides. The study of lncRNA involves in genetics, molecular biology, gene regulation, diseases (cancer, neurological diseases, etc. ) and other fields. miRNA and lncRNA form an interactive regulatory network, and lncRNA can be used as ceRNA to inhibit the function of miRNA, thereby affecting the expression of subsequent genes. In recent years, with the development of bioinformatics technology, researchers have found that the mechanism of ceRNA is not only related to human cancer diseases, but also plays an important regulatory role in the biological processes of muscle cell differentiation, adipocyte differentiation and granulosa cell apoptosis in various complex animals. The author reviewed the regulation mechanism of ceRNA, analyzed the influencing factors of ceRNA network regulation, and reviewed the research progress of lncRNA as ceRNA regulating miRNA in different animals, so as to provide reference for further research on the regulation network of lncRNA and miRNA, and provide new ideas for the development of animal husbandry and complex animal disease treatment.

The gut bacterial composition of grazing male yaks at different ages were compared and analyzed to provide a basis for the regulation of bacterial function. Rectal fecal samples were collected from 0.5-year-old (Ac group), 1.5-year-old (Bc group), 2.5-year-old (Cc group) and 3.5-year-old (Dc group) grazing male yaks in Xiewu town, Yushu prefecture, Qinghai province. 16S rRNA sequencing technology was used to analyze the bacterial composition of yaks at different ages. Alpha diversity and Beta diversity were calculated by Qiime software. PCoA diagram was drawn by R software. Anosim analysis was used to test the difference between groups. Mothur method and the SSUrRNA database of SILVA132 were used for species annotation analysis, and Tax4Fun was used for functional prediction of gut bacterial. The results showed that the increase of age had a significant effect on the number of OTUs and Simpson index (P<0.05), and simpson index of gut bacterial composition was the lowest in Cc group. The results of Beta diversity analysis showed that there were significant differences between Cc and Ac, Dc groups, and between Ac and Dc groups (P<0.05). At the phylum level, the relative abundance of Firmicutes in each group was the highest, and that of Bacteroidetes was the second. The relative abundance of Firmicutes in Bc and Cc groups were significantly higher than that in Ac and Dc groups (P<0.05). The relative abundance of Verrucomicrobia was significantly different among different age groups and the highest in Cc group (P<0.05). At the genus level, with the increase of age, the relative abundance of Alloprevotella increased gradually (P<0.05). There were significant differences in some non-dominant Romboutsia, Oscillibacter, Akkermansia and Mailhella between groups (P<0.05). In function prediction, the metabolic function of yaks gut bacterial showed the highest relative abundance, which was more than 44% in each group. The expression abundance of functional genes of amino acid metabolism, signal transduction and cell motility in Cc group were significantly higher than that in Ac and Dc groups (P<0.05). There was no significant difference in the expression abundance of functional genes among Ac, Bc and Dc groups (P>0.05). In summary, the gut bacterial composition of grazing male yaks changed in diversity with the increase of age. The changes of gut bacterial composition of 2.5-year-old yaks indicated that the protein digestion, metabolism and immunity of yaks were high at this stage, which provided a good foundation for fattening.

The purpose of this experiment was to study the effects of heat stress on physiological and blood biochemical indexes of Leizhou, Nubia and their hybrid F1 goats in Guangdong. Differences of heat adaptation performance were evaluated among the three groups. In this experiment, five-month-old wethers of each group were selected as the research objects. In a heat stress environment with the mean daily temperature humidity index (THI) of 84.30 in Guangdong summer (July 2019), four physiological indexes were measured at 08:00, 14:00 and 20:00 on the first two days of the experimental period, and 29 biochemical indexes were detected after blood collection on the third day of the experiment period. The results showed that skin temperature (ST), rectum temperature (RT), respiratory rate (RR) and heart rate (HR) of the three groups increased with the increase of THI from 08:00 to 14:00, and decreased with the decrease of THI from 14:00 to 20:00. When the mean THI was higher than 82 at 08:00 and 14:00, RT and RR of Leizhou goats were extremely significantly or significantly lower than those of Nubia goats (P<0.01;P<0.05). Among leukocyte classification indexes, neutrophil ratio (NEUTr) of Leizhou goats was the highest, and significantly higher than that of Nubia goats (P<0.05). On the contrary, lymphocyte ratio (LYMPHr) of Leizhou goats was the lowest among the three groups, and significantly lower than that of Nubia goats (P<0.05). Among blood electrolyte indexes, potassium ion (K⁺) of Leizhou goats was the highest among the three groups, and was extremely significantly higher than that of Nubia goats (P<0.01). Among serum enzyme indexes, lactate dehydrogenase (LDH) of Nubia goats was the highest among the three groups, and was significantly or extremely significantly higher than that of the other two groups (P<0.05;P<0.01). Alanine aminotransferase (ALT) of Nubia goats was higher than that of Leizhou goats and hybrid F1 goats, which was consistent with the trend of LDH, but it was only significantly higher than that of hybrid F1 goats (P<0.05). By comparing the two serum enzyme content indexes (ALT and LDH), hormone level of triiodothyronine (T₃) showed an opposite trend among the three groups, and the T₃ level of Nubia goats was the lowest, and significantly lower than that of Leizhou goats (P<0.05). The results of PCA cluster analysis showed that there was a significant population separation between Leizhou goats and Nubia goats, and hybrid F1 goats were between the two parent populations. Based on the above results, the experimental goats were in a heat stress state throughout the whole experiment period. Effects of heat stress on the physiological and blood biochemical indexes of the studied goats were identified, and which reflected the difference of heat resistance among groups. The heat resistance of Leizhou goats was better than that of Nubia goats, while the heat resistance of hybrid F1 goats was between the two parent goats.

This experiment was designed to investigate the effects of different doses of Sophora subprosrate polysaccharide (SSP) on the growth and immune function of Oreochromis niloticus. In the test, 400 tails of Oreochromis niloticus were randomly divided into 5 groups with 4 replicates per group and 20 Oreochromis niloticus per replicate. The animals were fed the basal diet supplemented with 0 (CK group), 1.0 (SSP 1 group), 2.0 (SSP 2 group), 4.0 g/kg (SSP 3 group) SSP and 20 mg/(kg·BW·d)(APS group), respectively. The experiment period was 30 d. At the end of the experiment, Astragalus polysaccharide the growth indices, such as weight gain rate (WGR), specific growth rate (SGR)and feed conversion ratio (FCR), blood physiological and biochemical indexes, serum and liver nonspecific immunity indexes of Oreochromis niloticus were determined. The results showed that compared with CK group, there were no significant changes in WGR, SGR and FCR in SSP 1 and SSP 2 groups (P>0.05). The levels of cholesterol (TC) and total protein (TP) in serum were significantly decreased (P<0.05), the organ indices of liver and spleen of Oreochromis niloticus in experimental groups (SSP 1, SSP 2, SSP 3 and APS groups) were no significent changes (P>0.05), IL-12 level was significantly increased (P<0.05). The levels of IL-2 were increased (P<0.05) and the levels of IFN-γ were decreased (P<0.05) in SSP 2 and SSP 3 groups. The activities of POD and GSH-Px of Oreochromis niloticus in SSP 1 and SSP 2 group were significantly higher than those in the CK group (P<0.01). In brief, SSP could promote the growth, antioxidant capacity and immune function of Oreochromis niloticus, and the optimal supplemental level of SSP was 2.0 g/kg.

