[Objective] The research was aimed to integrate and analyze the multiple transcription data sets of testis in sheep, reveal the differential genes and protein interaction network of testis in sheep, and explore the key genes affecting spermatogenesis in sheep, so as to provide theoretical reference for reproduction in sheep. [Method] 74 testis transcriptome in sheep were bioinformatic analyzed, differential genes were carried out by limma package, WGCNA was used to construct the weighted differential genes co-expression network of testis in sheep and MCODE was used to calculate the important genes in the network. Metascape was used for functional enrichment analysis, and Cytoscape plug-in AutoAnnotate was used to identify gene clusters. [Results] 11 884 genes were screened and 237 366 protein interactions were constructed. Protein interaction network were constructed from 2 058 differential genes, and 46 169 protein interaction relationship were generated. 4 gene sets with the highest scores were identified. At the same time, a weighted co-expression network was constructed for 2 058 differential genes, and a total of 7 modules were obtained, among which 929 genes in the Blue module were found to be significantly enriched in male gamete production, reproduction, spermatogenesis, flagellum motility and AMPK signaling pathway, and the enrichment results were highly connected and aggregated into a complete network. As a result, 25 key genes involved in spermatogenesis and sperm cell development. [Conclusion] This study revealed the protein interaction network of genes expressed in testis of sheep and the protein interaction network of multi-dimensional differential genes, finally 25 genes were associated with testis spermatogenesis and interaction relationship, it provided theoretical basis for breeding in sheep.

[Objective] The purpose of this study was to clone and analyze the sequence of fat mass and obesity-associated gene(FTO), detect the expression difference of FTO gene in various tissues of Guangling donkey, and explore the effect of the structure of FTO gene related to fat on the physiological and metabolic functions of Guangling donkey. [Method] RT-PCR method was used to amplify and clone the CDS sequence of FTO gene, and analyze the gene and protein function. Meanwhile, Real-time quantitative PCR was used to detect the differential expression of FTO gene in seven tissues (heart, liver, spleen, lung, kidney, longissimus dorsi muscle and subcutaneous fat) of Guangling donkey. [Result] The results showed that the CDS sequence of FTO gene in Guangling donkey was 1 518 bp and could encode 505 amino acids. The sequence was uploaded to NCBI and obtained the accession No.:MZ169553. The similarity of FTO gene in Guangling donkey were 99.3%, 90.3%, 89.5%, 90.8%, 90.7%, 89.2% and 89.2% with Equus caballus, Sus scrofa, Bos taurus, Homo sapients, Vicugna pacos, Ovis aries and Capra hircus, respectively. Phylogenetic tree analysis result showed that FTO gene of Guangling donkey was closely to Equus caballus. The molecular weight of FTO protein was 58.35 ku, the theoretical isoelectric point was 5.07, the fat index was 80.36, the instability coefficient was 48.82, and the average hydrophobic index was -0.550, the FTO protein of Guangling donkey was an unstable acidic hydrophilic protein. There was no signal peptide and transmembrane region of FTO protein, which were mainly located in the cytoplasm, and had 34 phosphorylation sites and 5 glycosylation sites. The secondary structure prediction of FTO protein showed that α-helix (43.96%) and random coil (37.82%) were the main structures. Real-time quantitative PCR analysis showed that FTO gene was all expressed in 7 kinds of tissues, the expression of FTO gene in lung and subcutaneous fat were extremely significantly higher than other tissues (P<0.01), and the expression was the lowest in longissimus dorsi muscle. [Conclusion] The results of this study provided a solid theoretical basis for the further research on gene expression, mechanism of fat deposition, and improved the meat quality of Guangling donkey.

[Objective] The genome-wide copy number variation in Longlin pigs was detected. [Method] Ear tissue samples from 33 Longlin pigs were collected. After extracting DNA by phenol-chloroform methods, the porcine 50K SNP beadchip was used to genotype. The raw dates were analyzed by Genomestudio software and Linux system. The copy number variation (CNV) was detected by CNVPartition and PennCNV softwares, then the Bedtools software was used to merge the CNVs as copy number variation region (CNVR). The Biomart was used to identify the genes in CNVR. The GO and KEGG enrichment analysis were completed by David website. The pig QTL database was used to annotate the QTL in common CNVR. [Result] 260 CNVs and 47 CNVRs were detected by CNVPartition, including 40 deletions, 5 duplications and 2 mixed. 84 genes were located, which were significantly enriched into 13 signal pathways. A total of 96 CNVs and 15 CNVRs were detected by PennCNV, including 9 deletions, 1 duplications and 5 mixed. 8 genes were located, which were significantly enriched into 8 signal pathways. The genes detected by the 2 software were mainly enriched in olfactory pathway and G-protein coupling pathway. INPP5B, NEURL1 and GAPDHS genes were significantly enriched in sperm motility pathway. 3 common CNVRs were obtained by Bedtools software, including 1 deletions, 1 duplications and 1 mixed. A total of 8 genes were located and significantly enriched into 7 signal pathways, including detection of chemical stimulus involved in sensory perception of smell pathway, olfactory receptor activity pathway, G-protein coupled receptor activity pathway, G-protein coupled receptor signaling pathway, olfactory transduction pathway, plasma membrane and integral component of membran pathway. A total of 130 QTLs were overlaid with 3 common CNVRs, including 11 backfat thickness QTLs, 9 meat quality QTLs and 6 teat number QTLs. [Conclusion] The CNV of Longlin pigs might be related to the olfactory function, reproduction ability, backfat thickness, meat quality and teat number.

[Objective] To understand the genome characteristics and variation of pigeon circovirus (PiCV) circulating in Beijing. [Method] Used the liver and spleen tissues of 3 diseased pigeons as templates, the pathogen was detected by PCR technology. Using the DNA of the positive liver tissue as a template, PCR technology was used to amplify the whole gene sequence of PiCV. DNAStar and Mega 7.0 softwares were used to splice and compare the nucleotide sequence of the amplified sequence, and construct evolutionary tree. The nucleotide sequence and amino acid sequence of the two open reading frames of the virus genome were compared, and the phylogenetic tree was constructed. [Result] The PCR test showed that PiCV positive was detected in the tissues of one of the three sick pigeons, but no other viruses were detected. The whole genome sequence of a PiCV strain was successfully obtained by PCR fragmented amplification, named PiCV BJ. The virus genome was 2 034 bp in size and contained 2 main open reading frames (ORFs), ORF-V1 encodes the Rep protein, ORF-C1 encodes the Cap protein. The similarity comparison results showed that the nucleotide similarity between the genome sequence of PiCV BJ strain and other reference sequences registered on GenBank was 86.0% to 97.0%, and the homology with PL53 isolated from Poland in 2011 was 97.0%. The genetic evolution results showed that the PiCV BJ strain was in the same branch as PL124 isolated from Poland in 2014, and was closely related;It was not in the same branch with other avian circoviruses, and was relatively distant. The start codon of Cap gene of PiCV BJ strain was ATG, which had 95.8% and 85.2% nucleotide and amino acid similarity with 11-08304 strain isolated from Belgium in 2011. The Rep gene had 94.6% and 96.2% nucleotide and amino acid similarity with PL53 isolated from Poland in 2011. The similarity of nucleotide and amino acid was 94.6% and 96.2%, respectively. The phylogenetic tree analysis of Cap and Rep genes also showed that PiCV BJ strains was in the same branch as PL53 and 11-08304, which was close to the results of similarity analysis. [Conclusion] PiCV BJ strain was from abroad and might be introduced into China by racing pigeons, which suggested that virus monitoring should be done well in external introduction. This study enriched the genetic research data of PiCV, provided a reference basis for further exploring the genetic variation and transmission mechanism of PiCV, and also provided an important theoretical basis for the prevention and control of PiCV.

[Objective] The Toll-like receptor 7 (TLR7) gene of Larus ridibundus was cloned, and its coding protein was analyzed by bioinformatics, so as to prepare for the study of antiviral activity of TLR7 protein in the later stage. [Method] The whole TLR7 gene sequence of Larus ridibundus was amplified by rapid amplification of cDNA ends (RACE). The TLR7 gene sequence, rare codon, similarity and physical and chemical properties, transmembrane region, signal peptide, N-glycosylation site, phosphorylation site and subcellular localization of TLR7 protein were analyzed by bioinformatics software. The secondary structure and tertiary structure of TLR7 protein were predicted by SOPMA and SWISS-MODEL softwares, respectively. [Result] The TLR7 gene of Larus ridibundus (GenBank accession No.:MZ668652) was successfully cloned. The total length of the gene sequence was 1 720 bp, the size of open reading frame (ORF) was 1 182 bp, and there were 39 rare codons, including 8 consecutive rare codons, encoding 393 amino acids. The similarity of TLR7 gene between Larus ridibundus and Gallus gallus, Tragopan temminckii, Coturnix coturnix, Anas platyrhynchos, Cygnus atratus, Paridae, Egretta garzetta, Aptenodytes forsteri, Gavia stellata, Pygoscelis papua in GenBank was 85.7%, 84.9%, 85.4%, 87.0%, 87.8%, 86.8%, 91.0%, 93.1%, 93.1% and 93.1%, respectively. The phylogenetic tree showed that it had the closest genetic relationship with Egretta garzetta. The molecular formula was C₂₀₈₀H₃₂₅₆N₅₅₆O₅₆₇S₁₉, the molecular weight of TLR7 protein was about 63 ku. The theoretical isoeletric point was 9.25. TLR7 protein had no transmembrane structure and signal peptide. It contained 6 N-glycosylation sites and 33 phosphorylation sites. It was a hydrophobic protein and mainly exists in the cytoplasm. The secondary structure of TLR7 protein was mainly alpha helix dominated, accounting for about 48.09%, followed by random coil (32.06%), extended chain (13.23%) and beta turn(6.62%). The tertiary structure of TLR7 protein was consistent with the secondary structure, and the similarity with the model protein human TLR7 protein was 68.78%. [Conclusion] The TLR7 gene of Larus ridibundus was closely related to the evolution of Egretta garzetta, it contained 8 consecutive rare codons and was difficult to express in vitro. The study provided an important reference for further exploring the antiviral immune mechanism of TLR7 protein in Larus ridibundus.

