Based on keyword co-occurrence analysis and literature co-citation analysis, a total of 1 026 international scientific papers about genetic mechanisms of heat stress in dairy cattle from Science Citation Index database from 1990 to 2020 were analyzed, including the authors, institutions and the annual number of publications. The co-occurrence network of co-cited literatures and important keywords in this field also were drawn. By the literatures with high citation frequency and the keywords with high frequency, the overall research situation and hotspots were showed, and the development context and trend were interpreted in this field. Furthermore, based on 52 Chinese scientific papers in this field from CNKI database, the research progress on genetic mechanism of heat stress in dairy cattle in China was summarized. By the co-occurrence of keywords and co-citation analysis of literatures at home and abroad, it was found that heat shock proteins, single nucleotide polymorphisms, oxidative stress and reproductive performance were research hotspots in this field. With the deepening of research in this field, it has gradually developed from the expression analysis of genes to identifying single nucleotide polymorphisms related to heat-resistance and exploring the regulation mechanisms of genes related to heat stress in dairy cattle. In the expression regulation of genes related to heat stress, epigenetics and miRNA played important roles. In the genetic mechanisms research for heat stress in dairy cattle, this paper could be beneficial for understanding research situation and knowledge structure in this field, and help quickly finding the primary focus and key literature.

As a kind of cell communication mediator, extracellular vesicles can interact with target cells/target tissues through miRNA, protein and other molecules carried by them, and influence the functions and phenotypes of target cells/target tissues. Recent studies have shown that extracellular vesicles from the embryo, endometrium and uterine microenvironment are involved in the process of mammalian embryo implantation, thereby ensuring successful embryo implantation, fetal growth and development by affecting endometrium receptivity and trophoblast cell adhesion. This article mainly discusses the occurrence, biological function and role of extracellular vesicles in the process of mammalian embryo implantation.

Exogenous hydrogen sulfide (H₂S), a malodorous gas, is released during decomposition of sulfur-containing organic matter in livestock and poultry production. Long-term exposure to H₂S damages central nervous system, irritates eye and respiratory organs, inhibits cytochrome oxidase activity, and even directly depresses the respiratory center, which causes acute and chronic poisoning. Moreover, high concentrations of hydrogen sulfide can quickly lead to asphyxia and death in human and animals. Thus, H₂S seriously affect the livestock and poultry production, animal welfare and human health. However, little is known about the prevention and treatment of hydrogen sulfide poisoning. Therefore, in-depth study of the absorption and metabolism of exogenous hydrogen sulfide in animals and clarification of its mechanism are of great significance to alleviate the harm of hydrogen sulfide in livestock and poultry production. This paper reviews the current research on the characteristics, absorption, metabolism and toxicity of H₂S for the purpose of proving a reference for further research on the mechanism by which H₂S affects animal health and a theoretical foundation for animal healthy breeding.

China has the world’s largest grassland resources. As the largest terrestrial ecosystem and the important green ecological barrier, natural grassland has lots of ecological functions. At the same time, it is also an important base for the development of animal husbandry and the most basic means of production and living for herdsmen. For a long time, people only paid attention to the productive function of grassland, while ignoring the protection of its ecosystem. As a result, it leads to degeneration of grassland production, decline of ecological service functions, and has further caused frequent occurrences of poisonous plants and other biological disasters, the decrease of forage species and yield, the imbalance of biodiversity and the deterioration of ecological environment. In this paper, the species and distribution of poisonous plants in natural grassland were briefly summarized, and then the economic losses and harms to natural grassland and animal husbandry caused by poisonous plants were introduced. This article gives an important overview of the research progress in toxicology and poisoning diseases of six main poisonous plants including locoweed, Aconitum, Ligularia, Stellera chamaejasme, Achnatherum inebrians and Ageratina adenophora and their prevention and control techniques, also gives several suggestions, aiming to provide theoretical guidance for the comprehensive prevention and control of toxic plants poisoning diseases and healthy breeding of grazing livestock in grassland pastoral areas.

Pig gut microbiota is an assembly of a large number of bacteria, viruses, fungi and archaea that colonize in the intestine of pigs. Previous studies have shown that many diseases and important economical traits of pigs are related to gut microbiota. Currently, 16S rRNA gene sequencing and shotgun metagenomic sequencing are the most commonly used approaches in the research of gut microbiota. However, the actual function capacities and physiological characterizations of gut bacterial strains can’t be fully illustrated by these sequencing technologies. The isolation and culture of gut bacteria are of special importance. In recent years, significant progress has been made in the area of cultivation of gut bacteria. Based on the diversity cultivation conditions, more and more gut bacterial strains can be isolated and cultured, which has significantly promoted the research and application of gut microbiota at the strain level. In this paper, we review and prospect the composition and structure of pig gut microbiota, the development of culturomics of gut microbiota, and the current status of pig gut bacterial culturomics, which will provide references for the subsequent studies on the function capacities of bacterial strains and the mechanism of gut bacteria affecting host phenotypes.