In order to determine the best supplementary feeding time by fence for lambs, the growth performance, wool quality and serum biochemical indexs of Dorper×Hu sheep hybrid lambs with different supplementary feeding time were tested. 180 healthy male lambs with uniform body weight and similar body conditions were selected and divided into 3 groups with 60 lambs in each group. Each group started supplementary feeding by fence at 15-day-old and ended at 40-day-old. The time of supplementary feeding by fence was 6 h per day for lambs in group Ⅰ and the time of group Ⅱ was transitioned from 6 to 10 h and from 6 to 12 h in group Ⅲ. Lambs were weighed at the beginning and ending of the experiment to determine their growth performance, at the end of the experiment, wool and blood samples were collected to determine wool quality and serum biochemical indexes. The results showed that with the prolongation of supplementary feeding time by fence, the average daily gain of lambs increased gradually, the feed intake decreased, and the ratio of feed to gain decreased, but there was no significant difference between groups (P>0.05). There were significant differences in the proportion of medullated and non medullated wool in lamb lateral wool among different groups (P<0.05). For proportion of medullated hairs, group Ⅰ>group Ⅱ>group Ⅲ, while for percentage of unmyelinated hairs, group Ⅲ>group Ⅱ>group Ⅰ, and the proportion of unmyelinated hair increased with the prolongation of supplementary feeding time by fence. There was no significant difference in other wool indexes among groups (P>0.05). The content of serum triglyceride in groups Ⅱ and Ⅲ was significantly higher than that in group Ⅰ(P<0.05), the content of serum albumin in groups Ⅰ and Ⅱ was significantly higher than that in group Ⅲ (P<0.05), the content of creatinine in group Ⅱ was significantly higher than that in group Ⅲ (P<0.05), and the content of creatine kinase in group Ⅱ was significantly higher than that in groups Ⅰ and Ⅲ (P<0.05). In conclusion, when lambs were gradually isolated from their mothers from 15 to 40 days of age, The growth performance of lambs was improved with the extension of feeding time. The results showed that group Ⅲ, which had the longest feeding time, had the best performance in average daily gain, feed to weight ratio and wool quality.

Vitamin A is a kind of fat-soluble vitamin, it's necessary for maintaining normal physiological function, biochemical metabolism and growth and development of beef cattle, it cannot be synthesized by the body and must be supplied by diet. Vitamin A not only affects the visual and skeletal development of beef cattle, but also plays an important role in the regulation of fat deposition and muscle marbling formation in beef cattle. The requirement of vitamin A is different at different physiological stages of beef cattle in production, vitamin A supplementation at the fetal and calf stages can enhance intramuscular adipocyte development and adipocyte proliferation and promote intramuscular fat deposition, and vitamin A restriction during the fattening period can improve intramuscular fat deposition and marbling grade in beef cattle. Marbling is closely related to the tenderness and flavor of beef, and is an important indicator to measure the quality of beef. Vitamin A promotes fat formation through retinol, retinaldehyde and retinoic acid in beef cattle, and can play important roles in each stage of adipogenesis, adipogenic differentiation and accumulation. The process of fat deposition is regulated by the transcription factors peroxisome proliferator-activated receptors (PPARs), CCAAT-erbinding protein (C/EBPs) and Janus kinase-signal transduction and activation of transcription (JAK-STAT) signal transduction pathways. Epigenetically modified DNA methylation and demethylation can also be involved in adipocyte differentiation and adipose tissue growth and development by regulating the expression of related genes during adipogenesis, thus affecting fat deposition in beef cattle. This review focused on the definition of vitamin A, its biological function and the process of adipose tissue formation, and emphatically expounded the mechanism of vitamin A supplementation and feeding restriction in beef cattle at different physiological stages, which affected the regulation of fat deposition by adipogenesis-related signaling pathways through the expression levels of transcription factors and epigenetic modifications, in order to promote vitamin A to improve beef cattle quality and high-grade beef production and provide a reference.

In order to study the differential expression of circRNA in Duroc and Yorkshire testis, a specific porcine testis circRNA library for sequencing was prepared using ribosomal RNA (rRNA) depletion and liner RNA digested method, and then sequenced on an Illumina PE150 platform. After quality control, comparison and splicing the sequencing data were obtained for subsequent analysis. The bioinformatics package find_circ and CIRI were used to identify circRNA and count the number of reads to obtain the cricRNA expression profile of porcine testis. Differential expression analysis of two groups was performed using the DESeq2 and then enrichment of those different expression circRNAs to screen which associate with male reproduction. The results showed that there was abundant expression of circRNA in testis. A total of 21 743 circRNAs were identified, and 632 circRNAs were significant differentially expressed in Duroc and Yorkshire pigs(|log₂(FoldChange)|>1, P<0.05), 281 circRNAs up regulated and 351 circRNAs down regulated in Duroc pigs. The enrichment of difference expressed circRNA showed that there were 52 GO terms related to reproduction, and 9 of them related to male reproduction. 6 circRNAs related to male reproduction (circ_0030058, circ_0009504, circ_00178101, circ_0019933, circ_0033379 and circ_0017932) were screened out and they might take part in spermatogonia differentiation, spermatogenesis and sperm cell development. In conclusion, testis of Duroc and Yorkshire boars possess differentially expressed circRNA, which might be involved in spermatogenesis, and could also be used as biomarkers to predict boar fertility.

Dong'e Black donkey was a new breed of skin and meat type as the basic group of Dezhou donkey. The aims of the present study was to explore the optimal combination of different breeding measures on the basis of the current breeding program, in order to establish a high-quality and efficient Dong'e Black donkey breeding system. Based on the production and breeding situation of Dong'e Black donkey, with selection index and gene flow methods, the ZPLAN software was used to analyze the breeding efficiency of current breeding program, and optimize the various factors affecting the breeding program of Dong'e Black donkey. The results showed that the genetic progress of the current breeding program was 271.78 ￥ per donkey and per year, the breeding profit was 1 123.70 ￥ per donkey and per year, and the current generation interval was 5.24 years. The improvement of each trait during the breeding period were as following:Weaning weight, 1.50 kg;18th months weight, 0.09 kg;First calving age, -0.02 days;Calving interval, -0.17 days;Skin production, 1.04 kg. The combination analysis of different levels of optimized factors showed:Based on the total herd size of base donkey population of 30 000, the proportion of breeding groups was 0.5, the service life of seed male donkey, test male donkey and breeding female donkey in breeding group was 1 year, the service life of seed female donkey was 2 years, and the service life of male and female donkey in production group was 1 year, the maximum genetic progress and breeding profits would be obtained, which were 320.40 ￥ per donkey and per year, 2 945.00￥ per donkey and per year. They were 17.9% and 162.0% higher than the current breeding programs. The current breeding program of Dong'e Black donkey had not reached the ideal breeding status, and more improvement was needed for it. After optimizing all the influencing factors, the maximum genetic progress and breeding profits could be obtained.