CRISPR/dCas9 is a system for regulating genome transcription and epigenetic modification based on the transformation and upgrading of the CRISPR/Cas9 gene editing system. It not only inherits the target accuracy of the CRISPR/Cas9 system, at the same time, it also showed a good effect. In this system, the dCas9 protein retains the ability of the Cas9 protein to bind DNA and the cleavage function no longer exists. Coupling dCas9 protein with different activation and inhibitory effector domains and epigenetic regulation enzymes makes it possible to precisely regulate gene expression and epigenetic modification. Epigenetic modification processes such as histone acetylation, histone methylation and DNA methylation are the basis of gene expression and have made a huge contribution to the entire life process. At the same time, epigenetics is causally related to many diseases and cancers. Different epigenetic modification systems based on the CRISPR/dCas9 system have unlimited future and research value in the field of human disease treatment and cancer research. Therefore, this article briefly introduces the discovery process and the principle of action of the CRISPR/Cas9 system. It mainly summarizes the application and optimization process of different regulatory systems based on the CRISPR/dCas9 system in the regulation of gene expression and epigenetic regulation. It hopes to provide some references for scientific research workers engaged in related fields.

[Objective] The purpose of this study was to perform cloning, identification and bioinformatics analysis of gamma-aminobutyric acid receptor type associated protein (GABARAP) gene, a member of the Atg8 family of autophagy-related proteins in Apis cerana cerana. Prokaryotic expression was carried out in E. coli BL21 (DE3) competent cell, hoping to lay the foundation for the subsequent exploration of the function of this gene. [Method] Primers were designed with reference to Apis mellifera GABARAP gene sequence in GenBank (accession number:XM_001120069.5). The nested PCR was used to amplify and clone the Apis cerana cerana GABARAP gene. The amino acid sequence similarity, secondary structure and tertiary structure were compared and predicted by bioinformatics software. The prokaryotic expression vector of GABARAP gene was constructed. The GABARAP protein was identified by Western blotting and induced by IPTG and then purified. [Result] The Apis cerana cerana GABARAP gene was amplified by nested PCR. The comparison of amino acid sequence similarity analysis using online BLAST tool showed that the GABARAP of Apis cerana cerana was 100% with that of Apis mellifera, Apis dorsata, Apis florea, Bombus terrestris and Bombus impatiens. The phylogenetic tree analysis of amino acid sequence showed that GABARAP of Apis cerana cerana was most closely related to Apis mellifera and farthest from Caenorhabditis elegans. The results of bioinformatics analysis showed that the CDS region of GABARAP gene fragment size was 354 bp, encoding 117 amino acids, the molecular weight of the protein was 13.99 ku and the theoretical isoelectric point was 9.48. The secondary structure of GABARAP protein was mainly composed of 3 alpha helices, 4 beta sheets and 6 peptide binding sites, the predicted results of secondary and tertiary structures were the same. SDS-PAGE analysis showed that the molecular weight of GABARAP protein induced by IPTG was 35 ku. The results of Western blotting showed that His monoclonal antibody could specifically recognize GABARAP protein. [Conclusion] In this study, the GABARAP gene of Apis cerana cerana was cloned successfully, and the GABARAP protein was obtained and analyzed by bioinformatics, which provided a reference for further study on the role of GABARAP gene and its protein in autophagy.

[Objective] By comparing the differences of proliferation rate, surface marker molecules, pluripotent gene expression and cartilage differentiation potential of umbilical cord mesenchymal stem cells (UC-MSCs) isolated from three different parts of the same umbilical cord of female equine fetus, the umbilical cord site which was suitable for isolation and culture of mesenchymal stem cells (MSCs) was identified. [Method] MSCs were isolated from the umbilical cord near the fetus (proximal end), the center of the umbilical cord (middle segment) and the placenta (distal end), and the proliferation ability of UC-MSCs was compared by plotting cell growth curve, the expression of multipotent genes and surface marker molecules were detected by Real-time quantitative PCR, and the potential of cartilage differentiation was detected by cartilage induction. [Result] The MSCs isolated from the proximal end, middle segment, and distal end attached to the wall and grew in a fibrous shape and the doubling time were 35.4, 25.5 and 34.9 h, respectively, and all of them expressed NANOG homeobox (NANOG), POU class 5 homebox 1 (POU5F1) and SRY-box transcription factor 2 (SOX2), overexpression of 5'-nucleotidase ecto (CD73), Thy-1 cell surface antigen (CD90), endoglin (CD105) and hematopoietic progenitor cell antigen (CD34), low expression of CD14 molecule (CD14), B-lymphocyte surface antigen B4 (CD19), protein tyrosine phosphatase receptor type C (CD45) and B cell antigen receptor complex associated protein α chain (CD79a). The relative expression of pluripotent gene NANOG in middle segment P1 cells was significantly higher than that in proximal end P1 cells (P<0.05), the relative expression of POU5F1 in P5 proximal end cells was significantly higher than that in middle segment cells (P<0.05), and it was extemely significantly higher than that of distal end cells (P<0.01). The relative expressions of CD14, CD19, CD73 and CD90 in P5 proximal end cells were significantly or extemely significantly higher than those in middle segment and distal end cells (P<0.05; P<0.01), the relative expressions of CD34 and CD79a in distal end cells of P5 generation was significantly or very significantly higher than that in proximal end and middle segment cells (P<0.05; P<0.01). The UC-MSCs derived from the proximal end, middle segment and distal end could be induced to differentiate into chondrocytes. On the 14th day of induced differentiation, the relative expression of SOX9 in proximal cells was extremely significantly higher than that in distal cells (P<0.05), and on the 21st day of induction, the relative expression of SOX9 in distal cells was extremely significantly higher than that in middle cells (P<0.01). The relative expressions of specific gene aggrecan (ACAN) and collagen type Ⅱ alpha 1 chain (COL2A1) in middle segment cells were extremely significantly higher than those in proximal and distal cells on the 7th and 14th day of induction (P<0.01), and significantly higher than those in proximal and distal cells on the 21st day (P<0.05). [Conclusion] The proximal end, middle segment and distal end of umbilical cord could be used to prepare MSCs, which could express pluripotent genes and MSCs surface marker molecules, and could be induced to differentiate into chondrocytes. The proliferation ability and cartilage differentiation potential of MSCs from the middle segment were better than those from the proximal and distal ends, so they were more suitable for isolating MSCs.

Oxidative stress is a malignant state characterized by redox imbalance, which is one of the important factor leading to tissue damage and disease. Oxidative stress is known to be closely associated with reproductive disturbance, decreased disease resistance, and reduced production performance and product quality of livestock and poultry. Nuclear factor erythroid 2 related factor (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway is one of the most important defense mechanism against oxidative stress. It maintains intracellular redox equilibrium, metabolism and protein homeostasis, and exerts biological functions such as anti-inflammation, anti-cancer and anti-aging by regulating the transcription of multiple downstream cytoprotective genes. Under normal physiological conditions, Nrf2, a transcription factor with a high sensitivity to oxidative stress, is negatively regulated by Keap1, which targets Nrf2 for ubiquitination and degradation by the ubiquitin proteasome system. Oxidative stress causes Nrf2 to dissociate from Keap1 and to subsequently translocate into the nucleus, which interacts with small Maf (sMaf) to form heterodimer and binds to ARE, leading to increased transcription of antioxidant genes. The Nrf2 regulatory network is complex, and its activity is subjected to the regulation at various levels, including transcription and post-transcription, protein stability, subcellular localization, and post-translation modification. The author introduces the molecular structural basis of Nrf2/Keap1-ARE signaling pathway, its biological functions and the current knowledge of its activity regulation, in order to acquire a greater depth of understanding Nrf2 regulatory mechanism and provide a theoretical basis for improving the health of livestock and devising Nrf2-based strategies for disease intervention.

Through epididymal transport, sperm undergo a series of biochemical and morphological changes and gradually acquire maturation. During the process of sperm maturation, most of its cytoplasmic components are lost, sperm are more sensitive to oxidative stress, which lead to the damage of sperm structure and function. Therefore, the role of antioxidant enzymes in the epididymal cavity microenvironment is very important. Reactive oxygen species(ROS) play a dual role in sperm function:Physiological levels of ROS can promote the pre-fertilization capacitation of sperm, while high levels of ROS can lead to oxidative damage of sperm. Antioxidant enzymes that remove excess ROS from the epididymis include superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GPXs) and peroxidase(PRDXs), however, the contents of different antioxidant enzymes in epididymis varied with the parts of epididymis and animal ages, and different antioxidant enzymes have different scavenging mechanisms to ROS. When the epididymis lacks a certain antioxidant enzyme, the sperm DNA is damaged, the sperm quality is reduced, and eventually leads to increased abnormal reproductive outcomes. Obviously antioxidant enzymes in mammalian epididymis need to coordinate with each other to maintain ROS at physiological level, however there are few reports on this topic at present. The antioxidant enzymes secreted by epididymal epithelial cells are transported to sperm in the form of epididymosome, which can eliminate ROS produced by their own aerobic metabolism and abnormal sperm, and ensure the normal maturation of sperm. This paper reviewed the oxidative stress of epididymal sperm and the protective effect of various antioxidant enzymes in epididymis on sperm.

