Genomic mating (GM) is a method of estimated breeding values, risk (usefulness) and coefficient of ancestry to optimize mating between parents. It focuses on mating instead of truncation selection, which can effectively control the rate of inbreeding and maintain genetic diversity in populations. GM has higher feasibility and effectiveness, and is the most prospective theoretical approach in the genomic era, which can achieve long-term and sustainable genetic gain. In this review, we summarized the principle of genomic mating, the methodology and usage of genomic mating, and the progress of its application in livestock and poultry. In addition, the pressing issue of genomic mating and future research directions are presented. The effectiveness of genomic mating will be further improved by exploring and studying these issues, so as to provide references for improving the long-term sustainable development of livestock and poultry.

As an important organ for male animals to produce sperm and secrete androgen, the temperature regulation of the testes is crucial to the maintenance of their fertility. A series of reactions induced by heat stress, such as oxidative stress, apoptosis, DNA damage, impairment of the blood-testis barrier, as well as abnormal secretion of androgen, will have adverse effects on testicular cells, sperm quality, the ability of sperm to fertilize eggs and support embryonic development. The purpose of this article is to review the temperature regulation mechanism of the testis, the adverse effect of heat stress on testicular cells and sperm quality, so as to provide a reference for the study of the influences of heat stress on male reproduction.

Negative energy balance (NEB) is a metabolic disease which often occurs in cows during the early postpartum lactation period, and the NEB status will be reached when the energy intake is not sufficient to meet the energy consumption. Cows in NEB status leads to the increase of cow disease rate, decrease of pregnancy rate, and decrease of production performance, which brings great economic losses to intensive dairy production. With the wide application of molecular and histological techniques, the mechanism of NEB effect on bovine follicular development in early lactation of cows has been further revealed. This paper reviewed the research progress related to the molecular mechanism of NEB on bovine follicular development in early lactation of dairy cows according to the studies in recent years and made an outlook, aiming to provide reference for the theory of reducing the adverse effects of NEB on the reproductive performance of dairy cows.

Branched-chain amino acids (BCAA) play important roles in poultry growth, production performance, immune function and intestinal health. They are involved in key processes such as organ development, immune response, muscle protein turnover and gene regulation. However, an excess, deficiency or imbalance of a single BCAA can affect poultry protein synthesis and intestinal health. Therefore, maintaining a balanced ratio of BCAA is critical for poultry performance. Although the NRC (1994) provides recommended amounts of BCAA for broilers, laying hens, and breeders, there are variations in BCAA requirements among poultry breeds, dietary protein levels, and growth stages. Meanwhile, the ban on antibiotic growth promoters and the challenges of multiple diseases in poultry farming necessitate a re-evaluation of the recommended levels of BCAA intake. Additionally, more research is needed to explore the mechanisms of action of BCAA in maintaining intestinal integrity and regulating gut microbial composition and enhancing protein turnover efficiency in the body. This review aims to describe the nutritional and physiological roles of BCAA in poultry, as well as their effects on production and health. And also provides BCAA requirements for different broiler breeds (broilers, laying hens and breeders) under various feeding conditions. These recommendations aim to provide theoretical references for the recommended levels of BCAA in low-protein diets for poultry.

The decline in the quality of eggshells can cause loss of economic benefits and food safety risks. The transport of inorganic calcium ions (Ca²⁺) in the uterus of laying hens involves different transmembrane pathways, ion homeostasis, and the expression of related transport genes and vectors, which is an important link in eggshell mineralization and regulation. This article reviews the effects of uterine Ca²⁺ transport pathway, Ca²⁺ transport on eggshell mineralization and eggshell quality, and the regulatory effect of nutritional factors on Ca²⁺ transport, aiming to provide reference for improving eggshell quality by regulating eggshell mineralization.

Rumen microorganisms are known as the "hidden organs" of ruminants, closely involved in the acquisition of host nutrients and the maintenance of physiological health; To date, metagenomic sequencing has identified nearly 5 800 genomes in the rumen, but more than 90% of the microorganisms have not been cultured and remain in the "biological information black box"^[1]. Culturomics is a culture method that uses multiple culture conditions combined with high-throughput sequencing technology to identify strains. The application of high-throughput and parallel culturomics techniques to rumen microorganisms provides a new perspective for studying the function of key strains and their interactions with the host at the strain level. At present, however, research into the application of culturomics to rumen microorganisms is still limited and at an early stage. In this review, the characteristics of rumen microorganisms, culturomics technology and its application status in rumen microbial culture, as well as the challenges faced, have been reviewed to provide ideas for continuously optimizing and standardizing culturomics research programs, expanding the cultivable strain resources in the rumen, and accelerating the cracking of the rumen biological information black box.

Cell death is an important component of host antiviral immunity response, viral infection can cause different forms of host cell death, including the lytic and non-lytic cell death mode. These types of cell death not only eliminate virus-infected cells, but also further promote the host’s innate and adaptive immune processes through release of inflammatory factors. On the contrary, viruses have developed different circumvention mechanisms to inhibit cell death and promote itself infection. This review summarized the research progress in the regulation mechanism of virus-host infection and anti-infection immunity struggle-apoptosis, necroptosis and pyroptosis, aiming to provide references for further understanding the viral molecules pathogenesis and developing a new antiviral strategy.

As messengers in the intercellular microenvironment, exosomes can mediate the transmission of signals between cells. Alveolar epithelial cells can regulate the body’s innate immune response in the form of exosomes. Under specific stimulation conditions, exosomes derived from alveolar epithelial cells target and regulate the gene expression pathway by delivering different effector active substances, and participate in the regulation of macrophage polarization, thereby controlling the pulmonary inflammatory response. This article reviews the latest research progress of alveolar epithelial cell-derived exosomes in regulating acute lung injury(ALI)by targeting macrophage polarization, provide reference for related research.

The study aimed to conduct an association analysis of GREB1L and MIB1 gene polymorphisms with rib number and carcass traits (carcass length, straight length, diagonal length and carcass weight), in order to screen for key functional loci. In this study, DNA was extracted from ear tissue of 317 healthy Beijing black pigs at the age of 215 days. The primers were designed for GREB1L and MIB1. The exon regions of two genes were genotyped by PCR and Sanger sequencing. The distribution of genotype frequency and allele frequency of the mutated loci was calculated, and their association analysis with rib number and carcass traits was performed. A total of 6 SNPs were detected in two genes, including 3 synonymous mutations in GREB1L, 2 synonymous mutations in MIB1, one splice donor 5th base variant-intron variant in MIB1. Statistical analysis using Duncan’s multiple test revealed that 3 synonymous mutations (6:106574972 G>A, 6:106648540 C>T, 6:106648558 A>G) in GREB1L and one synonymous mutation (6:107028660 G>A) in MIB1 were no significant association with traits (P>0.05). A synonymous mutation in MIB1 (6:106936590 C>T) was significant association with carcass length and one splice donor 5th base variant-intron variant in MIB1 (6:107016475 A>G) was significant association with the number of rib (P<0.05). In conclusion, the above two significant SNPs could be used as candidate functional loci for the variation of rib number and carcass length of Beijing black pigs.