The aim of this study was to use Real-time quantitative PCR technique to detect sexually controlled sperm of Duroc pigs, in order to establish a rapid, accurate and cost-effective method for sperm purity detection. The sex -determining region Y (SRY) gene located on the Y chromosome and A-kinase anchoring protein 4 (AKAP4) gene located on the X chromosome of pig were selected, using genomic DNA extracted from pig ear tissue samples as a template for PCR amplification for specific primer verification. The gel recovery kit was used to recover the gel, the plasmid were extracted. After testing, the obtained two plasmids were diluted to the same concentration (20 ng/μL), and mixed to construct plasmid templates containing different proportions of SRY and AKAP4 genes, which were used to construct the reaction template for the standard curve of sperm purity detection. The purity of 3 mixed X spermatozoa (P. x_gro1, P. x_gro2 and P. x_gro3) and 3 mixed Y spermatozoa (P. y_gro1, P. y_gro2 and P. y_gro3) were detected by SRY and AKAP4 genes specific primers, respectively. The results showed that the purity obtained using SRY primers of P. x_gro1, P. x_gro2 and P. x_gro3 sperm groups were 91.44%, 91.93% and 88.99%, and the purity of P. y_gro1, P. y_gro2 and P. y_gro3 sperm groups were 89.91%, 87.31% and 88.71%, respectively. The purity obtained by using AKAP4 primers of P. x_gro1, P. x_gro2, P. x_gro3 sperm groups were 91.44%, 91.93% and 88.99%, and the purity of P. y_gro1, P. y_gro2, P. y_gro3 sperm groups were 89.91%, 87.31% and 88.71%, respectively. Independent sample t-test analysis showed that no significant difference between the two test results, indicating that the results were accurate for sperm purity detection. In this experiment, Real-time quantitative PCR was used to accurately detect the purity of sperm sorted by flow cytometry, and established a new method for detecting the ratio of X sperm and Y sperm in porcine sperm samples by Real-time quantitative PCR.

The purpose of this study was to investigate the polymorphism of uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3) genes and its association with growth traits in Yanbian Yellow cattle. 120 Yanbian Yellow cattle aged 24 months were used as experimental subjects, construction of mixed DNA pool after extracting blood genomic DNA. UCP2 and UCP3 genes were sequenced and analyzed by DNA sequencing method, the genotyping of UCP2 and UCP3 genes were detected by high resolution melting curve, and the correlation analysis were conducted with growth traits. The results showed that UCP2 gene at 53 412 568 bp had C>G mutation (g. 53412568 C>G) and two alleles of C and G, which formed three genotypes of CG(0.358), GG(0.125) and CC(0.516). UCP3 gene at 53 443 536 bp had C>T mutation (g. 53443536 C>T) and two alleles of C and T, which formed three genotypes of CT(0.383), CC(0.450) and TT(0.167). Correlation analysis showed that the body weight and hip width of CC genotype of UCP2 gene g. 53412568 C>G were significantly higher than those of GG genotype, and the back height of CG genotype was significantly higher than that of GG genotype (P<0.05). The chest circumference of CC genotype individuals of UCP3 gene g. 53443536 C>T was significantly higher than that of TT genotype (P<0.05). In conclusion, UCP2 and UCP3 genes played a certain role in the growth and development of Yanbian Yellow cattle, which could be used as a reference for the research of modern molecular breeding of Yanbian Yellow cattle in the future.

The purpose of this study was to explore the effects of N-carbamylglutamate (NCG) feeding for different periods on superovulation and blood biochemical indicators in dairy cows. A total of 16 Holstein heifers were selected and randomly divided into two groups, each with 8 heifers. The control group was fed with a basal diet, and the test group was fed 20 g/(d·head) of NCG on the basis of the basal diet. The effect of feeding period of NCG on the superovulation effect of donor cattle was measured by the FSH method of decreasing injection for 4 consecutive days. Tail root vein blood were collected on the 0, 5, and 9 d of the 3 superovulation treatments to determine hormone indicators and serum biochemical indicators. The results showed that:①NCG was fed for 20 d (the frist superovulation), there were no significant differences in the number of recovered embryos, available embryos, degenerated embryos and unfertilized oocytes between test and control groups (P>0.05). When NCG was fed for 50 d(the second superovulation), the number of embryos recovered in test group was significantly higher than that in control group (P<0.05). When NCG was fed for 80 d (the third superovulation), the number of available embryos in test group was significantly higher than that in control group (P<0.05). In 3 superovulation tests, the number of embryos recovered in the test group was significantly higher than that in control group (P<0.05). ②The feeding period of NCG had no significant effect on the concentrations of FSH, LH, P₄ and E₂ in the blood of donor cattle (P>0.05). It showed that NCG feeding period had no effect or was a secondary influencing factor on the secretion of reproductive hormones, and there was no significant correlation between the change of superovulation effect and the change of reproductive hormones. ③The feeding period of NCG had no significant effect on the concentration of AST in the blood of the donor cattle (P>0.05). The concentration of GLU in the calf serum of the donors in test group was significantly higher than that in control group when NCG was fed for 13 d (P<0.05). The concentration of BUN in the calf serum of the donors in test group was significantly lower than that in control group when NCG was fed for 34 d (P<0.05). The concentration of NO in the calf serum of the donors in test group was significantly higher than that in control group when NCG was fed for 43 and 69 d (P<0.05). In conclusion, each donor heifer was fed with 20 g NCG per day for 3 consecutive superovulation, the number of recovered embryos and the number of available embryos could be increased by 4.98 and 1.8, respectively, and then reduce the production cost of embryos.

The aim of this study was to investigate the effect of tannin acid on porcine in vitro maturation (IVM) and their subsequent embryonic development. Thus, the porcine cumulus-oocyte complexes were supplemented with different concentration (0, 1, 10 and 100 μg/mL) of tannin acid during 42 h IVM, and then cumulus cells expansion, oocyte maturation rate, and the level of glutathione (GSH), reactive oxygen species (ROS) and growth differentiation factor 9 (GDF9) in oocytes were evaluated. Moreover, parthenogenetic activation (PA) and in vitro fertilization (IVF) were carried out, and cleavage rate, blastocyst formation rate and total cell number of blastocyst were evaluated. The results showed that when compared with the control group, 10 μg/mL tannin acid significantly increased the cumulus expansion index and 100 μg/mL tannin acid significantly decreased it (P<0.05). There was no significant difference in oocyte maturation rate between control, 1 and 10 μg/mL tannin acid groups (P>0.05), while 100 μg/mL tannin acid significantly decreased maturation rate (P<0.05). Moreover, the levels of GSH and GDF9 were dramatically enhanced (P<0.05), and the level of ROS was dramatically reduced by 1 and 10 μg/mL tannin acid supplementation (P<0.05). The results of development ability of parthenogenetic embryos and in vitro fertilization embryos showed that there was no significant difference in cleavage rate between tannic acid groups and control group (P>0.05). The blastocyst rate of parthenogenetic embryos and in vitro fertilization embryos in 10 μg/mL tannic acid group were significantly increased (P<0.05). The number of blastocyst cells in parthenogenetic embryos and in vitro fertilization embryos in 100 μg/mL tannic acid group was significantly lower than that in other groups (P<0.05). Taken together, 10 μg/mL tannin acid improved oocyte quality and the subsequent embryo developmental competence by increasing cumulus cells expansion and the GSH and GDF9 levels, and reducing the ROS level of the porcine oocyte.

Octamer-binding transcription factor 4 (OCT4) is a POU-family transcription factor, which is specifically expressed in early embryos, embryonic stem cells and germ cells. Since both embryonic stem cells and germ cells come from early embryos, especially inner cell mass (ICM) cells, OCT4 as a core development factor is involved in coordinating the maintenance of early embryo pluripotency and the formation of ICM. The exclusively expression of OCT4 and caudal type homeobox transcription factor 2 (CDX2) in mouse embryonic cells is crucial for ICM and trophectoderm (TE) differentiation and results in OCT4 specially expressed in ICM and CDX2 specially expressed in TE. However, in other large mammals represented by pigs, OCT4 continues to be expressed in blastocyst TE, indicating that OCT4 is highly conserved and has a certain species specificity. Researching the expression and regulation mechanism of OCT4 in early embryonic development of mammals is significant for us to understand the pluripotency and maintenance of embryonic stem cells. This review summarized the expression rules and expression regulation mechanism of OCT4 in mammalian early embryonic development, hoping to provide a reference for in-depth comprehending of mammalian early embryonic development regulation.