[Objective] To study the effects of compound probiotics added to drinking water on growth performance and intestinal microflora in Lingnan Yellow feather broilers. [Method] 270 healthy and fast-sized Lingnan Yellow feather broilers at 21 days old were selected and randomly divided them into 3 groups. The control group was fed basal diet, the antibiotic group was fed basal diet+300 g/t 15% chlortetracycline and 40 g/t 50% virginiamycin, and the probiotics group fed with basic diet+drinking water supplemented with compound probiotics. Among them, the compound probiotic preparation was 1:1:1 compatible Lactobacillus reuteri, Lactobacillus plantarum and Saccharomyces cerevisiae, and the addition amount of each strain was 1.0×10⁶ CFU/mL. The test period was 5 weeks, and the whole process was free to eat and drink. [Results] ①At 22-42 days of age, compared with control group, the feed-to-weight ratio (F/G) of probiotic and antibiotic groups were significantly reduced (P<0.05);At 22-56 days of age, compared with control group, the average daily gain (ADG) of probiotic and antibiotic groups were significantly increased (P<0.05), and the probiotic group had the lowest mortality, and the uniformity was better than that of control and antibiotic groups. ②At 42 days of age, the ileal Observed Species index, Shannon index and Ace index of antibiotic group were significantly higher than that of control and probiotic groups (P<0.05), and the cecal Observed Species index of antibiotic group was significantly lower than that of control and probiotics groups (P<0.05). ③In terms of relative abundance of species, the probiotic group had an advantage in the relative abundance of Firmicutes and Lactobacillus, while the antibiotic group in the other phylums and genus all showed higher relative abundance, the probiotic and control groups showed the same trend. [Conclusion] Adding compound probiotics to drinking water could significantly improve the growth performance of Lingnan Yellow feather broilers at 22-56 days of age, and effectively reduce mortality, the overall effect was significantly better than that of control group, closed to the antibiotic group, and had part of the high-efficiency replacement function. In terms of the intestinal microbiome, the probiotic group showed greater similarity with control group as a whole, indicated that the addition of compound probiotics in drinking water had little effect on the intestinal microbes.

[Objective] This experiment was conducted to investigate the effects of dietary calcium (Ca) and non-phytate phosphorus (NPP) levels on growth performance and tibia traits of fast growth Yellow-feathered broilers. [Method] A total of 4 050 one-day-old male broilers were randomly divided into 9 groups with 6 replicates per group and 75 broilers per replicate. A 3 (Ca level)×3 (NPP level) two-factor design was used. The experiment lasted for 63 days. The Ca levels were 1.00%, 0.90% and 0.85%, and the NPP levels were 0.46%, 0.38% and 0.30% from 1 to 21 days. The Ca levels were 0.90%, 0.75% and 0.65%, and the NPP levels were 0.42%, 0.35% and 0.28% from 22 to 42 days. The Ca levels were 0.80%, 0.70% and 0.60%, and the NPP levels were 0.39%, 0.31% and 0.23% from 43 to 63 days. Feed and gain ratio (F/G) was calculated for 0~21, 22~42, 43~63 and 0~63 d days by using feed intake and body weight gain. At the end of the feeding experiment, 2 chicks were selected from each replication of each treatment group and slaughtered for chemical analysis of chick tibia and serum level of Ca and P. [Result] The decrease of Ca level had no effect on the growth performance of broilers (P>0.05). The decrease of NPP level had no effect on the growth performance of broilers (P>0.05), but in the starter phase that low NPP level had a higher F/G (P<0.05). Ca level had no effect on tibia traits, the levels of Ca and P in serum of broiler (P>0.05). The contents of Ash and P in tibia, and the level of P in serum of broilers at 63 days of age were all significantly decreased (P<0.05) with the decrease of NPP level. There was no significant interaction between dietary Ca and NPP levels on growth performance and tibia traits of broilers (P>0.05). [Conclusion] Ca requirements of Yellow-feathered broilers seemed to be lower than the nutritional standards of Yellow-feathered broilers recommendations, and it could be reduced by 20%-30%, were 0.85%, 0.65% and 0.60% in the small, medium and large chicken stage, respectively. The decrease of NPP level affects growth performance of chicks and reduces the deposition of calcium and phosphorus in the tibia of broilers. NPP should be maintained at the current level, were 0.46%, 0.42% and 0.39%, respectively.

[Objective] This study was aimed to investigate the effect of crude polysaccharide from Melastoma dodecandrum Lour. on lipid metabolism in obese mice induced by high-fat diet. [Method] The mice were randomly divided into 6 groups, including blank group, model group, positive group (0.025 mg/g orlistat), high, medium and low dose of crude polysaccharide from Melastoma dodecandrum Lour. groups (1.2, 0.6 and 0.3 mg/g), with 12 mice in each group. The mice in blank group were fed with normal maintenance diet, the mice in the other groups were fed with high-fat diet, the mice in blank group and model group were given normal saline, the mice in positive group were given 0.025 mg/g orlistat, and the mice in high, medium and low dose groups were given 1.2, 0.6 and 0.3 mg/g crude polysaccharides of Melastoma dodecandrum Lour., once a day for 30 days. The weight of mice, epididymis, fat around kidney and liver tissuea were measured. The pathological changes of fat and liver were observed by HE staining. Serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C) and liver TC and TG were detected by biochemical method. The relative expression of acetyl CoA carboxylase 1 (ACC1) gene in liver was determined by Real-time quantitative PCR. [Result] Compared with model group, the body weight, Lee's index, food intake, the weight of liver, epididymis fat and kidney fat were significantly decreased in the high and medium dose groups (P<0.05). The body weight and fat weight of mice in low dose group had no significant change (P>0.05), the food intake and liver weight of mice in positive group had no significant change (P>0.05). The fat index of mice in positive and high dose groups was significantly decreased (P<0.05). The contents of serum TC, TG, LDL-C and liver TC, TG in positive and high dose groups were significantly decreased (P<0.05), but there was no significant difference of serum HDL-C (P>0.05). The contents of TC, TG in serum and TC in liver were significantly decreased in middle dose group (P<0.05). In positive group, high, medium and low dose groups, the adipocytes were reduced in varying degrees, and the lipid droplets and vacuoles in hepatocytes were significantly reduced (P<0.05), and the relative expression of ACC1 gene was significantly down regulated (P<0.05) by 20%, 42%, 15% and 11%, respectively. [Conclusion] The crude polysaccharide of Melastoma dodecandrum Lour. could intervene the occurrence of obesity by regulating the level of lipid metabolism in obese mice induced by high-fat diet.

Fish have an imperfect digestive system and low digestive enzyme activity compared with livestock and poultry animals. Plant raw materials often contain anti-nutritional factors and toxic components, resulting in intestinal injury, enteritis, and other diseases. Short-chain fatty acids such as butyric acid can significantly improve fish growth performance, enhance fish health, antioxidant and anti-stress ability. Due to the poor smell of butyric acid affecting the palatability of animals, sodium butyrate, as a substitute for butyric acid, is solid and non-volatile, easy to add and use. Sodium butyrate can promote the development of intestinal epithelial cells, improve the intestinal morphological structure, maintain the integrity of intestinal mucosal barrier, repair the damaged intestinal mucosa, stimulate the synthesis of mRNA and protein in epithelial cells, accelerate the proliferation of intestinal epithelial cells, increase the height of villi, and promote digestion and absorption. Sodium butyrate can destroy the DNA structure of harmful bacteria, and protein can not be synthesized, so that harmful bacteria can not normally divide, inhibit the growth and reproduction of harmful intestinal bacteria, promote the proliferation of beneficial bacteria, maintain the balance of intestinal flora, and significantly improve the diversity of flora. Sodium butyrate can improve fish digestion by stimulating the secretion of digestive enzymes, reduce the intestinal damage caused by poor protein raw materials, inhibit the apoptosis of fish intestinal cells, reduce the occurrence of intestinal inflammation, and significantly improve the function of fish intestinal barrier. Sodium butyrate can be used as a growth promoter and immune stimulant, reduce cost in low fish meal feed and improve fish growth and development. Therefore, sodium butyrate is a green and safe additive that can replace antibiotics, and plant protein can appropriately replace fish meals to improve economic benefits. The author focuses on the effect of sodium butyrate on intestinal digestion and absorption to provide a scientific reference for the application of sodium butyrate in fish and other aquaculture.

[Objective] This study was to explore the preventive mechanism of docosahexaenoic acid (DHA) on liver fat accumulation induced by high fat diet (HFD). [Method] 32 male mice were divided into 4 groups. Control group (Con) mice were fed with control diet, model group (Model) mice were feed with high fat diet, DHA groups were supplemented with 0.2 g DHA (DHAL) and 1.0 g DHA (DHAH) in high fat diet, respectively. The feeding cycle was 20 weeks. The body weight and diet weight were weighed during feeding, and the food intake was calculated. At the end of feeding, liver and blood were collected, and the contents of liver adiponectin and serum triglyceride (TG) were detected by ELISA. The expression of key enzymes of newborn lipogenesis (SREBP-1c, FAS), key genes of fatty acid oxidation (PPARα, PPARγ, CPT-1A and ACOX), mitochondrial gene (PGC-1α) and brown fatty genes (PRDM16, UCP1) were detected by Real-time quantitative PCR. The expression of the hepatic phosphorylated ACC, AMPK and AKT proteins were determined by the Western blotting. [Result] Compared with Con group, the final body weight, body fat weight and TG content were significantly increased (P<0.05), and hepatic adiponectin level was significantly decreased in Model group (P<0.05). When compared with Model group, the final body weight, body fat weight and TG content of DHAL and DHAH groups were significantly decreased (P<0.05), and liver adiponectin level was significantly increased(P<0.05). The results of Real-time quantitative PCR showed that compared with Con group, SREBP-1c and FAS mRNA level of Model group were significantly increased (P<0.05), PPARα, CPT-1A, ACOX, PGC-1α and UCP1 mRNA level of Model group were significantly decreased (P<0.05). While compared with Model group, SREBP-1c and FAS mRNA levels of both DHAH and DHAL groups were significantly decreased (P<0.05), and the level of DHAH group was lower than that of DHAL group (P<0.05), PPARα, CPT-1A, ACOX, PGC-1α, Prdm16 and UCP1 mRNA expression level of DHAL and DHAH groups were significantly increased (P<0.05), CPT-1A and ACOX mRNA level of DHAH group was higher than that of DAHL group (P<0.05), and there was no significant difference in PPARγ mRNA expression level among the four groups(P>0.05). The results of Western blotting showed that p-ACC in Model group was significantly higher than that in Con and DHAH groups, but significantly lower than that in DHAL group, p-AMPK/AMPK and p-AKT/AKT of Model group were smaller than those of Con group (P<0.05), p-AMPK/AMPK and p-AKT/AKT of DHAH group were bigger than those of Model and DHAL groups (P<0.05). [Conclusion] DHA could reduce the increase of final body weight, body fat and TG content in C57BL/6 mice caused by high fat diet, promote fatty acid oxidation and browning of white adipocytes, so as to prevent fat accumulation in liver.