This study aimed to explore the gene polymorphisms of aquaporins9 (AQP9) and ribosomal protein S10 (RPS10) and their correlation with backfat thickness in Beijing black pigs, in order to obtain effective markers of backfat thickness in Beijing black pigs. The backfat thickness of 413 Beijing black pigs was measured at different parts (shoulder, 6-7 intercostal, thoracolumbar, lumbosacral). The promoter and exon sequences of AQP9 and RPS10 genes were amplified by PCR to screen SNP sites, and the association between SNPs and backfat thickness was analyzed. Differential gene expression was detected by fluorescence quantitative PCR. The results showed that there were 4 mutation sites in the promoter region of AQP9 gene, among which one SNP,rs332699245 A>C, was significantly associated with backfat thickness at the thoracolumbar (P<0.05), and the mutation of this site predicted the change of binding transcription factors. There were 8 mutation sites of RPS10 gene in 3'UTR and CDS region, 5 of which were significantly associated with backfat thickness: rs80795904 C>G and rs80932213 T>C were significantly correlated with 6-7 intercostal backfat thickness (P<0.05), rs3469834461 C>T were significantly correlated with shoulders backfat thickness and 4 regions average backfat thickness (P<0.05),rs80862457 C>T was significantly associated with backfat thickness at the thoracolumbar and lumbosacral (P<0.05), and rs336938272 A>C was significantly associated with 6-7 intercostal backfat thickness, backfat thickness at lumbosacral and 4 regions average backfat thickness (P<0.05). One SNP (rs80862457 C>T) was detected in the 3'UTR region, which was located at the binding target of small RNA (miRNA). Analysis of gene expression differences showed that the AQP9 mRNA expression of AA type individuals in rs332699245 A>C was significantly higher than that of AC type individuals (P<0.05), and the RPS10 mRNA expression of TT type individuals in rs80862457 C>T was significantly higher than that of CC type individuals (P<0.05). In summary, AQP9 and RPS10 genes were significantly associated with backfat thickness of Beijing black pigs, and rs332699245 A>C and rs80862457 C>T sites can be used as potential molecular markers for backfat thickness breeding of Beijing black pigs, providing theoretical support for breeding of Beijing black pigs.

This study aimed to reveal the genetic mechanism of leg disease in broilers, with the objective of mitigating the incidence of leg disease in broilers. This study employed a comprehensive approach involving 2 331 white-feathered broilers of multi-strain and multi-generation of "Guangming No. 2" independently bred in China. The assessment of leg health and diseases was performed by X-ray analysis at 42 days of age. For genotyping, the "Jingxin No.1" 55K SNP microarray was used for all individuals, and the genome-wide association study (GWAS) of leg health phenotypes was performed utilizing a logistic regression model tailored for dichotomous traits via plink software. After the integration and meticulous quality control of microarray data across diverse populations, a total of 2 330 individuals (including 2 256 healthy and 74 leg disease subjects) and 30 414 SNPs loci were deemed suitable for subsequent analysis. Through the GWAS analysis, 5 potential SNPs loci, distributed on chromosomes 6 and 18, were identified. Additionally, the investigation led to the annotation of 3 functional candidate genes, namely TBCD, SIRT1, and PBLD, which are directly related to leg disease. This study results provide important genetic loci and candidate genes for the healthy leg phenotype of white-feathered broilers, and provides a genetic basis for advancing the breeding process of broiler disease resistance.

The aim of this experiment was to compare the meat quality characteristics of three-way hybrid sheep Charolais×Duper×Hu (CDH) and Charolais×Australian white×Hu (CAH) and Hu sheep. Healthy male lambs with similar birth weight ((3.61±0.65) kg)were selected and followed by mothers until 45 days, followed by intensive weaning. The weaned lambs were then divided into 3 treatment groups (Hu, CAH and CAH groups) with 15 lambs in each treatment. In addition, a total of 45 lambs from 3 treatments were mixed fed until 6 months of age for slaughter. Subsequently, the tissue morphology, meat quality and nutritional composition of longissimus dorsi were analyzed. During the experiment period, the whole mixed basal diet was fed. The results showed as follows: 1) Compared with Hu sheep, longissimus dorsi muscle L* was significantly decreased, and cooked meat rate was significantly increased (P<0.05), but there was no significant difference in longissimus dorsi muscle pH, shear force, water loss rate, a* and b* in three-way hybrid sheep (P>0.05). 2) Compared with Hu sheep, the muscle fiber diameter and average muscle fiber area of longissimus dorsi muscle of three-way hybrid sheep were significantly increased, while the number and density of muscle fibers were significantly decreased (P<0.05). 3) Compared with Hu sheep, the contents of crude protein, essential and non-essential amino acids in longissimus dorsi muscle of ternary hybrid sheep were significantly increased (P<0.05), but the contents of total saturated fatty acids, unsaturated fatty acids and trace elements (Cu, Fe, Zn and Mn) were not significantly affected (P>0.05). 4) There were no significant differences in pH, shear force, water loss rate, cooked meat rate, meat color, muscle fiber quantity, muscle fiber area, muscle fiber density, conventional nutrients (moisture, crude protein, crude fat, crude ash), trace elements (Cu, Fe, Zn and Mn), essential and non-essential amino acids contents, and total saturated and unsaturated fatty acids contents in longissimus dorsi muscle between CAH and CDH (P>0.05). In conclusion, there was no significant difference in meat quality between the hybrid offspring of the same terminal father. Interestingly, three-way hybrid sheep can significantly improve meat quality, increase crude protein and amino acid levels in longissimus dorsi muscle, and have the potential to breed and produce high-quality mutton.

The study aimed to investigate the breeding selection efficiency of the Single Step Genome Best Linear Unbiased Prediction(SSGBLUP) method for Inner Mongolia cashmere goat. Based on the Illumina GGP_Goat_70K BeadChip sequencing data from 2 256 individuals of Inner Mongolia cashmere goats (Albas type), the phenotype data and pedigree records of cashmere traits were collected from individuals age 1 to 8 years old. Different matrix parameters of the H inverse matrix in the SSGBLUP method were set(ω,τ) to estimate the genome breeding value. At the same time, the accuracy of breeding value was estimated by five-fold cross-validation method. The results indicated that, as the increasing ω, SSGBLUP method had led to higher accuracy in estimating genomics breeding values for cashmere traits in Inner Mongolia cashmere goats. Combining the genetic parameter estimation results of ABLUP and GBLUP, it could be concluded that when τ was 0.3 and ω was 0.9, the accuracy of genome selection for cashmere traits in Inner Mongolia cashmere goats was relatively good. The accuracy of cashmere length was 0.702 8. The accuracy of cashmere diameter was 0.668 2. The accuracy of cashmere production was 0.713 1. Selecting reasonable scale parameters for the H matrix of SSGBLUP method can improve the accuracy of genetic breeding value estimation for cashmere traits in Inner Mongolia cashmere goats, and accelerate population genetic improvement, and shorten generation intervals.

This study aimed to design and screen the sgRNAs for efficiently editing SOCS2 gene, a growth suppressor factor, in the parthenogenetic embryos of goats, laying a technical foundation for the creation of fast-growing goat. In this study, two sgRNAs were designed for the SH2 domain conserved sequence of goat SOCS2 gene by online website, and the corresponding expression vector was constructed. sgRNA and Cas9 mRNA were obtained by in vitro transcription. The target DNA cleavage efficiency of sgRNA+Cas9 protein was detected in vitro. On this basis, 442 parthenogenetic embryos isolated and cultured from goat ovaries in slaughterhouse were divided into 3 groups. Two experimental groups were microinjected with sgRNA and Cas9 mRNA mixture, and the control group was injected with ultra-pure water. Using the whole genome DNA amplification product of a single blastocyst as a template, the target region was amplified by PCR and sequenced, and the edited samples were sequenced to test editing varieties by T-A clone. According to the prediction of online websites, each sgRNA selected 5 potential off-target sites with the least number of mismatches for off-target detection. The results showed that two sgRNAs were obtained near amino acid codon 83 and 96 of SOCS2 gene, and their expression vectors were successfully constructed. High quality sgRNA and Cas9 mRNA were obtained by in vitro transcription. Both sgRNAs could guide the Cas9 protein to completely cleave the target DNA in vitro. The editing efficiency of SOCS2-sg-83 to embryos was 94.1%. Among 160 clones, 152 were inserted, deleted or replaced at the target site, with a probability of 95.0%, of which 81.3% had frameshift mutation, 9.4% of the clones destroyed the SH2 domain, and accumulated 90.6% of the clones achieved functional knockout of SOCS2 gene. The editing efficiency of SOCS2-sg-96 to embryos was 50.0%, among the 80 clones, 70 clones were inserted, deleted or replaced at the target site, with a probability of 87.5%, of which 75.0% clones had frameshift mutation, 5.0% of the clones lost amino acid codon 96, and accumulated 80.0% of the clones achieved functional knockout of the SOCS2 gene. In addition, no off target was found in all edited embryos. In conclusion, SOCS2-sg-83 can achieve efficient and accurate editing and knockout of SOCS2 gene in goat embryos, which laying a technical foundation for the efficient preparation of SOCS2 gene knockout goat and breeding of fast-growing mutton goat.