The purpose of this study was to establish an efficient timed artificial insemination (TAI) technology for multiparous sows. The experiment studied the effects of timed insemination on the reproductive performance of multiparous sows, the interval between weaning and farrowing, the farrowing performance of sows with different parities and the serum reproductive hormone levels within 7 days after weaning. A total of 309 binary (Landrace×Large White) sows of 2-8 parity randomly divided into control group and test group. The sows of control group were subjected to conventional artificial insemination (AI), and the sows of test group were given TAI by PMSG (1 000 IU) treatment for 24 h after weaning and GnRH (100 μg) treatment at 72 h interval, then followed by inseminations at 24 and 40 h after GnRH injection. The effects of timing insemination on reproductive performance of multiparous sows were evaluated by counting estrus rate in one week after weaning, conception rate, delivery rate and litter size. The effect of regular insemination on weaning delivery interval of multiparous sows was detected through the statistics of weaning time and delivery time. The radioimmunoassay (RIA) method was used to detect the serum E₂, LH, FSH and P₄ levels of sows with 2 to 4 parities within one week of weaning to study the effect of timed insemination on the reproductive hormones of sows. The results showed that the estrus rate of sows in the test group was significantly higher than that in the control group (P<0.05), but the conception rate and delivery rate were not significantly different between the two groups (P>0.05). Litter size per litter, qualified litter size per litter and reproductive efficiency increased, but the difference was not significant (P>0.05). Timed insemination significantly shortened the interval between weaning and farrowing (P<0.05). The estrus rate, conception rate and delivery rate of 3-4 litter sows were significantly higher than those of the control group (P<0.05). In terms of reproductive hormone, the E₂ level of the test group rose rapidly after PMSG injection, and was continuously higher than that of the control group (P<0.05) within 66 to 96 h after the timed insemination treatment. The level of P₄ in the experimental group was significantly lower than that in the control group from weaning to mating (P<0.05), but increased rapidly after mating, and higher than the control group. There was no significant difference in the content of LH and FSH between the two groups. In summary, timed insemination could effectively increase the rate of weaning and estrus of multiparous sows, reduce the number of non-productive days, and could significantly improve the reproductive performance of sows with 3 to 4 litters.

This study was aimed to investigate the effects of crocin on the in vitro maturation and subsequent embryonic development of mouse oocytes. Different concentrations of crocin (0, 5, 10, 15, 20, 25, 30 μmol/L) were added to the in vitro mature (IVM) culture medium, after IVM for 12 h, the first polar body extrution, the contents of reactive oxygen species (ROS) and glutathione (GSH) in the cytoplasm of oocytes were detected and then in vitro fertilization (IVF)was performed. Fertilization rate, cleavage rate and blastocyst rate were counted at 6, 24 and 96 h after IVF, respectively. The results showed that when compared with control group, the addition of 10 and 15 μmol/L crocin significantly increased the polar body extrution rate (P<0.05), and when the concentration of crocin further increased, the polar body extrution rate decreased, 30 μmol/L crocin significantly decreased the polar body extrution rate (P<0.05). 5, 10 and 15 μmol/L crocin could significantly reduce the content of ROS (P<0.05), and 10 and 15 μmol/L crocin increased the content of GSH of oocytes (P<0.05). Compared with control group, 10 μmol/L crocin had no significant difference in fertilization rate, cleavage rate and blastocyst rate (P>0.05), 15, 20, 25 and 30 μmol/L crocin all significantly decreased fertilization rate and blastocyst rate (P<0.05), and had no significant difference on cleavage rate (P>0.05). The results indicated that the addition of 10 μmol/L crocin to mouse oocytes in vitro maturation medium could significantly increase the rate of the first polar body extrution, reduce the content of ROS, and increase the content of GSH in oocytes, but did not affect the embryonic development ability of oocytes.

To understand the epidemic and genetic variation of porcine Pseudorabies virus (PRV) in Tianjin area, gB, gC and gE genes of 20 isolated strains from Tianjin during 2015 to 2020 were amplified, sequenced and analyzed with the sequences of reference strains. Similarity analysis showed that when compared with the Chinese strain after 2012, the homology of nucleotide and amino acid of the three genes were as follows:gB gene were both 99.9%-100%;gC gene were 99.7%-100% and 99.4%-100%, respectively; gE gene were 99.7%-100% and 99.6%-100%, respectively. Phylogenetic and sequence alignment analysis showed that according to gB, gC, gE genes phylogenetic trees, PRV strains could be divided into GⅠ and GⅡ genotypes, isolated strains from Tianjin were clustered in GⅡ genotype. 19 isolated strains showed a closer genetic relationship with the PRV variant strains, distributed in the same gene subtype, and had the same amino acid variation characteristic. The gB and gE genes of the other strain (TJBD6) showed a closer genetic relationship with the PRV variant strains, and the amino acid variation positions were the same as those of the PRV variant strains, however, its gC gene was closely related with the classical strain Ea, and the nucleotide and amino acid sequence similarity was 100%. These results indicated that there were two types of PRV strains prevalent in Tianjin since 2015:Classical strains and variant strains, and the variant strains were the main prevalent strains. This study preliminarily investigated the molecular epidemiological characteristics of PRV in Tianjin area, which could provide a basis for the prevention and control of pseudorabies.

In order to establish a recombinase polymerase amplification (RPA) diagnostic method for Pasteurella multocida from goat, the pET-28a(+)-KMT1 plasmid standard was constructed based on the KMT1 gene sequence of Pasteurella multocida from goat. The specific primers and RPA fluorescent probe based on RPA technology were designed to establish the optimal reaction system of Real-time RPA. The sensitivity of the method was detected and the correlation curve was drawn by 10 fold gradient diluted plasmid standard. The genomic DNA of 10 different strains were used as templates to verify the specificity of the method. Finally, the reliability of the method was verified by the tissue samples of goats and mice infected with Pasteurella multocida. The results showed that the optimal reaction temperature was 39 ℃, the optimal primer was KMT1-Fe1, and the sensitivity was 100 copies/μL. The detection limit was 10 copies/μL. There was no cross reaction with Escherichia coli, Staphylococcus aureus, Salmonella Paratyphi, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Klebsiella acidogenesis, Brucella S2 and Acinetobacter baumannii. The positive rate of 13 tissue samples was 76.9%, and 92.3% of them were consistent with Real-time PCR. In conclusion, the Real-time RPA of Pasteurella multocida from goat had the characteristics of strong specificity, high sensitivity, good reliability, fast and convenient, which was suitable for clinical molecular diagnosis of Pasteurella multocida.