The intestinal barrier is an important defense against infection. Diarrhea in piglets, mainly caused by intestinal biological imbalance and damage of barrier function, has brought extreme losses to the porcine industry. Due to the unique advantages in various intestinal diseases related to intestinal microbiota, faecal microbiota transplantation has been introduced into exploratory researches on the improvement of the intestinal barrier function in piglets as a new microbiota manipulation strategy. This article reviews the structures and functions of the intestinal barrier and the effects of faecal microbiota transplantation on it in piglets, then discusses the possible inner mechanisms, with the expectation to provide some references in this field.

African swine fever (ASF) is a highly fatal infectious disease caused by African swine fever virus (ASFV) in pigs. The proteins p30, p54 and p72 encoded by ASFV have high immunogenicity and some amino acid sequences are conserved, so they are often used as serological diagnostic targets to evaluate the changes of antibody levels through different stages or degrees of ASFV infection. In-depth study of ASF serological diagnostic targets would be helpful to explore its infection detection, pathogenic mechanism and immune system response. This paper systematically reviewed the progress in the detection of main candidate proteins and their targets for serological diagnosis of ASF, analyzed the antigenic epitopes (regions) of candidate targets and the characteristics of antibody binding, and discussed their application potential as new targets for serological diagnosis of ASF.

Outbreaks and circulation of H9N2 avian influenza viruses (AIVs) not only cause huge economic losses to poultry industry but also pose a potential threat to public health. In order to illustrate the current epidemiology of H9N2 AIVs in China, their antigenicity, receptor-binding feature and pathogenicity were summarized and viruses that circulated in 2016-2020 were analyzed. The results showed that H9N2 AIVs circulated in more than 20 provinces or cities of China, of which outbreaks occurred more in Jiangxi, Guangdong, Guizhou and Jiangsu. H9N2 AIVs were mainly isolated from chickens, and a few were isolated form waterfowl or small poultry. H9N2 AIVs were sporadically isolated from humans. Most of H9N2 AIVs fell into the clade of h9.4.2.5, and a few isolates belonged to the clade of h9.4.2.1. Current H9N2 AIVs exhibit dual receptor-binding tropism or preferentially binding to α-2-6 SA receptors. The amino acids at the antigen-related sites are polymorphic and the antigenicity is undergoing changes. Some adaptation mutations in PB2, PA and HA protein which enhancing viruses’ replication in mammalian cells and their pathogenicity to mice, were acquired, and those increased the risk of viruses to break through species-barriers to infect other mammals including human. Taken together, the surveillance of H9N2 AIVs and monitoring their variation of antigenicity and pathogenicity should be strengthened.

Animal mycoplasmas contribute to some important diseases including pneumonia, mastitis, and arthritis, characterized by chronic and persistent infection, which causes serious economic losses to the breeding industry. Subunit vaccine has the advantages of safety, high efficiency, low cost, etc. It is an important direction of mycoplasma vaccine research. This paper describes the research advances in immunogenicity of related proteins of important animal mycoplasmas. It would be helpful to provide references for research of subunit vaccine of animal mycoplasmas.

The aim of this study was to explore the differentially expressed proteins(DEPs) of hypoxic adaptation in kidney tissues of Tibetan pigs and Yorkshire pigs under high altitude hypoxic environment. Unrelated castrated boars from Tibetan pig and Yorkshire pig were selected as experimental animals, and fed ad libitum on the plateau with altitude of 3 000 m, up to 180-day-old, 9 pigs with similar growth and body condition were randomly selected from each breed to collect kidney tissue. In this study, isobaric tags for relative and absolute quantification(iTARQ) technique was used to analyze the difference of protein expression in kidney tissues between Tibetan pigs and Yorkshire pigs, the differentially expressed proteins were analyzed by GO functional annotation and KEGG pathway. The results showed that, a total of 4 370 proteins were obtained from kidney tissues of two pig breeds, FC>1.2 or <0.833 and P<0.05 as the criterion, a total of 181 DEPs were screened out, among them, 138 proteins were up-regulated and 43 proteins were down-regulated in the Tibetan pigs. In the functional analysis, DEPs were mainly enriched in items and pathways related to hypoxic adaptation, such as HIF-1 signaling pathway, PPAR signaling pathway, complement and coagulation cascades signaling pathway, oxidoreductase activity, carbon metabolism, regulation of blood pressure, mitochondria, etc., after further comprehensive analysis, 5 important DEPs related to hypoxic adaptation were screened out(CD59, A2M, eNOS, CDKN1B and PGK1). These DEPs play an important role in maintaining hemodynamics, regulating energy metabolism and physiological environment homeostasis, the result of this study provides a theoretical basis for further study on the adaptability of pigs to hypoxic at high altitude.