This study was conducted to investigate the effect of exogenous leucine supplementation on the proliferation of bovine myogenic cells and its mechanism. The effects of different concentrations of Leu on the proliferation of bovine myoblasts were examined by CCK-8 and EdU staining, and the optimal concentration of Leu was determined; transcriptome sequencing (RNA-Seq) was performed with the optimal concentration of Leu as the treatment group and 0 mmol·L^-1 Leu as the control group to screen the key pathways of Leu regulating the proliferation of bovine myoblasts. Key regulatory genes in the key pathway were screened using qRT-PCR and validated by adding inhibitors or activators. Addition of Leu increased bovine myogenic cell viability, with 0.5 mmol·L^-1 Leu significantly increasing myogenic cell viability and EdU positive rate (P<0.05). There was 1 290 differentially expressed genes between Leu group and control group, of which 688 were upregulated and 602 were downregulated. The 309 up-regulated genes annotated by KEGG enrichment analysis were involved in 47 significantly enriched pathways, among which the most enriched pathway related to skeletal muscle growth and development was the PI3K-AKT signaling pathway, containing 34 up-regulated genes. Six differentially expressed genes, including PIK3R1, FOXO3, IRS1, RPTOR, MTOR and MET, were randomly selected for qRT-PCR validation, and the results were consistent with the result of RNA-Seq. The addition of LY294002, an inhibitor of PI3K in the PI3K-AKT signaling pathway, blocked the proliferation-promoting effect of Leu on bovine myogenic cells. In conclusion, Leu can promote the proliferation of bovine myogenic cells through PI3K-AKT signaling pathway.

This study aimed to compare the changes in abomasum index and transcriptome expression profile of yaks at different ages, and to explore the signaling pathways and key genes that affect the development and metabolism of abomasum, so as to provide theoretical basis for further study of development mechanism of abomasum in yaks. Abomasum tissues of yaks at 1 day, 20 days, 60 days, 15 months and 3 years old were selected for transcriptome sequencing. The RNA-Seq data were performed quality-controlled, alignment, differential genes screening; GO and KEGG analysis were conducted for differentially expressed genes(DEGs). To further verify the reliability of sequencing data, 5 DEGs were randomly selected for qRT-PCR verification. The results showed that rumen weight increased the fastest, followed by omasum, reticulum and abomasum during development. Compared with 1 day, 1 310, 1 715, 1 931 and 2 199 DEGs were identified at 20 days, 60 days, 15 months and 3 years old, respectively. A total of 565 DEGs were defined as common DEGs among 4 closed groups. Compared with the previous time point, 1 310, 861, 569 and 597 DEGs were identified at 20 days, 60 days, 15 months and 3 years old, respectively, with a total of 9 common DEGs in the 4 consecutive groups. The GO functional annotation found that, taking 1-day-old group as the control, 1 191, 2 578, 1 117 and 2 835 significant items were enriched in 20-day-old, 60-day-old, 15-month-old and 3-year-old groups, respectively. Among the top 10 significant GO items in different age groups, there were common ones, as well as unique ones in each age group. KEGG enrichment analysis showed that there were 4, 1, 3 and 4 unique signaling pathways in the top 30 significant pathways at 20 days old, 60 days old, 15 months old and 3 years old, respectively, including ECM receptor interaction, cytochrome P450 metabolism of exogenous substances, drug metabolism-cytochrome P450 pathways and other pathways related to abomasum development. The qRT-PCR results were basically consistent with sequencing results, indicating that the sequencing results were reliable. Through transcriptome sequencing and bioinformatics analysis of abomasum tissues at different developmental stages of yaks, the differentially expressed candidate genes related to development and metabolism of abomasum were screened, and these genes were mainly involved in the proliferation and differentiation of gastric epithelial cells, cells differentiation and immune regulation. Among them, GKN1, CXCL17, SCNN1B, SCNN1G, CCL5 and IGF2BP3 may play an important role in the development of abomasum in yaks. The signal pathways and candidate genes involved in glucose metabolism, glucose transport, fatty acid transport and peptide transport in abomasum were further screened. Among them, GANAB, GBA2, SLC2A1, SLC2A3, SLC2A4, CPT1B and SLC15A1 were important candidate genes related to nutrient metabolism and absorption in abomasum of yaks.

This study was conducted to investigate the effects of miR-24-3p on estradiol synthesis in porcine granulosa cells. In this experiment, ovarian tissue of 180-day old healthy sows (n=20) was collected, and 20 pairs of ovaries were collected for isolation and culture of granulosa cells in each experiment, and mimics and inhibitor of miR-24-3p were transfected into granulosa cells. The ELISA, RT-qPCR, Western blot, and double luciferase reporting tests were used to explore the effect of miR-24-3p on estradiol synthesis in porcine granulosa cells. The results showed that overexpression of miR-24-3p could significantly promote estradiol synthesis (P<0.01), and up-regulate the transcription and translation of StAR, CYP19A1 and CYP11A1 (P<0.05). Interfering with miR-24-3p significantly inhibited the synthesis of estradiol (P<0.05) and significantly down-regulated the mRNA and protein levels of CYP11A1 and CYP19A1 (P<0.05). Further studies showed that TOP1 was the direct target gene of miR-24-3p, overexpression of miR-24-3p could significantly inhibit the expression of TOP1 (P<0.05), and interference with miR-24-3p could significantly up-regulate the expression of TOP1 (P<0.05). What’s more, overexpression of TOP1 could weaken the promoting effect of miR-24-3p on estradiol synthesis in granulosa cells (P<0.05). In summary, miR-24-3p inhibits mRNA and protein levels of TOP1, and promotes the expression level of genes related to estradiol synthesis, thus promoting estradiol synthesis in granulosus cells by targeting TOP1. Since the estradiol synthesis capacity of granulosa cells directly affects the developmental state of ovarian follicles, this study provides a theoretical basis for screening miRNA that can improve reproductive performance of sows.

This study was conducted to investigate the effects of L-cysteine (L-Cys) on proliferation, apoptosis and the secretion of steroid hormones (progesterone and estrogen, P₄ and E₂) in ovine ovarian granulosa cells (GCs). Thirty sexually mature ewes aged between 1-1.5 years with the similar body weight and the same feeding condition were selected, and the fresh ovaries were collected. Then, GCs were collected from the dominant follicles (2-8 mm), and the GCs were randomly divided into 5 groups with 6 replicates per group. GCs were treated with 0, 100, 300, 500 and 700 μmol· L^-1 L-Cys for 48 h. The cell proliferation, apoptosis assay and the secretion of steroid hormones were measured using the CCK-8 kit, Annexin V-FITC/PI flow cytometry and ELISA, respectively. Real-time quantitative PCR and Western blot were used to analyze the mRNA and protein expression of the cell proliferation related genes (PCNA, CCND2, CCNB1), apoptosis-related genes (BAX, Caspase-3, Bcl-2) and steroid-secretion-related genes (STAR, 3β-HSD, CYP11A1, CYP19A1). The results showed that 100, 300 and 500 μmol·L^-1 L-Cys significantly promoted the cell viability (P<0.05) and significantly inhibited the apoptotic rate (P<0.05) of GCs. The addition of 300 μmol·L^-1 L-Cys significantly inhibited the secretion of P₄ (P<0.05). The mRNA expression of proliferation-related genes (PCNA, CCND2, CCNB1) were upregulated in the 300 μmol·L^-1 and 500 μmol·L^-1 L-Cys groups, while the protein expression of proliferation-related proteins (PCNA, CCND2, CCNB1) were increased in the 300 μmol·L^-1 L-Cys group (P<0.05). The mRNA and protein expressions of apoptosis-related genes (BAX, Caspase-3) were significantly downregulated in 100 μmol·L^-1 and 300 μmol·L^-1 L-Cys groups (P<0.05). The mRNA and protein expressions of the steroid-related genes (STAR, 3β-HSD) were significantly decreased in 300 μmol·L^-1 L-Cys group (P<0.05), but the mRNA and protein expressions of the steroid-related genes (CYP11A1, CYP19A1) were significantly increased in 100 μmol·L^-1 and 300 μmol·L^-1 L-Cys groups (P<0.05). In summary, L-Cys promoted cell proliferation and inhibited the apoptosis of ovine ovarian GCs by upregulating the mRNA and protein expressions of PCNA, CCNB1, CCND2, Bcl-2 and downregulating the mRNA and protein expressions of BAX, Caspase-3; L-Cys inhibited secretion of P₄ by downregulating the mRNA and protein expressions of STAR, 3β-HSD and upregulating the mRNA and protein expressions of CYP11A1.