In order to establish a rapid immunochromatographic assay for H9N2 subtype Avian influenza virus (AIV), BALB/c mice were immunized with H9N2 subtype AIV purified by differential centrifugation. The splenocytes of immunized mice were fused with myeloma cell SP2/0 and selectively cultured with HAT. MDCK cells were infected with H9N2 subtype AIV to establish a monoclonal antibody detection method of heterologous immunoperoxidase monolayer assay (IPMA). The neutralizing monoclonal antibody against H9N2 subtype AIV was screened and identified by IPMA screening and continuous cloning of hybridoma cells. Using colloidal gold labeled HA monoclonal antibody, pairing HA monoclonal antibody and sheep anti mouse IgG as detection line and quality control line, a rapid detection strip for H9N2 subtype AIV was prepared, and its specificity and sensitivity were determined. The results showed that 11 hybridoma cells stably secreting monoclonal antibody against H9N2 subtype AIV were obtained, and the titers of monoclonal antibodies ascites IPMA were 1.28×10^-4 to 2.56×10^-5. Monoclonal antibodies 3A2, 5H6, 6B8, 7E10 and 9G12 showed hemagglutination inhibitory activity in hemagglutination inhibition test (HI), and their HI titers ranged from 6log2 to 9log2. Monoclonal antibodies 3A2, 6B8 and 9G12 had significant virus neutralizing activity against H9N2 subtype AIV in virus neutralization test. The neutralizing titers were 1:6 400, 1:25 600 and 1:25 600 respectively. Western blotting showed that the neutralizing monoclonal antibody recognized the linear epitope of HA protein. The titer of H9N2 subtype AIV allantoic fluid detected by paired monoclonal antibodies 3A2 and 9G12 was 9log2, the sensitivity was equivalent to that of classical hemagglutination test (HA), and there was no cross reaction with other subtypes of AIV (H1, H3, H5, H7), Newcastle disease virus and chicken infectious bursal disease virus. In this study, the monoclonal antibody against H9N2 subtype AIV with virus neutralizing activity was prepared, and the H9N2 subtype AIV detection strip was preliminarily developed, which laid a good research foundation for the new vaccine development and rapid detection of H9N2 subtype AIV.

The aim of this study was to obtain a highly purified antigenic peptide of Batai virus (BATV) G1 protein and to establish an indirect ELISA method for detecting sheep serum antibodies against BATV. Firstly, the codon of 189-239 amino acids strong antigen peptide encoded by 51 amino acids of G1 protein were optimized. After gene synthesis, a recombinant expression plasmids pET-32a-G1_189aa-239aa was constructed, and transformed into E. coli BL21 competent cells to induce expression. A large number of rHis-G1_189aa-239aa peptides were obtained by optimizing the expression conditions, which were purified by nickel column affinity chromatography, and the protein concentration was determined by BCA kit. Then the antigen specificity of rHis-G1_189aa-239aa peptide was analyzed by Western blotting. Using rHis-G1_189aa-239aa peptide as antigen and positive serum of BATV sheep as antibody, the reaction conditions were optimized by square titration, and the indirect ELISA detection method of BATV was established. SDS-PAGE result showed that the rHis-G1_189aa-239aa peptide existed mainly in the form of inclusion bodies in E. coli, the protein molecular weight was 24.5 ku, and the expression of rHis-G1_189aa-239aa peptide reached the peak after induced by 0.1 mmol/L IPTG for 5 h. After purification by nickel column affinity chromatography, the protein concentration was 0.296 mg/mL. Western blotting results showed that the specificity of rHis-G1_189aa-239aa peptide was good. The indirect ELISA method using rHis-G1_189aa-239aa peptide as antigen was established. Through the optimization of conditions, the optimum antigen coating concentration was 2.5 μg/mL, serum and the dilution of the second antibody were 1:60 and 1:10 000, respectively. When the sample D_{450 nm} value ≥ 0.367 was positive, D_{450 nm} value ≤ 0.319 was negative. This method had no cross reaction with the positive serum of Goatpox virus and Brucella abortus, the coefficient of variation within and between batches was less than 10%, and the positive serum could be diluted to 1 600 times at most. The method had the characteristics of strong specificity, high sensitivity and good repeatability. This method was used to detect 120 sheep serum samples in Yunnan, and the results showed that the serum positive rate was 21.67%. The rHis-G1_189aa-239aapeptide with high specificity and purity was obtained in this study, and the indirect ELISA method was established, which could be applied to the seroepidemiological investigation of sheep BATV and laid a foundation for the study of epidemic prevention and control and pathogenic mechanism.

The aim of this study was to obtain major royal jelly protein 2 (MRJP2) by insect cell-baculovirus expression system, so as to get basic materials for subsequent in-depth study of functional research on MRJP2. According to the MRJP2 gene sequence of Apis mellifera L. in GenBank, MRJP2 gene was amplified by PCR and cloned into the eukaryotic expression vector pFastBac1. The recombinant baculovirus MRJP2-Bacmid plasmid was constructed and transfected into Sf9 insect cells, with P2 generation baculovirus infects Sf9 cells to induce expression. The expressed product was purified by chromatography column and ion exchange column, and the target protein was analyzed and identified by SDS-PAGE, Western blotting and Q Exactive. The results showed that this experiment successfully constructed the baculovirus plasmid MRJP2-Bacmid, transfected into Sf9 insect cells and obtained the expression product. The verification results of SDS-PAGE and Western blotting showed that the MRJP2 recombinant protein (52 ku) with high purity was successfully expressed in insect cell-baculovirus expression system. The results of mass spectrometry analysis showed that the recombinant protein matched 39 unique peptides of MRJP2 in the honeybee protein database, with a sequence coverage of 61%, and had the highest matching score with MRJP2. It was further confirmed that the recombinant protein was MRJP2. In this study, the insect cell-baculovirus system was used to successfully express MRJP2, which laid a foundation for the subsequent in-depth study of the biological function of the protein.

This study was designed to investigate and compare the immune effect of different immunosuppressive mice models which were injected with cyclophosphamide (CTX) at different doses and adminstration times. 200 male Kunming mice aged 7 weeks were randomly divided into 5 groups, including the control group (normal saline, 0 mg/g CTX), groups injected with low dose (0.04 mg/g) and high dose (0.08 mg/g) CTX for three times, groups injected with low dose (0.10 mg/g) and high dose (0.16 mg/g) CTX for once, respectively. The mice in 3 injections groups were given intraperitoneal CTX for 3 days and the mice in the control group were given equal volume normal saline in the same way. In the single injection groups, mice were given intraperitoneal CTX injection once only on the 1st day of the experiment. All the mice were weighted every day. On the 1st, 2nd, 4th, 6th, 9th, 10th and 11th day of the experiment, 8 mice of each group were selected to test tail vein blood. On the 1st, 4th, 6th, 9th and 11th day, 8 mice of each group were executed and measured nucleated cells of femur marrow, thymus indexes and spleen indexes. The results showed that the immunosuppressive model could be established successfully at all doses. In terms of inhibition effect, 0.08 mg/g three injections had the best immunosuppression effects on white blood cells, bone marrow nucleated cells, weight gain, spleen and thymus, and the 0.16 mg/g once injection had the best immunosuppression effect on red blood cells. In terms of inhibition cycle, the duration of inhibition was different in various immune sites, the inhibition cycle of cyclophosphamide were shorter on leukocytes, bone marrow nucleated cells and spleen than that on erythrocyte and thymus. The multiple injections groups showed earlier hyperimmunity in leukocyte than once injection groups, while the effect on spleen was just on the contrary. In conclusion, different doses and adminstraion times of CTX administration had different effects on the immune status and immunosuppression cycle of different immune sites. This study could provide appropriate detection dose and time reference for different immune indexes in pharmacological experiments.