The intestinal tract is the main part of digestion and absorption of nutrients, but it is easy to be stimulated by the external environment to cause pathological changes. The frequent occurrence of animal intestinal diseases seriously affects the growth and development process of animals, increases the feeding cost of animals, and has a huge impact on the economic benefits of breeding industry. Although antibiotics are an effective strategy to alleviate the occurrence and development of intestinal diseases, people have to develop new environment-friendly treatments because of their drug resistance and drug residues. Plant polysaccharides widely exist in plants in nature, and have the characteristics of high efficiency, non-toxic side effects and no residue. As a green feed additive, plant polysaccharides can prevent intestinal diseases and improve animal production performance. It is mainly through the regulation of nuclear factor-κB(nuclear factor kappaB, NF-κB), mitogen activated protein kinase (MAPK), Janus kinase/signal transducer and activator of transcription (JAK/STAT), and transforming growth factor-β (TGF-β), phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and other signaling pathways to give full play to the physiological function of intestinal barrier, strengthen the interaction between various barriers, prevent animal intestinal diseases, regulate the level of intestinal inflammation, maintain the balance of intestinal microflora, enhance the body's antioxidant capacity and the immune function of animal body, and then, improve the quality of animal products to a certain extent, enhance the economic value created by animals. In this paper, the authors summarize the protective effects of plant polysaccharides on animal intestine and its molecular mechanism and expound its application and effect in animal production, in order to provide reference for the application of plant polysaccharides in animal production.

[Objective] The purpose of this experiment was to investigate the effects of compound plant extracts on carcass traits, muscle quality indexes and serum indexes in finishing Sujiang pigs. [Method] 200 healthy Sujiang castrated boars with body mass of 55 kg of 120 days old were randomly divided into 5 groups with 4 replicates in each group and 10 pigs in each replicate. The control group was fed corn-soybean meal basal diet, and the test groups A, B, C, and D were supplemented with 100, 200, 300, and 400 mg/kg plant extracts in the basal diet, respectively. The main ingredients of the plant extracts were Eucommia, Ligustrum lucidum and Schisandra (the ratio was 4:3:1). The main active ingredients of the plant extracts were chlorogenic acid, oleanolic acid and lignans, etc. The whole trial was divided into pre-feeding period (7 d) and formal period (45 d). After the experiment, blood was collected and the experimental pigs were slaughtered. And carcass traits, muscle composition, meat quality, serum biochemical indexes, antioxidant indexes and immune indexes of finishing pigs were determined. [Result] The average backfat thickness of groups B and C were significantly lower than that of control group (P<0.05), and the loin-eye muscle area of group B was significantly higher than that of control group and group A (P<0.05). The normal muscle compositions such as crude protein, crude fat and crude ash did not show difference among the groups (P>0.05). The marbling score of group B was significantly higher than that of control group, groups A and C (P<0.05). The meat color score of groups B and D were significantly higher than that of control group (P<0.05). The a^* value of group B was significantly higher than that of control group and group A (P<0.05). Compared with control group, the total serum protein of group B was significantly increased (P<0.05). The serum albumin of group D was significantly higher than that of control group (P<0.05). The serum MDA of groups A and D were significantly lower than that of control group (P<0.05). The IgA of group B was significantly higher than that of control group and group A (P<0.05). There was no significant difference in other indicators among the groups. [Conclusion] The addition of 200 mg/kg compound plant extracts to the basic diet of Sujiang pigs during the fattening period was beneficial to improve carcass traits, muscle quality and serum indexes, and could be used in the production of Sujiang pigs.

Fermentation is a traditional method of Chinese medicine processing method, which has the function of increasing the content of active ingredients, enhancing or producing new effects, reducing toxicity, and regulating intestinal flora. Modern fermentation technology overcomes the shortcomings of traditional fermentation technology, such as the complicated bacteria strains, uncontrolled temperature and humidity. The modern fermentation technology of Chinese medicine includes solid-state fermentation, submerged fermentation and bidirectional solid fermentation. In recent years, the state vigorously promotes reduced use of antibiotics in livestock and poultry industry. As a microecological preparation, fermented Chinese medicine has become one of the alternatives of antibiotic in livestock and poultry industry with its unique advantages in improving animal production performance, prevention and treatment of diseases. It has great potential and market in livestock and poultry industry and aquaculture industry. However, fermented Chinese medicine still has many problems, such as unclear fermentation mechanisms, unknown changes in composition, single fermentation bacteria, and lack of specific quality standards. The author introduces the advantages of fermented Chinese medicine and compares the advantages and disadvantages of various fermentation techniques, summarizes the application of fermented Chinese medicine in livestock and poultry industry as well as aquaculture industry, analyzes the potential of fermented Chinese medicine and the shortcomings of current research, intending to provide ideas and references for the in-depth study of fermented Chinese medicine in the future.

[Objective] In this experiment, lactic acid bacteria with high antioxidant activity were screened and identified in order to provide a new antioxidant microecological agent for alleviating oxidative stress in the process of livestock and poultry breeding. [Method] Lactic acid bacteria with significant antioxidant capacity were screened from 27 strains, by establishing a primary screening method for tolerance to hydrogen peroxide oxidative stress, and taking the tolerance survival rate to hydrogen peroxide as the re-screening index. Then the antioxidant activity of lactic acid bacteria was evaluated by DPPH free radical (DPPH·) and hydroxyl free radical (HO·) scavenging ability, reducing activity, anti lipid peroxidation ability, total superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC) in vitro. [Result] A Lactobacillus C2-0327 with excellent antioxidant activity was screened from the 27 strains. Scavenging rates of DPPH· and HO· of this isolate C2-0327 were 95.2% and 141.0%, respectively. The reducing activities of this isolate reached 3 147 μmol/L (L-cysteine equivalent), and inhibition rate of lipid peroxidation was up to 82.9%. T-SOD activity and T-AOC were 81.32 and 20.10 U/mL, respectively. The strain was identified as Pediococcus pentosus C2-0327 by 16S rDNA molecular identification. [Conclusion] A strain of Lactobacillus with high antioxidant activity was screened and identified, which was Pediococcus pentosus C2-0327. The high antioxidant Pediococcus pentosus could be used for the development of new antioxidant probiotics.

[Objective] The aim of this study was to explore the single nucleotide polymorphism (SNP) of enolase 3 (ENO3) gene, and analyze the linkage imbalance between SNPs and its correlation with growth traits in Yorkshire pigs. [Method] A total of 316 sows were selected, the SNP of ENO3 gene were identified by PCR amplification product sequencing of mixed DNA samples. GenoPlexs typing technique was used to genotype the target SNP of each individual, the linkage disequilibrium and correlation analysis were conducted with daily gain, days, backfat thickness, loin-eye area and loin-eye thickness at 100 kg body weight, and explored the correlation between ENO3 gene SNP and growth traits of Yorkshire pigs. [Result] The results showed that 9 SNPs were identified, among which rs196953768 was completely linked to rs324943047, rs345530479 and rs329283992 (D'=1, r²=1), and strongly linked to rs342598032 (D'=1, r²=0.99), rs327882211 was strongly linked with rs341541240 and rs325279236 (D'=1, r²>0.7). ENO3 gene rs196953768 and its complete linkage locus were significantly correlated with days and daily gain at 100 kg body weight (P<0.05), rs327882211 and rs341541240 were significantly correlated with loin-eye area and loin-eye thickness at 100 kg body weight (P<0.05);rs325279236 was significantly correlated with loin-eye area at 100 kg body weight (P<0.05);rs786427749 and rs342598032 had no significant correlation with the above growth traits (P>0.05). [Conclusion] In this study, a total of 9 SNPs were excavated, 4 fully linked SNPs were identified on ENO3 gene, 7 SNPs significantly correlated with growth traits were screened, it provided reference data for molecular marker assisted breeding of Yorkshire pigs.

N⁶-methyladenosine (m⁶A) is the most prevalent epigenetic modification of eukaryotic RNA, it is highly conserved among eukaryotes and plays important roles in gene expression and cell fate determination. Meanwhile, it has great influence on mRNA processing, including alternative splicing, localization, translation efficiency and stability. The existing m⁶A modification detection technology can accurately and efficiently detect the m⁶A modification abundance in biological samples, quickly and easily carry out high-throughput sequencing of m⁶A modification, and detect the position of m⁶A modification on RNA at single base resolution. Although there are a few reports on m⁶A-related proteins regulating animal complex economic traits, the mechanisms are still unclear. A lots of studies on humans and model organisms showed that m⁶A-related proteins play the vital roles in lots of biological process, such as growth and development, reproduction, heat stress, inflammation and cancer, and these ideas and discoveries can provide a great reference for exploring the effect of m⁶A on domestic animals' complex economic traits. The author mainly expounds m⁶A methylation modification related proteins (writers, erasers and readers), m⁶A detection technology, the regulation mechanism of m⁶A on complex traits of mammals, and the interaction mechanism between m⁶A and other eoigenetic modifications, so as to provide new insights for the application of m⁶A in livestock and poultry genetic breeding.