The purpose of this study was to explore the proteomic characteristics of yak serum during pregnancy, and to screen and analyze the role of differentially expressed proteins(DEPs) in the maintenance of yak pregnancy. In this study, nine natural estrus female yaks aged from 4.5 to 6 years old with good health and no calving in the current year were selected for artificial insemination(AI). Serum samples were collected on the 30, 60 and 90 days after AI, and pregnancy diagnosis was performed on the mated female yaks. According to the diagnostic results, the collected samples were retrospectively grouped into the first, second, third month of pregnancy group and non-pregnant group(non-pregnant group samples were from non-pregnant yak individuals diagnosed by pregnancy diagnosis 90 days after AI), with 3 biological replicates in each group. The protein expression of yak serum samples was quantitatively analyzed by tandem mass spectrometry(TMT) technology, and the differentially expressed proteins were screened. Bioinformatics analysis such as GO function enrichment and KEGG pathway were carried out to further screen potential proteins related to yak pregnancy maintenance. The results showed that a total of 497 proteins were obtained from the four groups of yak serum protein samples, and a total of 68 differential proteins were obtained by screening with a fold change ≥1.2 or ≤0.83, and P<0.05. GO functional enrichment and KEGG pathway analysis showed that these DEPs were significantly enriched in biological processes such as metabolism, immune system, adhesion, and biological regulation, as well as pathways related to inflammation and immune pathways such as complement and coagulation cascades and Staphylococcus aureus infection. It is worth noting that the differential protein enrichment pathways between the groups during pregnancy include PI3K-Akt, Rap1, MAPK and other reproductive-related signaling pathways. The expression of IGFBP2, FGG, B2M, PDIA4 and AZGP1 in DEPs had a tendency to be up-regulated with the prolongation of pregnancy time, and the expression of these proteins in the third month of pregnancy was significantly higher than that in the non-pregnant state. The results showed that there were obvious changes in serum proteome in early pregnancy of yak. These DEPs play an important role in promoting cell adhesion and proliferation, maternal-fetal angiogenesis, and maintaining maternal metabolism and immune balance. In particular, IGFBP2, FGG, B2M, PDIA4 and AZGP1 may play an important role in the maintenance of yak pregnancy by participating in the regulation of placental growth and development, immune response and lipid metabolism. The results provide basic data for further exploring the regulatory mechanism of yak in maintaining pregnancy.

The aim of this study was to compare the distribution of endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) in the normal testes and cryptorchidism of yak, and analyze the role of ET-1/eNOS differences in cryptorchidism of yak. The 20 pairs of healthy and pathological adult (4-year-old) male yak testes were collected and divided into three groups: the normal group (10 pairs), the unilateral descent group (6 pairs), and the cryptorchidism group (4 pairs). The H.E, Masson’s and Gomori’s staining were used to compare the histochemical characteristics; Immunohistochemical methods, immunofluorescence techniques and quantitative real-time PCR (qPCR) were used to detect the expression levels of ET-1 and eNOS in different groups testes, and morphometric statistical software was used to compare the differences.The results showed that, compared with the normal group, there was no significant difference in the ratio of interstitial to luminal area in the unilateral descent group of yaks (P>0.05); In the cryptorchidism group, the luminal area was significantly reduced, and the content of collagen and reticular fibers were increased. Immunohistochemistry and immunofluorescence results showed: The ET-1 was expressed in leydig cells, spermatogenic cells at different stages, while mainly expressed in leydig cells and no significant expression was observed in the cryptorchidic epithelium, the difference in average optical density of ET-1 between the normal and unilateral descent groups was significant (P<0.05), also the difference between the normal and cryptorchidism groups was extremely significant (P<0.01). The eNOS was expressed in seminiferous tubules and leydig cells. Compared with the normal group, the difference in eNOS expression was extremely significant in the unilateral descent group (P<0.001) and significant in the cryptorchidism group (P<0.05). qPCR results showed that the relative expression of ET-1 in normal group was significantly higher than that in unilateral descent and cryptorchidism group (P<0.01), the relative expression of eNOS in unilateral descent group was higher than that in normal group (P<0.05). Yaks living in a hypoxic environment on the plateau, the seminiferous tubules are atrophied and interstitial cells decreased in the cryptorchidism testes of yak, ultimately leading to the local secretion regulation inhibited and sperm development obstructed. ET-1 and eNOS are co-located in leydig cells, the expression imbalance in interstitial cells in the unilateral descent group and cryptorchidism group should be caused by abnormal distribution in interstitial cells and blood vessels.

The aim of this study was to investigate the effects of dietary supplementation with manganese glycine (MG) on gut microbiota and laying performance in aged laying hens. A total of 720 70-week-old Hy-Line Brown hens were assigned equally to four groups with six replicates of 30 birds each. No additional manganese (Mn) was added to the basal diet (the measured Mn content was 21.77 mg·kg^-1). The hens were fed basal diets supplemented with 120 mg·kg^-1 of Mn from manganese sulfate monohydrate (MSM), or 40, 80, or 120 mg·kg^-1 Mn from MG. The measured Mn contents in the experimental diets were 144.46, 57.84, 96.97 and 135.59 mg·kg^-1, respectively. The experimental period was 12 weeks (70-82 weeks). The results showed that, dietary supplementation with 40 mg·kg^-1 Mn from MG resulted in higher laying rate than those observed in the 120 mg·kg^-1 MSM group and 80 mg·kg^-1 MG groups from 77 to 80 weeks (P <0.05). The laying rate of growps supplemented with MG from 70 to 82 weeks of age tended to increase (P= 0.071), while no significant difference was observed in other indexes of laying performance among the treatment groups (P>0.05). The Chao1 and Shannon indexes of cecal microflora in layers fed with 40 mg·kg^-1 MG were significantly higher than those in 120 mg·kg^-1 MSM group (P<0.05),and has the highest OTUs number, which indicated the highest diversity and richness of the microbial community. At the genus level, the top 10 bacteria were traced back to the phylum level, the Firmicutes relative abundance in the 120 mg·kg^-1 MSM group was significantly higher than that in the 80 mg·kg^-1 MG group (P<0.05). The microbial groups that played an important role in the 120 mg·kg^-1 MSM group were concentrated in the Firmicutes and Selenomonadaceae, the microbial groups that played an important role in the 80 mg·kg^-1 MG group were concentrated in the Spirochaetota phylum Spirochaetaceae family. The results of differential bacteria analysis showed that the relative abundance of Lactobacillus in the 40 mg·kg^-1 MG group was significantly higher than that of the 120 mg·kg^-1 MSM group (P<0.05), and the relative abundance of Paenibacillus, Massilia and Campylobacter in 80 mg·kg^-1 MG group was significantly higher than that of 120 mg·kg^-1 MSM group (P<0.05); Compared with the 40 mg·kg^-1 MG group, the 80 mg·kg^-1 MG group and 120 mg·kg^-1 MG group showed a decrease in the relative abundance of beneficial bacteria Megamonas, Lactobacillus, and Romboutsia (P<0.05), while the relative abundance of harmful bacteria Campylobacter increased (P<0.05). In conclusion, dietary MG supplementation with 40 mg·kg^-1 could improve the species richness, diversity index, increase the abundance of beneficial bacteria and the flora involved in the metabolic process of the intestinal microflora in aged laying hens, and improve the laying performance of laying hens in the later stage of the experiment (77 to 82 weeks of age).