In order to explore infection of Porcine reproductive and respiratory syndrome virus (PRRSV) in native pig breeding farms of Hunan province, 287 blood samples were collected from two native pig breeding farms during 2019 to 2020. PRRSV were detected by RT-PCR in 41 mixed seral samples. Open reading frame 5 (ORF5) gene of PRRSV were amplified from PRRSV positive samples by high fidelity PCR. DNAStar software was performed to analyze the genetic evolution of ORF5 gene and GP5 amino acid between these PRRSV strains and different PRRSV strains from China and abroad after sequencing. Finally, PRRSV positive seral were inoculated in Marc-145 cells followed by blind passaging. The titer of the isolated PRRSV was determined by Reed-Muench method. The results showed that 3 samples from the 41 mixed samples were positive for PRRSV. Six ORF5 sequences were amplified from PRRSV positive mixed samples, all of which belonged to branch lineage 8 of PRRSV-2 strain, and the nucleotide homology was 99.2% to 99.8%. The corresponding GP5 protein acid sequences encoded by six ORF5 gene sequences were different in the signal peptide region (position 23), potential N-glycosylation site (position 33) and epitope C (position 59). Obvious cytopathic effect (CPE) was observed in the Marc-145 cells inoculated the PRRSV positive sera after five blind passages. A stain of PRRSV designated as NX-1 was obtained with a TCID₅₀ of 4×10⁵/mL. The data indicated that there existed infection of PRRSV in Hunan native pig breeding farms. The prevalent PRRSV isolate belonged to lineage 8 of PRRSV-2 with several variations in the amino acid sequence of GP5, which might be associated with immune failure. The present study could supply a foundation for immune prevention and control in Hunan native pig breeding farms.

Brucella canis is one of the six classical species of Brucella. It is a natural rough type with weak virulence. For a long time, the harm of Brucella canis has been ignored because of its low pathogenicity to human. Since it was found in the United States in 1966, it has spread to many places in the world. With the continuous development of the kennel industry and the increasing number of pet dogs, the incidence rate of canine brucellosis caused by Brucella canis has been increasing. In some areas, it has become an epidemic disease, and the threat to the public health security of human society is also increasing. There is no reliable diagnosis and effective vaccine for canine brucellosis caused by Brucella canis. In view of this, on the basis of consulting the relevant literature, this paper reviewed the etiology, epidemiology, pathogenic mechanism, test method, vaccine of Brucella canis, in order to provide meaningful reference for the prevention and control of canine brucellosis caused by Brucella canis.

The purpose of this study was to express bovine interferon regulatory factor 7 (IRF7) protein by prokaryotic expression system, and purify the protein, so as to prepare high purity bovine IRF7 rabbit polyclonal antibody. Bioinformatics software was used to predict and analyze the potential biological functions of bovine IRF7, with reference to the bovine IRF7 gene sequence (GenBank accession No. :NM_001105040.1), primers for bovine IRF7 were designed and PCR was used to amplify bovine IRF7 gene from Bovine viral diarrhea virus (BVDV) infected MDBK cells. The bovine IRF7 gene was attached to the pMD19-T clone vector, and then attached to the prokaryotic expression vector pET-28a(+) to transform into Escherichia coli DH5α competent cells, recombination plasmid pET-28a-IRF7 was constructed. The recombinant plasmid pET-28a-IRF7 was transformed into Escherichia coli BL21 (DE3) competent cells. After induction by IPTG, the expression products were analyzed and identified by SDS-PAGE. The specificity of the polyclonal antibody was identified by Western blotting, and the titer of rabbit anti-bovine IRF7 polyclonal antibody was determined by indirect ELISA. After BVDV infected cells, the expression of the antiviral factor IRF7 was detected by Real-time quantitative PCR. Bioinformatics analysis showed that IRF7 protein had no transmembrane domain, no signal peptide, and many phosphorylation sites. The secondary structure was mainly random coil, which was consistent with the three-dimensional model of the tertiary structure, indicating that this protein might have a good antigenic potential. A 1 497 bp fragment of IRF7 gene was successfully amplified by PCR, and the bovine IRF7 protein was successfully expressed with a molecular weight of about 60 ku. The titer of rabbit anti-bovine IRF7 polyclonal antibody was 1:128 000 by indirect ELISA. The expression of IRF7 was decreased in MDBK cells infected with BVDV strain, and the expression of IFN-β was significantly promoted by BVDV infection of cells overexpressing IRF7 (P<0.05). In conclusion, bovine IRF7 protein was successfully expressed and purified in this study, and rabbit polyclonal antibody against bovine IRF7 protein was prepared, which provided materials for elucidating the molecular mechanism of bovine IRF7 in innate immune antiviral response.

The purpose of this research was to select the expression vector of lactic acid bacteria (LAB) with excellent performance, and lay the foundation for the new live vaccine of LAB in the later period. The biological characteristics of the two types invasive LAB NC8-pSIP409-FnBPA and NC8-pLc23-FnBPA with constitutive and inducible expression were researched from acid tolerance, bile salt tolerance, growth curve, plasmid stability, bacteriostasis test and drug susceptibility test. The results showed that the survival rate of the two strains under the acid stress condition of pH 2.5 reached 125%, and the survival rate was as high as 200% in the high bile salt solution with a mass fraction of 0.5%. The plateau of the two types strains were reached at 9 h that denoted excellent growth performance. The D_{600 nm} value of the constitutively expressed strain NC8-pLc23-FnBPA was 1.3 when reached it's plateau, which was higher than that of the inducible expression strain and showed better growth performance. The growth curve of pH results showed that there was no significant difference in the acid production performance of LAB and the pH in the solution dropped to 3.5 at 10 h which indicated the acid production capacity was strong. The bacterial cells and supernatants of the two types of strains had different degrees of antibacterial effect on common pathogenic bacteria. Among them, the antibacterial effect of the bacteria was better than that of the supernatant, and the antibacterial effect of the constitutive bacteria expressing FnBPA protein was stronger than the other one. Both of the invasive lactic acid bacteria were highly sensitive to most antibiotics and had certain biological safety. The plasmid stability of the two strains were relatively strong even greater than 90%. This study found that the inducible LAB NC8-pSIP409-FnBPA strain and the constitutive NC8-pLc23-FnBPA strain had similar biological characteristics. Among them, it was more convenient for the constitutive LAB to express protein even without adding inducing peptides, which was more suitable to be the recipient for a new LAB strain of live vaccines and provided convenience for the later development of live LAB vaccines.

The study was aimed to establish the oxidative stress model of Kunming mouse induced by Salmonella Typhimurium, and explore its molecular mechanism. 18 mice were randomly divided into 3 groups with 6 mice in each group. Normal saline (control group), low dose Salmonella solution (group 1) and high dose Salmonella solution (group 2) were given by gavage respectively. Mice were dissected and samples were collected after gavage for 24 h. Serum oxidative stress indexes were detected. HE sections of liver, duodenum, jejunum and ileum were observed and scored. The mRNA expression of liver and duodenal antioxidant enzymes, key proteins in the antioxidant signal pathway, and tight junction proteins were detected. The results showed that:Compared with the control group, superoxide dismutase (SOD) and glutathione peroxidase (GPx1) activity in the serum of the group 2 decreased significantly or extremely significantly (P<0.05;P<0.1), while malondialdehyde (MDA) increased extremely significantly (P<0.01). The duodenal villi were broken and dropped off, and ulceronecrotic of the intestinal lamina propria, and the liver mainly showed inflammatory cell infiltration, hepatic cord disorders and punctate necrosis in groups 1 and 2. Compared with the control group, the expression of manganese superoxide dismutase (SOD2), GPx1, heme oxygenase-1 (HO-1), sequestosome 1 (p62) and nuclear factor E2-related factor 2 (Nrf2) mRNA decreased significantly or extremely significantly in duodenum and liver (P<0.05;P<0.01), while the expression of ZO-1 and OCLN mRNA in duodenal, and the expression of ZO-1 and claudin-8 (CLDN8) mRNA in liver decreased extremely significantly (P<0.01) in group 2. The above results showed that the oxidative stress model of mice was successfully established by high dose Salmonella solution, and it was found that high dose Salmonella solution might inhibit the expression of antioxidant enzymes in duodenum and liver by reducing the transcription of p62 and Nrf2, resulting in the damage of cell barrier function in duodenum and liver.