[Objective] The experiment was to study the effect of resveratrol(RES) on the proliferation activity, hormone secretion, cumulus expansion as well as apoptosis and antioxidase of buffalo cumulus cells cultured in vitro. [Method] Cumulus cells were cultured in medium supplemented with different concentrations (0(control group), 1, 10, 20, 30, 40, 50 and 60 μmol/L) of RES, cell proliferation activity was measured using CCK-8 kit. The best RES treatment concentration and time were selected for subsequent tests. The concentrations of estradiol (E₂)and progesterone (P₄)in the culture medium were measured by ELISA method, and the expression level of genes related to cumulus expansion, apoptosis and antioxidase were determined by Real-time quantitative PCR method. [Result] Compared with control group the proliferation activities of cumulus cells cultured in medium supplemented with 1, 10 and 20 μmol/L RES within 24-36 h were significantly promoted. The cell viability was the strongest after 36 h treatment with 10 μmol/L RES, so it was used for cell treatment in subsequent experiments. When cultured in vitro for 36 h, the cell proliferation ability and the secretion of E₂ and P₄ of 10 μmol/L RES group were significantly increased (P<0.05). The relative mRNA expression of PTX3, PTGS2, Bcl-2 and SOD1 genes of 10 μmol/L group was significantly or extremely significantly increased (P<0.05;P<0.01), and the expression of Bax, Caspase-3 and p21 genes was significantly or extremely significantly decreased (P<0.05;P<0.01), while there was no significant difference in the expression of HAS2 and CAT genes (P>0.05). [Conclusion] 10 μmol/L RES could increase the proliferation activity of cumulus cells, promote hormone secretion and the expansion of cumulus cells, and enhance the antioxidant capacity and reduce apoptosis of cumulus cells in buffalo.

X chromosome inactivation (XCI) is an epigenetic mechanism of dosage compensation between two sexes in mammals during mammalian embryonic development, which leads to the lifelong transcriptional silencing of one randomly chosen X chromosome in females. X chromosome inactivation center (XIC) is the main regulatory region of XCI, X-inactive specific transcript (Xist) is located in this region, and it is a key part of XCI. Xist long chain non-coding RNA(lncRNA) is an inactive specific transcript, which can recruit a series of auxiliary factors such as RNA interaction binding protein (RBP) and chromatin modification factors to change the spatial structure of chromosome and nucleus, chromatin composition and X-linked gene transcription level, so as to transform active chromosomes into inactive chromosomes. At present, studies in mice, humans and other species have found that the establishment and maintenance of appropriate econometric compensation are very important and widespread for mammals to survive in the embryonic development process. However, there are certain specific differences in the biological phenomenon of embryos in different species and genders, and some human diseases are also related to XCI, such as cancer and tumor. At present, the research on the mechanism of XCI is relatively scattered. The author summarizes the main molecular mechanism, regulatory mechanism and research in different species of XCI mediated by Xist lncRNA.

[Objective] The study was aimed to evaluate the effect of trehalose as extracellular cryoprotectant on the morphology, active mitochondrial distribution and nuclear maturation of vitrified-thawed bovine immature oocytes. [Method] The immature oocytes were randomly divided into fresh group, different concentrations (0.25, 0.5 and 1 mol/L) of trehalose and 0.5 mol/L sucrose groups. The fresh group was used as the control group of vitrification, and the trehalose and sucrose groups were vitrified and thawed by two steps. Trehalose and sucrose were not added in the first step of freezing, trehalose and sucrose of corresponding concentration were added in the second step of freezing and the first step of thawing respectively, and trehalose and sucrose concentrations were halved in the second step of thawing. After warming, the normal rate of oocyte morphology was counted, oocytes from trehalose, the distribution area and fluorescence intensity of active mitochondria in oocytes were detected by JC-1 staining, and the nuclear maturation rate of oocytes was counted after 24 h of in vitro maturation culture. [Result] The morphological normal and nuclear maturation rate of oocytes in vitrification groups were significantly lower than those in fresh group (P<0.05), the percentage of morphological normal and nuclear maturation rate of oocytes vitrified in the presence of 0.5 mol/L trehalose were significantly higher than those of 0.5 mol/L sucrose, 0.25 and 1 mol/L trehalose (P<0.05). The active mitochondria of oocytes in 0.5 mol/L trehalose group were evenly distributed in the inner region of cell membrane, which showed strong red fluorescence and yellow green fluorescence under green and blue light, respectively. The fluorescence intensity in 0.5 mol/L trehalose group was higher than that of other vitrified oocytes, but still lower than that of fresh oocytes. The distribution area and fluorescence intensity of active mitochondria in 0.25 mol/L trehalose group were lower than those in 0.5 mol/L trehalose group, and its fluorescence was green and showed discontinuous scattered distribution under blue light. The distribution area of oocyte active mitochondria in 0.5 mol/L sucrose group and 1 mol/L trehalose group was very few, and its fluorescence was dark green under blue light. [Conlusion] 0.5 mol/L was considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of bovine immature oocytes.

The terminally differentiated mammalian gametes are converted into totipotent fertilized embryo after fertilization. The embryo's development progress is initially guided by oocyte's products stored in the embryo. When the maternal-to-zygotic transition (MZT) completes, maternal mRNA is degraded, the zygotic genome begins to be transcribed, and the development of the zygote is regulated by itself. With the development of embryos, the epigenetic genome has undergone drastic reprogramming, and epigenetic modification plays an important role in the process of embryonic development. Notably, chromatin remodeling regulates zygotic gene expression by adjusting the genome accessibility of DNA-binding proteins. Chromatin remodeling refers to the sharp changes in chromatin structure and genome accessibility. The uneven distribution of nucleosomes leads to different loose degrees in different regions of the genome, and the regions with high chromatin accessibility are usually important regulatory regions and easier to combine with transcription factors. Chromatin remodeling participates in the regulation of zygotic gene expression by regulating the genomic accessibility of DNA binding proteins, and plays an important role in the process of zygotic genome activation (ZGA). In the review, the authors discuss the dynamic changes in the chromatin structure and local chromatin accessibility during zygotic genome activation, and their role in zygotic genome activation, which provides a reference for understanding the regulatory mechanism of zygotic gene expression.

[Objective] This study was to explore the effects of different oocyte's origin on developmental ability of reconstructed embryos after somatic cell nuclear transfer (SCNT) and the expression levels of key development related proteins. [Method] The experiment was divided into two groups:Ovum pick up (OPU) and slaughterhouse (SLH) groups. In the OPU group, the follicles of 10 non-lactating buffalo were punctured and aspirated by ultrasonic in vivo to collect oocytes. In SLH group, follicles were drawn from the ovaries of slaughterhouses to collect oocytes. All the COCs were matured in vitro for 22-24 h, respectively. The cumulus cells were removed by blowing, the oocytes with the first polar body were selected and enuvleated for SCNT using ear fibroblasts as donor cells, and the fusion rate, cleavage rate and blastocyst rate of SCNT reconstructed embryos were recorded respectively. Furthermore, the protein expression levels of E-cadherin and transcription factor Sox2 in two SCNT reconstructed embryos were determinated by immunofluorescence. [Result] The maturation rate of oocytes and the blastocyst rate of reconstructed embryos in OPU group was significantly higher than that of SLH group (P<0.05), but there were no significant differences in fusion rate and blastocyst rate of two SCNT reconstructed embryos (P>0.05). According to the analysis of immunofluorescence images, E-cadherin protein was located on the cell membrane, Sox2 protein was distributed in the cell nuclear membrane and cytoplasm, and the expression level of E-cadherin and Sox2 protein in OPU group SCNT embryos was significantly higher than that of SLH-SCNT embryos (P<0.05). [Conlusion] Buffalo oocytes collected in vivo were more suitable for the construction of SCNT reconstructed embryos.

The rabbit is the one of Chinese traditional domestic animals, and also is an important part of animal husbandry in China. In recent years, with the industry upgrading, the developing rapidly of biotechnology, great progress has been made in rabbit genetic breeding, both at home and abroad. In this review, the research progress on genetic breeding and reproduction of rabbits in 2020 are reviewed from the traditional breeding, molecular breeding and reproduction of rabbits. For abroad, lots of researches involved in traditional breeding and molecular breeding. Traditional breeding mainly focuses on the effects of selection and environment on the growth and reproductive performance of rabbits. In molecular breeding, the related genes of growth, meat quality and genetic diversity were studied. In China, there are more studies on rabbit genetic breeding and reproduction than abroad, and the research concentrate mainly on molecular breeding, including functional genes related to fur traits, meat quality traits and reproductive performance. In addition to, there are lots of studies on the effects of selection and environmental effects on growth and reproductive performance of rabbits in traditional breeding. This review could provide basis and ideas for the protection, development and utilization of the rabbit.