The purpose of this study was to analyze the differentially expressed mRNA, lncRNA and its target genes in liver transcripome sequencing of Chuanzhong Black Goats fed silage Neolamarckia Cadamba instead of 50% silage corn, and to find out the key genes affecting liver metabolism of Chuanzhong Black Goats fed silage Neolamarckia Cadamba, so as to explore the feeding effect of silage Neolamarckia Cadamba. A total of 6 healthy male Chuanzhong Black Goats [5 months old, average body weight (21.07±4.05) kg] were selected and randomly divided into two groups: experimental group was fed silage Neolamarckia Cadamba replaced 50% silage corn, and control group was fed silage corn. Total mixed diet (TMR) was used for feeding during the experiment. On the last day of the experiment period, goats were slaughtered and their livers were taken. RNA from the liver tissues was extracted to construct a library. High-throughput sequencing technology was used for transcriptome sequencing. Differential mRNA and lncRNA were screened at a threshold of P<0.05, and biological function enrichment analysis was conducted to construct a targeted relationship network. Finally, 3 genes were randomly selected from differentially expressed lncRNA and mRNA separately, and verified by RT-qPCR. The results showed that a total of 1 696 mRNA were differentially expressed, of which 943 were up-regulated and 753 were down-regulated. A total of 33 lncRNA were differentially expressed, of which 22 were up-regulated and 11 down-regulated. Combined with the enrichment analysis of target genes of differentially expressed lncRNA and intersection genes of differentially expressed mRNA, it was found that NDUFA10, NDUFB11 and NDUFS5 were involved in signal pathways related to liver function, such as polysaccharide metabolism, glucan catabolism and cellulose catabolism. Combined with lncRNA target gene prediction, it was found that GSTA3 and GSTA4 genes were involved in signal pathways related to liver function, such as drug metabolism-cytochrome P450 and cytochrome P450 metabolism of heterologous substances. In conclusion, NDUFA10, NDUFB11, NDUFS5, GSTA3 and GSTA4 may affect the liver function from the aspects of nutrient metabolism and toxin metabolism in the liver tissue of Chuanzhong Black Goats fed with silage Neolamarckia Cadamba.

The aim of this study was to investigate the effects of the full length, fragmentation and GTPase activity of guanylate-binding protein-1 (GBP1) and guanylate-binding protein-2 (GBP2) on the replication of porcine rotavirus (PoRV). The full-length and truncations (G, M, E, ME regions) of GBP1 and GBP2 genes were amplified by PCR using porcine kidney cell cDNA and GBP1 and GBP2 plasmids as templates and cloned into pCDNA3.1(+) eukaryotic expression vector. Overexpression or silencing of GBP1 and GBP2 genes on rhesus monkey cells (MA104) cells was performed to explore the effect on PoRV replication. Different time variables were set to screen the time points at which GBP1 and GBP2 exerted their effects on PoRV replication. Apply pan-GTPase enzyme inhibitor (CID-1067700) to probe the effect of GTPase enzyme activity of GBP1 and GBP2 on PoRV. The expression of GBP1 and GBP2 proteins were detected by Western blot, quantitative PCR and viral TCID₅₀ assays, and their antiviral functions were investigated. The results showed that pCDNA3.1 (+)-GBP1-full-length-HIS and pCDNA3.1(+)-GBP2(47aa-592aa), (131aa-592aa)-HIS recombinant plasmids were successfully constructed and verified to be expressd. PoRV significantly promoted up-regulations of GBP1 and GBP2 at protein and mRNA expressions. Overexpression of GBP1 and GBP2 significantly inhibited PoRV replication, and silencing the expression of GBP1 and GBP2 genes significantly promoted PoRV replication. The pCDNA3.1 (+)-GBP1/GBP2-G, M, E, ME-HIS structural domain truncations of GBP1 and GBP2 proteins were successfully constructed and verified to be expressed. Overexpression of the truncator gene revealed that the G region of GBP1 and GBP2 exhibited an inhibitory effect on PoRV replication. It was verified by pan-GTPase inhibitory enzyme activity that GTPase enzyme activity is required for GBP1 and GBP2 to inhibit PoRV replication. The results suggest that the GBP1 and GBP2 proteins are able to inhibit PoRV replication at replication phase of the virus, and the inhibitory effect dependents on its G region containing the GTPase enzyme active sites. The results of the study provide a theoretical basis for finding new drug targets for PoRV and developing novel vaccines.

In order to obtain specific nanobodies against GP5 protein of porcine reproductive and respiratory syndrome virus (PRRSV) and explore their inhibitory effect on virus replication. The PRRSV-GP5 protein was expressed in a large amount by prokaryotic expression system and purified by nickel column affinity chromatography. The obtained recombinant GP5 protein was used to immunize alpacas, and blood was collected at the 0th, 14th, 28th, and 42nd days. After detecting the antibody titer in alpacas, the whole blood lymphocytes were isolated, and the total cell RNA was extracted. After reverse transcription, the heavy chain antibody variable region (VHH) fragment was amplified by nested PCR, and connected with pCANTAB-5E vector, and then transformed into TG1 receptor cells, The GP5-VHH phage antibody display library was constructed using phage display technology. After three rounds of screening and enrichment of specific phages, nanobodies with high affinity to PRRSV-GP5 protein were screened out, and their reactivity was identified by indirect ELISA. To investigate the effects of screened nanobodies targeting PRRSV-GP5 protein on virus replication and transcription in the cell,the obtained nanobody genes were cloned into eukaryotic expression vector pcDNA3.1 (-) and transfected into Marc-145 cells using Lipofectamine3000. They were incubated with PRRSV for 0, 24, 36, 48, 60, and 72 hours, respectively. The results showed that the PRRSV-GP5 protein was successfully expressed and purified in prokaryotic form, and the recombinant protein with a concentration of 2.16 mg·mL^-1 was obtained. After 14 days of three immunizations, the antibody titer in alpaca was as high as 1∶819 200. The GP5-VHH phage antibody display library was constructed, with a capacity of 2.93×10⁷ CFU·mL^-1, the insertion rate was 98%. After three consecutive rounds of panning and enrichment of phage antibody library, two nanobodies with different amino acid sequences were finally obtained. The results of indirect ELISA showed that the two nanobodies had good affinity with PRRSV-GP5 protein. Two nanobodies were transfected into Marc-145 cells, and they significantly hindered the replication and transcription of PRRSV at 36 and 60 hours,respectively.They demonstrated good ability to inhibit virus replication. In this study, the specific nanobodies against PRRSV-GP5 protein was screened for the first time and it has been verified that the screened nanobodies have the ability to inhibit PRRSV replication in cells. The research results could lay the experimental and material foundation for the development of new drugs against PRRSV.

Homologous recombination mediated antigenic variation plays an important role in the frequent persistent infection of Mycoplasma hyopneumoniae of pigs. Allosteric regulation of Holliday junctions by helicase RuvA is a key step involved in homologous recombination. Given that, this study aims at expressing RuvA_Mhp recombinant protein, identifying its DNA binding activity and preparing polyclonal antibody against RuvA_Mhp. Prokaryotic expression plasmid pET21a-RuvA was constructed by molecular cloning. Following expression induction and purification, the RuvA_Mhp recombinant protein was obtained; The polyclonal antibody against RuvA_Mhp was prepared by immunizing rabbit, and the titer and specificity were determined by indirect ELISA and Western blot; The DNA binding activity of RuvA_Mhp was analyzed by electrophoretic mobility shift assay and surface plasmon resonance technique; The oligomerization of RuvA_Mhp was determined by electrophoretic mobility shift assay combined Western blot. RuvA_Mhp recombinant protein expressed by prokaryotic expression was about 26 ku; The prepared polyclonal antibody against RuvA_Mhp had good specificity and high titer of 1:256 000; RuvA_Mhp has strong DNA binding activity with an affinity of 624.4 pmol·L^-1 (K_D) for Holliday junctions, and forms stable complexes with it mainly in octamers. In this study, the prokaryotic expression, polyclonal antibody preparation and activity identification of RuvA_Mhp were achieved, which laid a foundation for further exploration of the molecular mechanism of helicase RuvA mediating antigenic variation of Mycoplasma hyopneumoniae by homologous recombination regulation.