This study was aimed to explore the protective effect and potential mechanism of Forsythiae suspensa extract (FSE) preventive intervention on acetaminophen (APAP)-induced liver injury in mice. The semi-bionic enzyme method was used to extract the active components of Forsythia suspense, and the mouse liver injury model was constructed by APAP in this experiment. 60 female Kunming mice were randomly divided into 6 groups:normal control(NC) group, APAP liver injury model (LD) group, Forsythia suspense extract (FSE) group, high-dose Forsythia suspense extract (HFSE+LD) group, medium-dose Forsythia suspense extract (MFSE+LD) group and low-dose Forsythia suspense extract (LFSE+LD) group. 10 mice for each group. The HFSE+LD group, the MFSE+LD group, and the LFSE+LD group were intragastrically administrated with Forsythia suspense extract 200, 100, and 50 μg/g, respectively, twice a day, for 6 consecutive days. The NC group and the LD group were given the same amount of normal saline. After preventive administration with FSE for 3 days, APAP was injected intraperitoneally once a day. Blood samples were collected from the eyeball, and the liver was quickly taken out 12 h after the last administration. Serum aminotransferase (ALT) and aspartate aminotransferase (AST) were detected to evaluate the degree of liver injury. The homogenates of liver glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were detected to evaluate the degree of liver oxidative stress of liver. Liver samples were visualized by hematoxylin and eosin staining. Furthermore, liver mitochondrial cytochrome P4502E1 (CYP2E1) activity were measured by probe drug method. The expression level of liver mitochondrial CYP2E1 mRNA was detected by Real-time quantitative PCR and the liver CYP2E1 protein expression was detected by Western blotting. The results showed that:Compared with NC group, the ALT and AST activities in serum were increased significantly (P<0.05), the SOD activity and GSH content in liver were significantly reduced (P<0.05), the MDA and H₂O₂ contents, and the expression of CYP2E1 mRNA and protein in liver were significantly increased (P<0.05) in LD group. The mouse liver injury model was successfully established. 200, 100, and 50 μg/g FSE treatment significantly reduced the serum ALT and AST activities in mice with liver injury (P<0.05), and significantly increased the liver SOD avtivity (P<0.05). 200 and 100 μg/g FSE significantly increased the GSH level in liver (P<0.05), and significantly reduced the level of MDA, H₂O₂ and the expression of CYP2E1 mRNA and CYP2E1 protein in liver (P<0.05). The results showed that FSE reduced APAP-induced mice liver injury in a manner of dose-dependent, and its potential mechanism was related to the antioxidant effect and the inhibition of CYP2E1 enzyme activity and expression of the active substances.

This study was aimed to establish a fast, efficient and sensitive duplex Real-time quantitative PCR method for differential diagnosis of Porcine deltacoronavirus (PDCoV) and Transmissible gastroenteritis virus (TGEV). By drawing the standard curve of duplex Real-time quantitative PCR, the specificity, sensitivity and repeatability of the method were tested, and clinical samples were tested. The results showed that there was a good linear relationship between the cycle threshold of this method and the logarithm of the copy number of PDCoV and TGEV plasmids, and the corresponding correlation coefficients were R_(P)²=0.9994 and R_(T)²=0.996, respectively. It could specifically detect PDCoV and TGEV, but had nocross-react with PEDV, PRV, PRRSV, CSFV and RV, and had strong specificity. The minimum detection limits of the PDCoV and TGEV plasmid standard products reached 2 and 20 copies/μL, respectively, and were 1 000 and 100 times higher than conventional RT-PCR, respectively, with higher sensitivity. The average Ct values of intra-assay and inter-assay reproducibility of PDCoV and TGEV were basically the same, and the coefficient of variation (CV) was less than 2%, which had good reproducibility. The results of 114 piglet diarrhea samples tested by this method showed that the positive rates of PDCoV and TGEV were 5.6% (6/114) and 8.8% (10/114), respectively, and the detection rate of mixed infection was 4.6% (5/114), duplex Real-time quantitative PCR had a higher detection rate and sensitivity than conventional RT-PCR. The result indicated that the duplex Real-time quantitative PCR had the advantages of strong specificity, high sensitivity, good repeatability and stability, it was suitable for early virus diagnosis and batch clinical sample detection, and provided technical support and data reference for disease prevention and control, epidemiological investigation and correlation research.

The aim of this study was to investigate the protective effect and mechanism of Ganoderma leucocontextum (GLAE) aqueous extracts on hippocampal neurons after cerebral ischemia. Fifty rats were divided into control, model, low-(0.05 mg/(g·BW)), middle-(0.1 mg/(g·BW)) and high-dose (0.2 mg/(g·BW)) GLAE groups. The cerebral ischemia model of rats was established by bilateral common carotid artery occlusion, and then treated intragastrically with different doses of GLAE once a day for two weeks, and rats of control and model groups were administered the same volume of normal saline. Step down test was used to assess memory acquisition, memory consodilidation and memory reappearance disorder in rats learning and memory ability. The rats pathological changes in hippocampus were observed by HE staining. The nitric oxide synthase(NOS) activity and nitric oxide (NO) content were determined by colorimetric method. The levels of growth associated protein-43 (GAP-43) and brain derived neurotrophic factor (BDNF) in hippocampus were detected by Western blotting and Real-time quantitative PCR, respectively. The results showed that when compared to control group, in the model group, the escape latency of rats in step down test were significantly shortened and the number of electric shocks were significantly increased (P<0.05), hippocampal neurons exhibited obviously degenerative changes with pyknosis and a loose and disorderly arrangement, and a decrease in cell number (P<0.05), NOS activity and NO content were significantly decreased (P<0.05), the level of GAP-43 protein of hippocampal tissue was significantly increased (P<0.05), BDNF mRNA level of hippocampal tissue was significantly decreased (P<0.05). When compared to model group, GLAE administration could obviously prolong the escape latency of rats and decrease significantly the number of electric shocks (P<0.05), the morphology of neurons in GLAE high-dose group of CA1 area and dentate gyrus was significantly improved, and the number of neurons was significantly increased (P<0.05), GLAE low-dose group had no significant effect on NOS activity (P>0.05), but significantly increased the content of NO (P<0.05), while the activity of NOS and the content of NO were significantly increased in middle- and high-dose groups of GLAE (P<0.05). The expression of GAP-43 protein in hippocampus of GLAE low-, middle- and high-dose groups were significantly increased (P<0.05), the level of BDNF mRNA of GLAE low-, middle- and high-dose groups were significantly increased (P<0.05). The above results indicated that GLAE had a certain protective effect on hippocampal neurons injury after cerebral ischemia by increasing the activity of NOS and the content of NO, promoting the hippocampal neurogenesis and neuro-functional recovery, and could improve cognitive function of rats. The effect of 0.2 mg/g GLAE was the best.