[Objective] This study was aimed to explore whether the concentration of glycerol in cryoprotectant could be reduced by supplementing trehalose, so as to improve the quality of frozen-thawed sperm. [Method] Sperm were treated with 6% glycerol (group Ⅰ) and 3% glycerol+0, 50, 100 and 150 mmol/L trehalose (Ⅱ, Ⅲ, Ⅳ and Ⅴ groups), respectively. The motility, plasma membrane integrity, acrosome integrity and dynamic parameters of frozen-thawed sperms were detected by the dynamic (static) image detection system (CASA), and the best trehalose treatment concentration was selected for subsequent experiments. Tyrosine phosphorylation of sperm protein was detected by Western blotting, the viability and mitochondrial membrane potential of sperm were detected by Hoechst 33342/PI/JC-1 combined staining, sperm DNA integrity was detected by chromomycin A₃ (CMA₃) staining, and the level of lipid disorder in sperm membrane was detected by cyanine 540 (M540) and Yo-Pro-1 staining. [Result] Compared with Ⅰ group, sperm motility, plasma membrane and acrosomal membrane integrity, VCL (curvilinear velocity) and STR (straightness) of Ⅱ group were significantly decreased (P<0.05), the sperm motility, VCL, VSL (straight line velocity), VAP (average path velocity), LIN (linearity) and STR of Ⅲ, Ⅳ and Ⅴ groups were significantly increased (P<0.05). Therefore, 100 mmol/L trehalose was used to treat sperm in the follow-up test. Compared with fresh sperm, the protein tyrosine phosphorylation bands of frozen-thawed sperm was decreased significantly after capacitation (P<0.05), and the viability and mitochondrial membrane potential of frozen-thawed sperm was decreased significantly (P<0.05). The level of high membrane lipid disorder of frozen-thawed live sperm and dead sperm was increased significantly (P<0.05), and 100 mmol/L trehalose could significantly reduce the level of high membrane lipid disorder (P<0.05). The protamine deletion rate of frozen-thawed sperm was significantly increased (P<0.05), and the protamine deletion rate of Ⅱ and 100 mmol/L trehalose groups was significantly lower than that of Ⅰ group (P<0.05). [Conclusion] Trehalose could be used as a new cryoprotectant to reduce the toxicity of glycerol. Replacing part of glycerol with 100 mmol/L trehalose could significantly improve the sperm quality of Yanbian Yellow cattle after freezing and thawing.

The main function of Leydig cells (LCs) is to synthesize and secrete testosterone. In Leydig cells, steroidogenic acute regulatory protein (StAR) located on the outer mitochondrial membrane with cholesterol as raw material promotes the transport of cholesterol to the inner mitochondrial membrane, and pregnenolone is produced under the catalysis of cholesterol side chain cleavage cytochrome (P450scc) in the inner mitochondrial membrane, then hydroxyl steroid dehydrogenase 3 through smooth endoplasmic reticulum β-hydroxysteroid dehydrogenase(3β-HSD) and transporter protein (TSPO) to synthesize testosterone. Therefore, the synthesis and secretion of testosterone by Leydig cells are closely related to mitochondria. The integrity of mitochondrial structure and function directly affects the biosynthesis of testosterone, and StAR and P450scc located on mitochondria are the key regulatory factors of testosterone synthesis. Testosterone can promote the development and maturation of male reproductive organs and maintain their function. It is of great significance to promote protein synthesis (such as protein synthesis in muscle, bone and reproductive organs). In recent years, promoting testosterone synthesis by maintaining mitochondrial structural integrity and improving mitochondrial oxidative damage and mitochondrial biogenesis has become a research hotspot of testosterone synthesis mechanism, which has attracted extensive attention of scholars at home and abroad. In this review, the molecular mechanism of testosterone synthesis in Leydig cells and the important factors affecting testosterone synthesis were introduced, the effects of mitochondrial structure, mitochondrial oxidative damage, mitochondrial regulated cell death and mitochondrial biogenesis on testosterone synthesis in Leydig cells were reviewed, the relationship between mitochondria and testosterone synthesis was expounded. It can provide a basis for improving the structure and function of mitochondria in Leydig cells and promoting testosterone synthesis, and it is of great significance to deeply understand the regulation of testosterone synthesis and improve the reproductive performance in male animals.

[Objective] The interferon stimulating gene 15 (ISG15) gene of Tupaia belangeri yaoshanensis (T. b. yaoshanensis) was cloned, the ISG15 protein was highly expressed and purify in Escherichia coli, then its polyclonal antibody was prepared, which laid the foundation for its biological application and the establishment of detection methods. [Method] Total RNA was extracted from the peripheral lymphocytes of T. b. yaoshanensis, and ISG15 gene was amplified by RT-PCR. ISG15 gene was subcloned into the eukaryotic expression vector to construct the recombinant eukaryotic expression plasmid pcDNA3.1-ISG15, and transiently transfected into renal cells of Cricetidae baby hamster syrian kidney (BHK-21) cells. At the same time, it was subcloned into the prokaryotic expression vector pET-28a(+) and the recombinant expression plasmid pET-28a-ISG15 was constructed. The pET-28a-ISG15 was transformed into E. coli BL21(DE3), and the ISG15 protein of T. b. yaoshanensis was induced by IPTG. The recombinant protein was purified by nickel ion affinity chromatography and immunized with mice to obtain mouse anti-ISG15 polyclonal antibody, and its reactivity was detected by Western blotting and IFA. [Result] ISG15 gene of T. b. yaoshanensis was successfully cloned, and its eukaryotic and prokaryotic expression vectors were constructed. The eukaryotic expression vector could be highly expressed in BHK-21 cells. The prokaryotic expression vector was induced with 0.5 mmol/L IPTG at 30 ℃ for 6 h to obtain the recombinant protein with molecular weight at approximately 22 ku. The protein was expressed in the form of inclusion body and purified by nickel ion affinity chromatography. The mice were immunized with the emulsified recombinant protein to prepare the polyclonal antibody against ISG15 of T. b. yaoshanensis. Western blotting showed that the mouse-anti-T. b. yaoshanensis ISG15 polyclonal antibody could still bind to 0.01 μg of ISG15 recombinant protein at the dilution of 1:8 000. IFA tests showed that the antibody could react with the proteins overexpressed by eukaryotic cells, with good reactivity. [Conclusion] The constructed prokaryotic expression vector highly expressed ISG15 protein of T. b. yaoshanensis in E. coli, and the recombinant protein had good immunogenicity after purification and renaturation. The obtained polyclonal antibody laid a good foundation for further study on the antiviral infection immunity of T. belangeri.

[Objective] The purpose of this experiment was to investigate the effect of mitochondrial fusion protein 2 (MFN2) gene interference and overexpression on mouse macrophages apoptosis induced by Brucella. [Method] Three siRNA sequences, siMFN2-450, siMFN2-1661, siMFN2-2275 and one negative interference sequence siMFN2-Negative specifically targeting MFN2 gene were designed by RNA interference techniques. A recombinant plasmid pcDNA3.1-EGFP-MFN2 was constructed by overexpression technique using empty plasmid pcDNA3.1-EGFP. The overexpression of recombinant plasmid pcDNA3.1-EGFP-MFN2 was identified by double enzyme digestion. The best interference sequence was screened and the overexpression efficiency of the recombinant plasmid was identified by Real-time quantitative PCR and Western blotting. The optimal interference sequence and the overexpressed recombinant plasmid were used to construct the macrophage model of MFN2 gene interference and overexpression, respectively. Macrophages were infected with Brucella bovis vaccine strain A19 for 24 hours according to the ratio of bacterial number to cell number (100:1). The expression of Bcl-2 related X protein (BAX) was detected by Real-time quantitative PCR and Western blotting, and cell apoptosis was detected by flow cytometry. [Result] The results of double enzyme digestion showed that the overexpression recombinant plasmid pcDNA3.1-EGFP-MFN2 was successfully constructed. Real-time quantitative PCR and Western blotting results showed that siMFN2-1661 could extremely significantly inhibit the expression of MFN2 (P<0.01), and pcDNA3.1- EGFP-MFN2 could extremely significantly enhance the expression of MFN2 (P<0.01). The transfected macrophage model with interference and overexpression of MFN2 gene was successfully constructed. After macrophages interfering with MFN2 gene expression were infected by Brucella, the protein expression levels of BAX was significantly higher than that in siMFN2-Negative group (P<0.05), transcription levels and the apoptosis rate of BAX were extremely significantly higher than that in siMFN2-Negative group (P<0.01). After macrophages overexpressing MFN2 gene were infected by Brucella, the transcription and protein expression of BAX were significantly lower than those in pcDNA3.1-EGFP group (P<0.05), and the apoptosis rate was extremely significantly lower than that in pcDNA3.1-EGFP group (P<0.01). [Conclusion] The study showed that MFN2 gene interference would enhance the ability of Brucella to induce cell apoptosis, while MFN2 gene overexpression would inhibit the ability of Brucella to induce cell apoptosis. The results could provide a reference for studying biological function of MFN2 gene and provide a theoretical basis for further analysis the pathogenic mechanism of Brucella.

The outbreak of animal epidemic diseases not only seriously affects the production and development of animal husbandry, causing economic losses, but also seriously endangers human health. The increase of viral diseases makes animal diseases more complex and the epidemic trend more severe. Sensitive, specific, rapid diagnosis of animal epidemic diseases is the key link to implement disease prevention and control measures. Molecular biological diagnosis based on PCR technology and isothermal amplification technology has become a commonly used pathogen screening method, because it solves the problems of traditional pathogen diagnosis, such as time-consuming and laborious, window period of immune serology, and gets rid of the dependence on high-precision temperature control equipment. However, most detection methods can only detect one or several pathogens at one time, and the complex test process and high requirements for facilities reduce the ability of immediate intervention in the outbreak, and cannot meet some detection and analysis needs. In recent years, interdisciplinary high-throughput diagnostic technology has developed rapidly. It can monitor, detect and characterize hundreds of targets at the same time, which is suitable for the rapid detection of pathogens in various environments. It is an important biological detection method in the development of life science. In this review, we summarized several major viral animal disease detection methods including etiology, immune serology, molecular biology, and high-throughput diagnosis. Looking forward to the development of animal disease diagnosis and detection technology in the future, and these methods provide more capability for the rapid diagnosis of animal diseases and improve the prevention and control ability of animal diseases.