M. bovis infection and survival depend on various nutritional and metabolic factors provided by the external microenvironment. The study of the mechanism of exogenous biotin uptake by M. bovis will be of substantial significance to M. bovis prevention and control. An examination of the M. bovis PG45 MbBioY (MBOVPG45_0349) gene was used for phylogenetic analysis and molecular docking analysis; Screening of M. bovis PG45 ΔMbBioY mutant; to compare the biotin sensitivity of PG45 and PG45 ΔMbBioY the function of M.bovis was further verified by heterologous construction of MbBioY functional clone. The results showed that a novel biotin membrane transporter MbBioY was found on the chromosome of PG45. It was 996 bp in length, 30.95% GC content, and 332 amino acids in length. The protein consists of seven transmembrane domains. Further verification revealed that MbBioYloss affected M. bovis growth and showed phenotypic biotin metabolism deficiencies. By heterologous knock-in of biotin-metabolizing nutrient-deficient E. coli MG1655, it was shown that MbBioY alone could restore the physiological function of the nutrient-deficient E. coli strain with biotin metabolism. It has been preliminarily demonstrated that the S component of the energy coupling factor (ECF) transporter encoded by MbBioY can absorb exogenous biotin. This research result will contribute to the diversity and complexity of bacterial biotin metabolism regulation. M. bovis prevention and control will be provided from an innovative perspective.

The aim of this study was to isolate Mycoplasma gallisepticum (MG) from dead chicken embryos in a large-scale chicken farm in Guangdong, and genetic evolution analysis, pathogenicity and drug susceptibility tests were carried out. Isolation of MG were conducted, then the isolated strains were identified by colony observation, serological test and 16S rRNA sequencing. At the same time, the isolated strains were used to infect chicken embryos and SPF chickens, and were subjected to antimicrobial susceptibility tests. Under the microscope, the colonies showed typical ‘poached egg’. Plate agglutination test showed that it had agglutination reaction with MG positive serum and did not react with MS positive serum. The 16S rRNA sequencing showed that the homology of each isolate with MG was 99.9%, so the isolates were identified as MG strains. Each isolate was used to infect 7-day-old SPF chicken embryos, and the results showed that most of the infected chicken embryos died near hatching. After 3 weeks of infection in 3-week-old SPF chickens, significant pathological changes were found in the air sac of chickens, indicating that it had strong pathogenicity. The drug sensitivity test showed that the 5 isolates were highly sensitive to warnimulin hydrochloride, doxycycline, tywanmycin and tymectin, and were resistant to tilmicosin, tylosin, enrofloxacin, erythromycin, kitasamycin and lincomycin to varying degrees. Five MG isolates were successfully isolated from dead chicken embryos in a large-scale chicken farm in Guangdong province. All of the isolates could cause chicken embryo death and cause typical air sac inflammatory symptoms to SPF chickens, and they had varying degrees of resistance to multiple drugs. This study provides reference technical methods and guidance for the isolation, identification and drug selection for clinical MG, and also provides a research basis for the establishment of MG challenge model.

At present, there is a risk of cross-infection caused by blood collection in the commonly used serological detection methods of African swine fever virus (ASFV) antibodies, and the serological detection sensitivity is affected by the relatively delayed serological conversion of serum antibodies. Therefore, it is important to establish a non-invasive sampling serological method that can realize early diagnosis and monitoring of ASFV infection. In this study, pFastbac1-P30-His recombinant expression vector was constructed by artificial synthesis. The recombinant ASFV P30 protein (SUMO-P30) was expressed by baculovirus-insect cell eukaryotic expression system, purified by Ni column affinity and used as the coating antigen. After a series of conditions optimization, an indirect ELISA method of ASFV antibody targeting the mucosal sIgA antibody in porcine oral fluid was established. The results showed that the eukaryotic recombinant P30 protein (SUMO-P30) was about 47 ku, and Western blot analysis showed that it had good reactivity; The optimal reaction condition of ELISA was determined after optimization: the coating amount was 1.0 μg·mL^－1, 5% skimmed milk was the best sample dilution, the best mixing ratio with the oral liquid to be tested was 2∶8, the sample reaction time was 120 min, the best dilution of rat anti-pig IgA-HRP was 1∶5 000, the reaction time was 60 min, and the best color development time was 15 min. The antibody titer of ASFV infected oral fluid detected by this method could reach 1∶32, and there was no cross reaction with CSFV, PRV, PRRSV mucosal antibody positive oral fluid, which indicated its good sensitivity and specificity. By using this method and the ASFV commercialized ELISA serum antibody detection kit, the oral fluid and serum samples of the same pig infected with ASFV virulent or artificially attenuated strains were detected at different time points. The mucosal sIgA antibody in the oral fluid showed a significant increase in S/P values 3-5 days after infection, while serum antibodies were not detected to be positive during the monitoring period. Oral fluid and serum samples from challenged or indirectly infected piglet with natural variant ASFV strains were detected at different time points post infection. The positive coincidence rate of this method with the commercial ASFV antibody detection kit was 100%, the negative coincidence rate was 37.5%, and the total coincidence rate was 61.5%. In conclusion, this study has established an ELSIA detection method of ASFV mucosal antibody with oral fluid as sample without blood collection, which can realize non-invasive early diagnosis of ASFV infection. This method provides new technical support and supplement for ASFV monitoring, prevention and control.

The purpose of this study was to explore the regulatory effect of calcium-binding protein S100A4 on autophagy of human monocyte-macrophage THP-1 cells infected with Bacillus Calmette-Guérin (BCG). BCG infected THP-1 cells at different times (MOI=10). The expressions of S100A4 and LC3 proteins were detected by Western blot, and the number of autophagosomes was observed by transmission electron microscopy (TEM). After BCG infection alone or co-treatment with S100A4 small interfering RNA for 12 h, the mRNA expression of S100A4 and autophagy-related factors, LC3Ⅱ(microtubule-associated protein light chain 3 Ⅱ), Atg7 (autophagy-related gene 7), and Beclin1, were detected by qRT-PCR. The protein expressions of S100A4, LC3Ⅱ, Atg7, and Beclin-1 were detected by Western blot. Autophagy flow was detected by mRFP-GFP-LC3, and spot aggregation of mRFP-LC3 and mRFP-GFP-LC3 was observed by laser confocal microscopy. After BCG was infected with THP-1 cells at different times, the protein expression levels of S100A4 and LC3Ⅱ firstly increased and then decreased with the extension of infection time, and the expression levels were the highest at 12 h (P<0.001), and the number of autophagosomes increased significantly. mRNA levels of S100A4, LC3Ⅱ, Atg7, and Beclin-1 in the siNC+BCG infection group were significantly up-regulated compared with those in the siNC group (P<0.05), S100A4, LC3Ⅱ, Atg7, and Beclin-1 were significantly expressed at mRNA level after BCG and siS100A4 treatment (P<0.05) or very significantly (P<0.01, P<0.001) down-regulated; Protein levels of S100A4, LC3Ⅱ, Atg7, and Beclin-1 increased significantly compared with an uninfected group (P<0.05), the protein expression levels of S100A4, LC3Ⅱ, Atg7, and Beclin-1 were significantly reduced after BCG and siS100A4 treatment (P<0.001); Compared with the uninfected group, the number of autophagosomes in BCG group was significantly increased (P<0.01), the number of autophagosomes in siS100A4+BCG group was significantly reduced compared with siNC+BCG infection group (P<0.05). S100A4 has a regulatory effect on the autophagy of macrophages induced by BCG infection, and S100A4 can promote the BCG-induced autophagy of THP-1 cells.