The aim of this study was to evaluate the combined toxicity of enrofloxacin and sulfamethazine. SD rats were selected and divided into 6 groups:High dose group (500 mg/(kg·BW)), middle dose group (250 mg/(kg·BW)), low dose group (50 mg/(kg·BW)), enrofloxacin group (250 mg/(kg·BW)), sulfamethazine group (250 mg/(kg·BW)) and control group 0.5% CMC, each group contained 12 rats. The corresponding drugs were prepared for intragastric administration. 1 d after the last administration, rats were weighed, heart blood was collected and liver tissues were dissected. Then the weight gain rate, blood routine indicators, serum biochemical indicators and liver pathological changes of the rats were analyzed, and the combined effect of the two drugs was evaluated by SPSS 22.0 software. The results showed that, in female rats, a significant decline of the weight gain rate was found at high and middle dose groups (P<0.05), but there was no significant change in male rats (P>0.05). Leukocyte populations in male rats had a significant rise (P<0.05), neutrophil and lymphocyte ratios were significantly increased and decreased at high dose group, respectively (P<0.05). The content of peripheral blood AST of male rats was significantly increased at high and middle dose groups (P<0.05), and liver pathologic reports revealed different degrees of inflammatory cell infiltration with different degrees of injury at high and middle dose groups. This study showed that mixing enrofloxacin and sulfamethazine might increase toxicity, it had a certain effect on the rat immune system, and could cause liver damage, and the higher the dose, the greater the effect. This work provided data support for the joint toxicity mechanism of enrofloxacin and sulfamethazine, and provided references for their clinical application. In addition, it suggested that new food safety assessment should consider the effects caused by co-exposure to multiple drugs.

This study was aimed to investigate the acute toxicity, sub-chronic toxicity and clinical safety of Qinzhu Antai powder, and provide basis for its application for prevention and treatment of domestic cats threatened miscarriage. Acute toxicity test:Qinzhu Antai powder was prepared, sixty 6 weeks old healthy Kunming mice were randomly divided into 5 groups. The doses of groups 1-4 were 6 000, 4 800, 3 840 and 3 072 mg/kg, respectively, and the control group was given the same volume of purified water. After 10 days of feeding, the poisoning symptoms and mortality were observed, and the median lethal dose (LD₅₀) was calculated. Twenty 6 weeks old healthy mice were randomly divided into two groups:The experimental group was given 2.0 g/mL solution 3 times within 18 hours, 0.8 mL each time, and the control group was given the same volume purified water for 7 days, and the maximum tolerance dose (MTD) and tolerance multiple were calculated. Sub-chronic toxicity test:Twenty-four 7 weeks old female SD rats were randomly divided into high, middle and low dose groups and control group, the daily intragastric doses of Qinzhu Antai powder were 4 800, 2 400 and 1 200 mg/kg, respectively, the control group was given the same volume of pure water and fed rats for 30 days, the mental state, poisoning symptoms and mortality of rats in each group were observed and recorded every day. On the 31st day, the rats in each group were weighed and blood samples were collected for hematological examination, the pathological changes of the major organs were observed and pathological sections were made. Clinical safety test:Twenty 2-5 years old healthy female domestic cats were selected and fed adaptively for 10 days. They were randomly divided into 4 groups, the Qinzhu Antai powder dosage was 1, 3 and 5 times of the recommended clinical dose of domestic cats, respectively:1.15 g/kg in low dose group, 3.45 g/kg in middle dose group and 5.75 g/kg in high dose group. The drug was given orally once a day for 7 days. The blank control group was given empty capsule, and the appetite, mental state and defecation of domestic cats in each group were observed every day. Venous blood samples were collected from domestic cats in each group on the 8th day, and blood routine indexes and blood biochemical indexes were detected. The results showed that there was no death in mouse in acute toxicity test, LD₅₀>6 000 mg/kg, the MTD of Qinzhu Antai powder in mouse was 240 g/kg, indicating that the drug was no-toxic. In the sub-chronic toxicity test, there was no significant difference in growth, organ coefficient and blood routine index between each dose and control groups (P>0.05). The content of serum total cholesterol in high and middle dose groups was significantly lower than that in low dose and control groups (P<0.05). There was no significant difference in other biochemical indexes (P>0.05). The results of pathological examination and tissue section observation showed that there was no obvious abnormality in the main tissues and organs of high dose group compared with control group. In the clinical safety test, the mental state, coat glossiness and feces of domestic cats in different dose groups were normal, there were no significant difference in hematological indexes with control group (P>0.05). The results indicated that Qinzhu Antai powder had no obvious toxicity, according to the recommended clinical dose used for domestic cats were safety.

The unexplained death of sheep occurred in a sheep farm in Ordos city. In order to find the cause of the disease and formulate treatment and prevention measures, this study confirmed the disease by clinical examination, laboratory pathology and molecular biology diagnosis methods, and provided treatment plan. In this experiment, the dead sheep were used as the main materials. Firstly, the clinical examination was carried out, including autopsy and histopathological sampling. Then, the brain tissue was taken back to the laboratory by routine aseptic method for contact film, staining and microscopic examination, and bacteria isolation. Autopsy of the dead sheep showed congestion and consolidation of lung, punctate hemorrhage of epicardium, soft and muddy texture of kidney, severe brain edema. Under the microscope, the infiltration of neutrophils in cerebral cortex and the phenomenon of neutrophil vascular cannula were observed. After contact staining in deep brain tissue, Gram positive cocci were found in single or paired arrangement under microscope, and a suspicious bacterium was isolated and named NEF1. Based on the 16S rRNA gene of the isolate, the phylogenetic tree analysis by Mega 6.0 software showed that the isolate NEF1 was clustered on the same branch with the domestic isolate (MT197247.1) and the Iranian isolate (KM495939.1) of Enterococcus faecium, but was far away from other reference strains of Enterococcus faecium in GenBank. The drug sensitivity test showed that NEF1 was only moderately sensitive to ciprofloxacin, but obviously resistant to penicillin and other selected antibiotics. A strain of Enterococcus faecium NEF1 was successfully isolated and identified from the brain tissue of dead sheep in this experiment. Meanwhile, the sensitive drugs screened by the drug sensitivity test method effectively controlled the further spread of the disease in the diseased sheep farms. It provided effective experimental data for effective prevention and control of sheep bacterial diseases in this area.

In order to explain the clinical significance of tibial plateau angle (TPA) in dogs cranial cruciate ligament rupture (CCLR) and provide references to pathogenesis and risk, diagnosis and treatment of canine CCLR, 30 stifle joints of 15 CCLR dogs in China Agricultural University Veterinary Teaching Hospital from June 2018 to January 2019 were selected. TPA (R-TPA and CT-TPA) was measured on radiographs and CT respectively. The consistency, advantages and disadvantages of two methods were compared. The results showed that:The magnitude of R-TPA was not related to age, weight, fat and thin, sex and sterilization/castration or not (P>0.05). The difference in TPA between the stifles with or without cranial cruciate ligament rupture was not significant (P>0.05). The mean value of 30 CT-TPA in this study was 26.93° (range:19.03°-32.67°). The mean CT-TPA of the left stifle joint was 26.82° and that of the stifle knee joint was 27.04°. There was a high correlation between R-TPA and CT-TPA (r>0.75, P>0.05). Osteophytes was observed in 21 stifles (70%) on CT images, and the boundary between osteophyte and bony cortex could be identified more clearly on CT image than radiographs. These results indicated that there were no associations between TPA and the clinical features of the dogs with CCLR. Radiographs and CT had consistency in measuring TPA in dogs, but CT was more convenient, timesaving and comprehensive in acquiring image, and hasd higher accuracy in the osteoarthritis cases.