[Objective] The aim of this study was to purify SVV-CH-HB2016 strain and prepare monoclonal antibodies of Seneca Valley virus (SVV) specific to the structural proteins VP1, VP2 and VP3. [Method] SVV-CH-HB2016 virus concentrate was purified using sucrose density gradient centrifugation and used as antigen to immunize BALB/c mice. Immune spleen cells and myeloma cells (SP2/0) were used in cell fusion experiment. Both indirect immunofluorescence assay (IFA) and indirect ELISA were conducted to screen positive cell lines which could secrete hybridoma cell lines specific to structural proteins. The reactivity of monoclonal antibody with recombinant and natural structural proteins was tested using Western blotting and IFA, the neutralization and adsorption effects of monoclonal antibody were also tested. Then plaque assay and Real-time PCR were used to explore the effect of neutralized monoclonal antibody on the absorption process of SVV-CH-HB2016 strain. Finally, the antibody addition test was used to analyze the epitope of the monoclonal antibody. [Result] Both the purity and concentration of SVV-CH-HB2016 strain structure protein could keep a higher level when sucrose gradient was between 5%-45% (W/V). The serum antibody titer of the immunized mice reached 1:12 800, and successfully prepared 17 hybridoma cell lines capable of stably secreting specific monoclonal antibody. It was found that 14 monoclonal antibody strains could react with recombinant structural proteins in Western blotting, and 17 monoclonal antibody strains could react with the virus in IFA. Strains 2G6, 4A3 and 4C11 not only had a significant neutralizing and protective effect on BHK-21 cells infected by SVV-CH-HB2016 strain but also could effectively inhibit the adsorption of SVV-CH-HB2016 strain on 293T cells. It was found that in addition to 1F5 and 2E1, 4B8 and 4F11, the other 10 monoclonal antibody strains were specific to different epitopes. [Conclusion] In this study, the purification method of SVV was preliminarily established, and 17 monoclonal antibody strains specific to SVV-CH-HB2016 strain were prepared, which laid a foundation for the further development of inactivated SVV vaccine, the establishment of ELISA detection method and the identification of protective antigen epitope in the future.

[Objective] The purpose of this study was to establish an indirect ELISA method based on Mycoplasma mycoides subsp. capri (Mmc) membrane lipoprotein LPPA. [Method] In this study, the Mmc LPPA gene was amplified and cloned into pCold-Ⅰ vector. The recombinant plasmid pCold-Ⅰ-LPPA was constructed. After sequencing identification, it was transformed into E. coli DH5α competent cells. The recombinant protein of Mmc LPPA was purified after induced expression by IPTG. The expression of the recombinant protein was verified by SDS-PAGE. Its reactivity with Mmc positive serum was analyzed by Western blotting test and traditional classical agar diffusion serological test. Using the recombinant protein as the coating antigen, an indirect ELISA antibody detection method for Mmc recombinant LPPA protein was established. After the reaction conditions were optimized, the specificity and repeatability tests were carried out, and it was preliminarily applied to 184 goat serum samples. [Result] The Mmc LPPA gene was amplified by PCR, and the recombinant plasmid pCold-Ⅰ-LPPA was successfully constructed. After induced expression, the recombinant protein Mmc LPPA was obtained. SDS-PAGE result showed that the recombinant fusion protein of LPPA with the size of about 23 ku was obtained. Agar diffusion and Western blotting showed that the protein had good reactivity. The optimized reaction conditions of ELISA showed that the coating concentration of LPPA antigen protein of 2.0 μg/100 μL, serum dilution of 1:300 and blocking with 3% BSA for 1 h were the optimum reaction conditions, and the critical value was 2.738. Intra assay and inter assay coefficients of variation of the indirect ELISA method established in this test were both less than 10%, which proved that the method had good repeatability. The standard positive sera of Mo, BTV, PPRV, FMDV, Toxoplasma gondii and GPV were negative, indicating that the method had strong specificity. The clinical application results of 184 clinical sera showed that the positive coincidence rate between the method and the positive indirect hemagglutination diagnostic kit was 93.33%, the negative coincidence rate was 75.23%, and the relative coincidence rate was 82.61%. [Conclusion] This study successfully established an indirect ELISA method for the detection of antibodies for Mmc LPPA protein, which laid a foundation for the monitoring of serum antibody level of clinical Mmc and the investigation of serum epidemiology of Mmc.

[Objective] The aim of the experiment was to construct a recombinant Swinepox virus (rSWPV) expressing green fluorescent protein (GFP) and prepare monoclonal antibody against GFP. [Method] Firstly, the enhanced green fluorescent protein (EGFP) sequence EGFP-His containing His-tag at 3'-end was synthesized and inserted into the basic plasmid vector pSW by double digestion to construct the recombinant transfer vector pSW-EGFP-His. The vector was homologously recombined with SWPV(SWPV-JX20G strain) by liposome transfection, and rSWPV-EGFP-His was obtained by plaque purification. The recombinant virus was identified by PCR and SDS-PAGE, and EGFP-His protein was amplified and purified to immunize BALB/c mice. The spleen cells of mice were fused with SP2/0 cells to screen hybridoma cells secreting anti-GFP specific antibodies, and ascites was prepared. The titer and specificity of anti-GFP monoclonal antibodies were identified. [Result] PCR and SDS-PAGE results showed that rSWPV-EGFP-His was successfully constructed and purified. The virus infected PK15 cells stably expressed EGFP-His protein, which was about 27 ku, and was soluble. The EGFP-His protein purified by Ni-agarose showed obvious green. The serum antibody titer of BALB/c mice immunized with EGFP-His protein was 1:256 000. Hybridoma techniques were employed to produce monoclonal antibodies. Nine hybridoma cell lines stably secreting monoclonal antibodies against EGFP-His were prepared by hybridoma technique. Indirect ELISA and Western blotting results showed that seven of the nine monoclonal antibodies were linear epitopes for GFP, and the other two were conformational epitopes. [Conclusion] In this study, EGFP-His protein and nine monoclonal antibodies against EGFP-His were successfully prepared, which provided necessary materials for the subsequent establishment of immunological detection methods for GFP.

Recombinant virus live vector vaccines are live vector vaccines constructed through genetic engineering methods, avoiding some of the shortcomings of traditional vaccines, and have great advantages and application prospects. Animal Herpesviruses currently widely used as recombinant virus vectors include Herpesvirus of turkey (HVT), Pseudorabies virus (PRV), Duck enteritis virus (DEV), and avian Infectious laryngotracheitis virus (ILTV), etc. Analysis of the Herpesvirus genome sequence shows that its genome is large, there are many unnecessary regions for replication, and the ability to accommodate exogenous gene is strong. At the same time, the Herpesvirus has the advantages of wide host range, long duration of immunity, good safety and mature related gene manipulation technology. This article summarized the main methods of constructing recombinant animal Herpesviruses, including traditional homologous recombination technology, bacterial artificial chromosome technology (BAC), Fosmid library and CRISPR/Cas9 gene editing technology, and briefly described the basic principles and technical characteristics of each method, compared the advantages and disadvantages of each method, analyzed the optimal application conditions of each method, so as to provide more accurate theoretical and technical references for research work. The expression of exogenous genes were analyzed, and examples were given to demonstrate the different regulatory effects of different promoters on the expression of exogenous genes. At the same time, the common insertion sites of different Herpesviruses were listed and summarized. The research progress and the registration application information of related biological products of the current recombinant viruses HVT, PRV, DEV and ILTV live vector vaccines were summarized, and the high-selectivity exogenous genes and high-success rate insertion sites were induced, so as to provide a theoretical reference for the development of related biological products.

[Objective] The study was aimed to obtain the antigenic 12L protein of African swine fever virus (ASFV) used to serological diagnosis of ASFV. [Method] The 360-12L gene was synthesized and inserted into pET-28a(+) vector to construct prokaryotic expression plasmid pET-28a-12L. The correct plasmid identified by sequencing was transferred into E. coli BL21(DE3) competent cells for induction. The optimal induction temperature was selected and its solubility was analyzed. The recombinant protein 12L was purified by affinity chromatography and analyzed by SDS-PAGE. The recombinant protein 12L was used as immunogen to immunize female BALB/c mice to obtain mouse polyclonal antibody against ASFV 12L protein. The titer of the polyclonal antibody was determined by indirect ELISA, and its specificity was verified by indirect immunofluorescence assay. [Result] The prokaryotic expression plasmid of ASFV 12L was successfully constructed, and the recombinant strain pET-28a-12L-BL21 had a high expression level at 37 ℃ for 6 h, mainly in the form of inclusion body, with an obvious band at 54.7 ku, which was consistent with the expectation. Indirect ELISA showed that the titer of the polyclonal antibody against ASFV 12L protein was higher than 1:512 000. Indirect immunofluorescence assay showed that the polyclonal antibody against ASFV 12L protein had good specificity and could specifically recognize recombinant protein 12L. [Conclusion] The recombinant protein 12L expressed in E. coli had good immunogenicity, and the polyclonal antibody against ASFV 12L protein prepared from mice had high reactivity and specificity, which could lay a foundation and provide biomaterials for further study on biological function of ASFV recombinant protein 12L, serological diagnosis technology of ASFV and vaccine research.