The aim of the present study was to isolate Bacillus subtilis and its bacteriocins, and to analyse the antibacterial activity and stability of the recombinant bacteriocin. The Bacillus spp. producing bacteriocins screened from alpaca feces were isolated by Oxford Cup diffusion method and identified by 16S rRNA. The amino acid sequence of bacteriocins were obtained by (NH₄)₂SO₄ precipitation, chloroform extraction, SDS-PAGE and mass spectrometry analysis. The target bacteriocins were expressed by E. coli expression system, and the antibacterial activity and stability of the recombinant bacteriocins were identified by Oxford Cup diffusion. The results showed that the isolated strain was Bacillus subtilis, and was named as Bacillus subtilis SXAU18, the bacteriocins it produced could inhibit the growth of S.aureus, S. epidermidis, Micrococcus luteus and L. monocytogene. Analysis of the purified bacteriocins showed that they were probably a 10-20 ku DarA protein and two unknown proteins, which belonged to the bacteriocins category. The unknown proteins were named as BLIS SXAU181 and 182, respectively. Recombinant BLIS SXAU181 and 182 were mainly expressed in the soluble form in E. coli expression system, with a single band after purification. Recombinant BLIS SXAU182 had good antibacterial activity, and exhibited high resistance to high temperature, high acidity and alkalini, artificial gastri and intestinal fruid, but recombinant BLIS SXAU181 had no antibacterial activity. In conclusion, a bacteriocin with inhibitory activity against Gram-positive bacteria was isolated from Bacillus subtilis SXAU18 in this study, and the recombinant BLIS SXAU182 showed good antibacterial activity and stability.

The aim of this study was to investigate the effect of Chinese herbal monomers in combination with antibiotics on Clostridium perfringens (CP) infection in mice. In this study, licorice chalcone A (LCA) was used as a representative drug to investigate the in vitro and in vivo therapeutic effects of LCA in combination with antibiotics. Some antibiotics were identified by combined drug sensitivity tests as having synergistic effects with the monomeric LCA of Chinese medicine. The therapeutic effect of the three antibiotics in combination with LCA at different concentrations was compared by measuring the bactericidal profile of Clostridium perfringens at different concentrations, the hemolysis test, the sliding test and therapeutic effect on gas gangrene in mice. Three antibiotics, clindamycin (CLDM), tetracycline (TCN), and tilmicosin (TMS), which have synergistic effects with the LCA, were identified by combined drug sensitivity tests for follow-up trials. According to the bactericidal curve results, LCA at 8 μg·mL^-1 and TMS at 16 μg·mL^-1 can almost completely kill Clostridium perfringens. The combination of LCA and TMS (both 2 μg·mL^-1) significantly reduced bacterial hemolysis and hemolysis was almost completely inhibited (hemolysis quantified at 9.08%). It is worth noting that LCA has a facilitating effect on bacterial mobility and can improve hair-mediated motility, and its facilitating effect is significantly enhanced when combined with TMS. The results of the animal studies showed that CLDM alone had a good inhibitory effect on Clostridium perfringens and was clinically effective; TCN and TMS each showed a synergistic effect (FIC index of 0.375) when combined with LCA, and also showed a good synergistic effect in treatment. In practice, therefore, TMS can be used in combination with LCA in the treatment of Clostridium perfringens infections to reduce the amount of medication used while improving the therapeutic effect.

This study aimed to explore the effect of Astragalus membranaceus on the cell activity of osteogenic differentiation of BMSCs in hypoxic environments based on the PI3K/AKT signaling pathway. BMSCs cultured in osteogenic differentiation at low oxygen concentration (10%) were intervened with astragalus lyophilized powder solution at low, medium and high doses (100, 200 and 300 μg·mL^-1), respectively, and the dynamic proliferation and differentiation generations of BMSCs were observed by high-intrinsic real-time imaging system; label-free tracing was performed to simulate cell movement trajectories and analyze the cell movement speed, displacement and movement distance of each group. The cellular mitochondrial membrane potential was observed by laser confocal microscopy, and the changes of PI3K/AKT signaling pathway-related proteins and gene expression levels were detected by immunofluorescence and RT-PCR. Results were as follows: Compared with the control group, 10% hypoxia concentration inhibited the proliferation of BMSCs, reduced their differentiation generations, slowed down the motility, displacement and movement distance of BMSCs; at the same time, the mitochondrial membrane potential activity decreased, p-PI3K and p-AKT protein expression decreased, PI3K and AKT gene expression level decreased, the difference was statistically significant (P<0.01). Compared with the hypoxia group, astragalus lyophilized powder solution intervention significantly maintained the proliferation and differentiation activity of BMSCs in hypoxic environment, increased motor viability, increased mitochondrial membrane potential activity, and increased expression of p-JAK2, p-STAT3 proteins and PI3K and AKT genes, with statistically significant differences (P<0.05 or 0.01). Astragalus maybe maintain the proliferative activity of BMSCs under hypoxic conditions by activating PI3K/AKT signaling pathway.

The purpose of this study was to investigate the effect of adipose derived mesenchymal stem cells (ADSCs) on pyroptosis of miniature pigs with liver Ischemia-reperfusion injury (IRI) combined with hepatectomy. Eighteen Bama mini pigs were randomly divided into three groups, 6 in each group, which were Sham surgery group (sham group), model group (IRI group) and ADSCs intervention group (ADSCs group, dose is 1×10⁶ cells·kg^-1). The model of liver IRI combined with hepatectomy injury was established by laparoscopic minimally invasive technique. The IRI group and ADSCs group mini pigs were injected with normal saline and ADSCs into the liver parenchyma immediately after operation. Blood and liver tissue samples were collected 1, 3, and 7 d after surgery. Application of ELISA method to detect pro-inflammatory factors IL-18 and IL-1β in serum. The content of pyroptosis related genes and proteins were detected by RT-qPCR and Western blot. The results showed that on the 1 and 3 d, compared to the sham group, the serum pro-inflammatory factors IL-18 and IL-1β in the IRI group were significantly higher than those in the sham group (P<0.01). The expression levels of IL-18 and IL-1β genes and proteins in liver tissue were also significantly increased (P<0.01); The levels of the pyroptosis related factors NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1, GSDMD and nuclear factor kappa-Bp65 (NF-κBp65) genes and proteins in liver tissue were significantly elevated (P<0.05). After intervention with ADSCs, the expression levels of IL-18 and IL-1β in serum and liver tissue were significantly reduced (P<0.05). The gene and protein expression of pyroptosis related factors NLRP3, ASC, Caspase-1, GSDMD and NF-κBp65 in liver tissue was significantly reduced (P<0.05). Therefore, this study proves that laparoscopic liver IRI combined with hepatectomy injury can induce pyroptosis in miniature pigs, and ADSCs can reduce and improve the pyroptosis injury caused by this liver injury.

The effect of Huiyangjiuzhen acupuncture technique (HAT) on the quality of recovery in female cats undergoing spaying surgery was investigated to understand the effect of applying HAT in the perioperative period, and to provide reference for acupuncture intervention in the perioperative period to ensure anesthesia safety. In this study, 21 healthy female cats undergoing routine spay surgery were randomly divided into a control group and two acupuncture groups. The acupuncture groups were further divided into a positive sequence acupuncture group and a disordered sequence acupuncture group, with 7 animals in each group, and the animals were grouped for acupuncture treatment, then indexes of the cardiovascular and respiratory system were monitored after completion of surgery. After positive sequence acupuncture, the time to restore eyelid reflex, to regain spontaneous breathing, to recover swallowing reflex, to head up for the first time and stand for the first time, compared with the control group, were significantly shortened (P<0.05). Compared with the control group, the systolic blood pressure, diastolic blood pressure, mean blood pressure of the cats in the positive sequence acupuncture group were significantly reduced, the respiratory rate of the cats in the positive sequence acupuncture group were significantly decreased (P<0.05) and their body temperature and heart rate were not significant difference (P>0.05). Compared to the control group and positive sequence acupuncture group, there was no significant difference in the recovery time of cats after disordered sequence acupuncture (P>0.05). During the recovery period, compared with the control group, the systolic blood pressure, diastolic blood pressure and mean blood pressure in the disordered sequence acupuncture group were significantly reduced (P<0.05); compared with the positive sequence acupuncture group, partial pressure of carbon dioxide at the end of breathing was significantly reduced (P<0.05). It shows that HAT can promote the rapid recovery of the cats after surgery, maintain the stability of heart rate and blood pressure, maintain the temperature, and ensure the stability of the animal’s recovery process.

The pathogenesis of acute Endometritis in postpartum dairy cows was elucidated from the change characteristics of blood lipid oxide group. Seven Holstein dairy cows with endometritis within 7 days after delivery, which were similar body condition scoring and tire postpartum parity, were selected from a large-scale intensive dairy farm in Ningxia. Before treatment, they were regarded as test group (E), and after treatment, they were regarded as control group (C). Blood samples were collected before morning feeding and plasma was prepared. The content of oxidized lipids in plasma was quantitatively detected by high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The changes of plasma oxidized lipid metabolic profiles were analyzed by lipomics technology, and the differentially oxidized lipid metabolites were screened for metabolic pathway enrichment analysis. The plasma metabolic profile of E group was significantly different from that of C group (P<0.05, VIP≥1, FC ≥2 or FC≤0.5), of which 4 were up-regulated, including 12-HEPE(hydroxyeicosapentaenoic acid), 5-HEPE, 12-HETE(hydroxyeicosatetraenoic acid),14(S)-HDHA(hydroxydocosahexaenoic acid), and 6 were down-regulated, including 9,10-EpOME(epoxyoctadecenoic acid),12,13-EpOME,11,12-EET(epoxyeicosatrienoic acid),8,9-EET,14,15-EET and 17(18)-EpETE(epoxyeicosatetraenoic acid); The differential metabolites were enriched in arachidonic acid metabolism, inflammatory mediator regulation of TRP channel, PPAR signaling pathway, vascular smooth muscle contraction. The occurrence of acute endometritis in postpartum dairy cows is closely related to the changes of oxidized lipids in vivo. These oxidized lipids may participate in the occurrence and development of postpartum acute endometritis through arachidonic acid metabolic pathway, inflammatory mediator regulation of TRP channel, PPAR signaling pathway and vascular smooth muscle contraction.

This study aimed to express the African swine fever virus (ASFV) capsid protein pE120R and prepare the monoclonal antibody (mAb) for the further study of its function. The pE120R gene was amplified from pCAGGS-Flag-pE120R and the prokaryotic expression plasmid pGEX-6p1-pE120R was constructed. After transforming into BL21(DE3)competent cells, it was induced to express soluble pE120R protein by IPTG. pE120R protein was purified by GST Beads and immunized BALB/c mice, the spleen cells of the immunized mice were collected and perform cell fusion with SP2/0 cell, thus a hybridoma cell line secreting pE120R monoclonal antibody was obtained by indirect ELISA. The results showed that the prokaryotic expression plasmid pGEX-6p1-pE120R was successfully constructed and the high purity pE120R protein was obtained. The results of Western blot and indirect immunofluorescence assay (IFA) showed that pE120R mAb could specifically recognize Flag-pE120R protein expressed by HEK293T cells transfected with plasmid and pE120R protein expressed by ASFV infected alveolar macrophage cells (PAMs). The binding site of this mAb is in the 30-60 aa region, and the mAb subclass identifies the heavy chain as IgG2a. In this study, the soluble pE120R protein was successfully expressed and purified; the monoantibody against pE120R was prepared, which has good specificity and laid a foundation for exploring the biological function of ASFV pE120R protein.

The aim of present study was to develop serotyping real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods for serotype identification of Palyam virus (PALV) from clinical samples or insect vector. According to segment 2 (Seg-2) sequences of Chinese epidemic PALV strains, PALV serotype-specific qRT-PCR methods were established and their specificity, sensitivity and repeatability were evaluated. The reliability of the established qRT-PCR methods was assessed by 28 Chinese PALV strains and 90 PALV-positive blood samples, then the established methods were applied to identify the serotypes of PALV from the collected Culicoides samples. The established PALV serotyping qRT-PCR methods showed highly specificity and sensitivity, with the minimum copy number of detectable viral nucleic acid ranging from 22 to 28 copies·μL^-1. The qRT-PCR identification results of 28 PALV strains are consistent with the results of virus sequencing. Furthermore, serotype qRT-PCR identification results of 90 PALV-positive blood samples from infected sentinel animals were consistent with serotyping results of the isolated PALVs. The established method can accurately identify the serotype of PALV collected from Culicoides samples. With features of strong specificity, high sensitivity and positive repeatability, the PALV serotype-specific qRT-PCR methods established in this study could be used for rapid and accurate diagnosis of the serotypes of PALV in vectors and animals.

This study aimed to develop specific monoclonal antibodies for feline calicivirus (FCV) and provide materials for immunological diagnostic methods of FCV. In this study, BALB/c mice were immunized with FCV purified by sucrose gradient centrifugation and inactivated. The positive hybridoma cells were screened by an indirect ELISA method and characterized by a series of methods. One hybridoma 3B11 stably secreting monoclonal antibodies was successfully obtained. The antibody type of MAb 3B11 was identified as IgG1 for the heavy chain and κ chain for the light chain. MAb 3B11 specifically reacted with FCV-SH192. The mouse ascites MAb 3B11 showed high ELISA titers against five FCV strains; it had IFA and Western blot binding characteristics with five FCV strains. The MAb 3B11 developed in this study has good specificity, immunoreactivity and broad-spectrum recognition of FCV, which provides biological raw materials for the optimization of FCV diagnostic methods, screening of epidemic strains and basic research.

This study aimed to establish a recombinase aided amplification-lateral flow dipstick (RAA-LFD) method for simultaneous detection of bovine norovirus(BNoV) and Bovine rotavirus(BRV). According to the principle of RAA primer probe design, this test first designed primers and probes targeted the conserved regions of BNoV RdRp gene and BRV VP7 gene, second prepared the standard recombinant plasmids, then established and optimized the RAA-LFD reaction system, finally the specificity, sensitivity and repeatability of the detection method were also evaluated. At the same time, 168 clinical samples of calves with diarrhea were tested in parallel by the RAA-LFD, PCR and qPCR method. The results showed that the method could amplify the target fragment in 20 minutes at 39 ℃. The sensitivity of this method is 10³ copies·μL^-1 for both BNoV and BRV, and is basically consistent with the detection results of PCR. It has no cross-reaction with bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV). Although the test strip result may be lower than that of the qPCR test, it can be easily observed with the naked eye. In summary, the RAA-LFD method has high sensitivity, and is simple, fast, and does not require specialized equipment or operators, making it ideal for on-site detection.

The aim of this study was to develop a highly sensitive, specific and reproducible method for the quantitative detection of porcine epidemic diarrhea virus (PEDV) by droplet digital PCR (ddPCR). In this study, we designed and synthesized specific primer pairs and probes based on the conserved region of the M gene sequence of PEDV (MK862249.1) registered in GenBank, and successfully established a fluorescent quantitative real-time PCR (FQ-PCR) for the detection of PEDV by optimizing the reaction system and reaction conditions. By optimizing the reaction system and reaction conditions, a fluorescent quantitative real-time PCR (FQ-PCR) method for PEDV detection was successfully established, and the primer pairs/probes of the self-established FQ-PCR method were used to determine the ddPCR method; sensitivity, specificity, reproducibility and preliminary application tests of the ddPCR method were performed. The results showed that the self-established FQ-PCR method outperformed the industrial standard FQ-PCR method (SN/T 1699—2017) in terms of sensitivity and reproducibility; the lowest detection limit of the ddPCR method based on the self-designed FQ-PCR method was 0.15 copies·μL^-1, which was more sensitive than the industrial standard FQ-PCR (1.0×10¹ copies·μL^-1); The linearity was good (R²>0.99) at template concentrations of 1.0×10⁰ to 1.0×10⁴ copies·μL^-1. The intra- and inter-batch coefficients of variation (CV%) ranged from 1.52% to 7.40%. The results were negative for 11 control viruses including porcine delta coronavirus and porcine pseudorabies virus. The PEDV nucleic acid test was performed on 150 clinical samples by the established ddPCR and FQ-PCR methods, and the coincidence rate of the ddPCR method with the FQ-PCR method in detecting the positive samples was 100%.The PEDV ddPCR assay successfully established in this study can be used for early detection and quantitative detection of PEDV infection in clinical settings, and provides a quantitative means for the development of PEDV nucleic acid standards.