[Objective] The best cut-off value of biomarkers for the diagnosis of fatty liver was screened by the receiver operating characteristic (ROC) curve. [Method] Serum was randomly collected from 77 Chinese Holstein dairy cows 7 days postpartum to determine physiological and biochemical indicators. According to the Y value calculated by the Reid equation, the cows were divided into normal group (Y>1) and fatty liver group (Y<1), and the differences in serum indexes of cows in two groups were compared, and the correlation analysis with Y value was performed to screen the group. The indicators with significant differences and correlations are used as biomarkers, and the receiver operating characteristic (ROC) curve was used to evaluate the AUC value, diagnostic sensitivity and specificity, and the best cut-off value of the biomarker. Finally, the accuracy of the Raid equation in diagnosing fatty liver was used as the standard, and the best cut-off value of NEFA and AST was used to detect the fatty liver of 461 dairy cows. [Result] Physiological and biochemical test results showed that NEFA, glucose(Glu), AST, alanine amino transferase(ALT) between two groups had extremely significant difference (P<0.01), total protein(TP) had significant difference (P<0.05), and other indicators had no significant difference (P>0.05). NEFA, Glu, AST, ALT, TP, triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), insulin(Ins), glucagon(Gln) were significantly correlated with Y value and could be used as biomarkers. The AUC value of NEFA, AST, ALT and Gln could be more than 0.5. Accurately diagnose fatty liver, the diagnostic sensitivity were 81.8%, 72.7%, 63.6% and 54.5%, the specificity were 92.8%, 89.4%, 84.8% and 69.7%, the best cut-off value were 471.74 μmol/L, 95.50 U/L, 46.50 U/L and 64.60 ng/mL, respectively. The accuracy of using NEFA, AST and NEFA+AST best cut-off values to diagnose fatty liver were 89.96%, 73.49% and 69.08%, respectively. The incidence rate diagnosed by the Y value was 54.01%, and the incidence rates diagnosed by NEFA, AST and NEFA+AST were 48.59%, 39.70% and 37.31%, respectively. [Conclusion] NEFA, AST, ALT and Gln could be used to quickly and accurately diagnose fatty liver.

[Objective] The purpose of this study was to express the structural protein ORF2 of Goose astrovirus 1 (GAstV-1) by prokaryotic expression system and prepare its polyclonal antibody. [Method] According to the gene sequence of GAstV-1 TZ03 strain, the ORF2 gene sequence was optimized and synthesized. It was cloned into prokaryotic expression vector pET-30a(+). The recombinant plasmid pET-ORF2 was constructed and transformed into E. coli BL21(DE3) competent cells. The recombinant protein was induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western blotting. The polyclonal antibody was prepared by immunizing rabbits with the purified recombinant protein, and the specificity of the polyclonal antibody was identified by indirect immunofluorescence assay (IFA). [Result] The results of enzyme digestion and sequencing showed that the recombinant plasmid pET-ORF2 was successfully obtained. SDS-PAGE showed that the molecular weight of the expressed recombinant protein was about 70 ku, mainly in the form of inclusion body. Western blotting showed that the protein could react specifically with mouse monoclonal antibody against His tag. The titer of rabbit polyclonal antibody against ORF2 protein detected by indirect ELISA was 1:128 000. IFA results showed that the polyclonal antibody could react specifically with GAstV-1. [Conclusion] The prokaryotic expression of GAstV-1 ORF2 recombinant protein had good immunogenicity, and the rabbit polyclonal antibody had high titer and specificity, which laid a foundation for the development of GAstV-1 related diagnostic reagents.

[Objective] The purpose of this study was to isolate and identify the isolated strains of ducks with clinical fungal infections in Baoding city, and screening the antibacterial drugs for the isolated strains in vitro. [Method] The strains were identified by morphological observation, PCR amplification, sequencing and animal regression test. Microdilution method was used to determine the minimum inhibitory concentration (MIC) and in vitro drug sensitivity test was used to calculate the colony count and diameters to investigate the in vitro antibacterial activities of Acorus gramineus, Phellodendron chinensis, Cassia obtusifolia, Sophora flavescens, Polygonum hydropiper, Pulsatilla adans and Herba taraxaci against Aspergillus fumigatus and Aspergillus flavus. [Result] Aspergillus fumigatus and Aspergillus flavus were successfully isolated. The results of the animal regression test showed that the chicks had the symptoms similar with diseased ducks, the liver had gray-white nodules on autopsy, and granulomatous lesions appeared in the diseased tissues, indicating that both pathogens could infect the chicks. The MIC₈₀ results of Aspergillus fumigatus showed that the MIC₈₀ of Acorus gramineus Phellodendron chinensis, Cassia obtusifolia and Sophora flavescens were 8, 16, 32 and 64 μg/μL, respectively, and Polygonum hydropiper, Pulsatilla adans and Herba taraxaci were all 128 μg/μL. The MIC₈₀ results of Aspergillus flavus showed that the MIC₈₀ of Acorus gramineus, Cassia obtusifolia and Phellodendron chinensis were 8, 16 and 32 μg/μL, respectively, and Sophora flavescens and Polygonum hydropiper were both 64 μg/μL, and Pulsatilla adans and Herba taraxaci were both 128 μg/μL. The results of drug sensitivity test in vitro showed that when the concentration of Acorus gramineus was 16 μg/μL, Aspergillus fumigatus and Aspergillus flavus colonies were grown on the medium, and when the concentration of Cassia obtusifolia was 32 μg/μL, colonies of Aspergillus flavus were grown on the medium, and the concentration of Phellodendron chinensis was 32 μg/μL, Aspergillus fumigatus colonies grew in the medium, while the larger diameter Aspergillus fumigatus and Aspergillus flavus colonies were grown in the medium with a concentration of 128 μg/μL of Sophora flavescens, Polygonum hydropiper, Pulsatilla adans and Herba taraxaci. [Conclusion] Acorus gramineus, Phellodendron chinensis, Cassia obtusifolia had significant inhibitory effects on the growth of Aspergillus fumigatus and Aspergillus flavus, among which Acorus gramineus had the most significant inhibitory effect. This experiment could provide new ideas for the research and development of traditional Chinese medicines against poultry aspergillosis.

[Objective] The purpose of the experiment was to study the bioequivalence of cephalexin test and reference formulations in Beagle dogs. [Method] Twenty-two Beagle dogs were randomly allocated to two equal-sized treatment groups in a randomized dual-cycle and dual-sequence cross-over design. The test (Trolevis^®300) and the reference (Rilexine^®300) formulations were orally administered in a single dose of 30 mg/kg BW. Blood samples were collected from brachiocephalic vein at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 17 and 24 h after administration. The specificity, linearity, detection limit, accuracy, precision, and stability of UPLC-MS/MS method were investigated. The concentration of cephalexin in plasma was determined by UPLC-MS/MS. The pharmacokinetic parameters were analyzed and calculated with WinNonlin^TM 8.1. [Result] The methodological results showed good correlation over the concentration range of 100-5 000 ng/mL with a correlation coefficient (R²) ≥ 0.99 and a standard curve equation of y=10.6828x-176.481. Relative recoveries were 105.63%, 104.35% and 102.40% for high, medium and low concentrations. Coefficient variations of intra-day and inter-day were all less than 15%, and the limit of detection and the limit of quantification were 50 and 100 ng/mL, respectively. The pharmacokinetic parameters of the two groups were as follows:T_max were (1.77±0.55) and (2.70+4.68) h, C_max were (28.09±5.09) and (26.82±7.94) μg/mL;T_1/2 were (3.39±1.43) and (3.12±1.05) h;AUC_0-t were (121.81±25.80) and (116.34±36.30) μg·h/mL. The plasma profiles of cephalexin following the administration of both formulations were similar. And the ratio of the average C_max, AUC_0-t and AUC_0-∞ of the test/reference preparation were 99.51%, 99.27% and 99.30% respectively, the 90% CI all fell between 80.00%-125.00%. [Conclusion] The UPLC-MS/MS method established in this experiment was accurate and reliable, and could be used to determine the concentration of cephalexin. The two formulations were bioequivalent for cephalexin, and could be used clinically for the treatment of related diseases.

[Objective] The suspected pathogenic Salmonella causing abortion in two large-scale donkey farms in Xinjiang was determined, and its pathogenicity and drug resistance were explored. [Method] In this experiment, the isolated bacteria were identified by bacterial isolation and culture, morphological observation, physiological and biochemical test, and the flagella gene FliC of the isolated bacteria was amplified by PCR and sequenced. The pathogenicity of the isolated bacteria was identified and analyzed by pathogenicity determination, bacterial load detection and histopathological observation, and its drug resistance was analyzed by drug sensitivity test. [Result] Through bacterial isolation and culture, morphological observation and physiological and biochemical test, it was determined that the two strains of bacteria were Salmonella abortus equi and named G1-1 and XD1-2, respectively. The genetic evolution analysis of flagellum gene FliC of the two isolates showed that the similarity of FliC amino acid sequences between the two isolates was 99.0%, and the similarity between FliC amino acid sequences of the two isolates and Salmonella abortus equi isolates Ireland-HE801373 and Ireland-HE801378 was the highest, both of which were 99.3%. The isolates G1-1 was closely related to Ireland-HE801373, Ireland-HE801378, China-KJ486797.1 and China-KJ486769.1, and the isolates XD1-2 was closely related to USA-EBQ1214032.1. The similarity analysis were consistent with the phylogenetic tree results. The mouse pathogenicity test showed that the two isolates had strong toxicity to mice and different pathogenicity. The pathogenicity of XD1-2 to mice was stronger than that of G1-1. The isolated strain XD1-2 was resistant to 11 kinds of antibiotics and sensitive to 5 kinds of antibiotics. The isolated strain G1-1 was resistant to 9 kinds of antibiotics and sensitive to 11 kinds of antibiotics. [Conclusion] In this study, two strains of Salmonella abortus equi from different donkey farms were successfully isolated and identified, which had different drug sensitivity and virulence. In addition, the genetic evolution analysis result indicated that they were genetic different, and the evolutionary analysis of flagella gene FLIC also showed differences. The results provided a reference for the prevention and treatment of Salmonella abortus equi in donkey.