The study aimed to explore the regulation and mechanism of miR-186-5p on the proliferation and adipogenic differentiation of porcine primary preadipocytes. The neck subcutaneous fat of 7-day-old healthy Mashen boar was collected and the primary preadipocytes were isolated and cultured. The primary preadipocytes were divided into 4 groups, which were transfected with miR-186-5p mimics (mimics) and its control group (mimics NC), miR-186-5p inhibitor (inhibitor) and its control group (inhibitor NC). CCK8 and scratch test were used to analyze the proliferation effect of porcine primary preadipocytes. Oil red O staining was used to detect the adipogenic differentiation ability. qPCR was used to detect the expression of proliferation and adipogenic differentiation related genes. The target genes of miR-186-5p were predicted, and the interaction between miR-186-5p and target genes was detected by dual luciferase reporter assay. When mimics were transfected into porcine primary preadipocytes, the expression of miR-186-5p significantly increased(P<0.01); meanwhile the cell proliferation ability and expression of proliferation related genes, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinases 4 (CDK4), and the target gene sirtuins 2 (SIRT2) significantly decreased(P<0.01); however, the adipogenic differentiation ability and expression of adipogenic differentiation related genes, peroxisome proliferators-activated receptor γ (PPARγ), sterol regulatory element binding transcription factor 1 (SREBF1), CCAAT enhancer binding protein β (C/EBPβ), fatty acid-binding protein 4 (FABP4) and lipoprotein lipase (LPL) significantly increased(P<0.01). When the primary preadipocytes were transfected with inhibitor, the expression of miR-186-5p significantly decreased(P<0.01); meanwhile the cell proliferation ability and expression of PCNA, CDK4 and SIRT2 significantly increased(P<0.05 or P<0.01); while the adipogenic differentiation ability and expression of PPARγ, SREBF1, C/EBPβ, FABP4 and LPL significantly decreased(P<0.01). Transfection of miR-186-5p mimics significantly inhibited the activity of luciferase in SIRT2-Wt-psiCHECK-2, but did not affect the activity of luciferase in SIRT2-Mut-psiCHECK-2. The results show that miR-186-5p can target the 3'UTR of SIRT2 and decrease the expression of SIRT2, which can inhibit the proliferation of Mashen pig primary preadipocytes and promote their adipogenic differentiation.

The aim of this research was to clone the goat NR4A1 gene CDS sequence, clarify its tissue and cell expression patterns, and explore the effect of overexpression of NR4A1 gene on goat subcutaneous adipocyte differentiation. In this experiment, the goat overexpression vector pcDNA3.1-NR4A1 was constructed by double digestion. Five healthy Jianzhou big ear goats of one-year-old were used as experimental animals. The coding region sequence of NR4A1 gene was cloned through RT-PCR. Relative expression level of NR4A1 gene in different tissues and subcutaneous adipocytes at different stages were detected by real-time quantitative PCR (qPCR). By transfecting pcDNA3.1-NR4A1 vector into subcutaneous adipocytes to overexpress NR4A1, and changes of lipid droplet aggregation after overexpression was detected by morphology, at the same time, the relative expression level of adipose differentiation marker genes was detected by qPCR. The results showed that the coding region sequence of goat NR4A1 gene was 1 797 bp, encoding 598 amino acids. NR4A1 widely expressed in different tissues of goats and had the highest expression level in the longissimus dorsi (P<0.01), and NR4A1 expression level was the highest in goat subcutaneous adipocytes at 60 h of differentiation (P<0.01). The overexpression of NR4A1 significantly promoted the accumulation of lipid droplets in subcutaneous adipocytes of goats, and significantly upregulated the relative expression level of C/EBPα, C/EBPβ, PPARγ, LPL, SREBP1 and AP2 (P<0.05). NR4A1 maybe a positive regulator for goat subcutaneous adipocytes differentiation by regulating the expression of adipocyte differentiation marker genes.

The objective of this study was to explore candidate genes which are significantly associated with growth and development traits of beef cattle by performing genome-wide association study (GWAS) for body weight longitudinal data of 808 Chinese Simmental beef cattle. The longitudinal body weight data of 808 Chinese Simmental beef bulls aged 0, 6, 12 and 18 months were used to fit the individual body weight prediction model using 3 nonlinear models (Gompertz model, Logistic model and Brody model), and the parameters A (mature body weight), b (time-scale parameter) and K (maturity rate) were estimated. Then, the parameter values were used as phenotypes. After the quality control using the BovineHD Beadchip (770K), 671 991 SNPs were generated. GAPIT was used for association analysis, combined with gene annotation to identify candidate genes associated with development traits of beef cattle. Gompertz model with the highest goodness of fit (R²=0.954) was selected to determine the parameter estimates. A total of 9, 49 and 7 significant SNPs associated with parameters A, b and K were identified by GWAS, respectively. These SNPs were mainly mapped on BTA 2, 3, 7, 9, 11, 14, 22 and 25. Gene annotation results showed that PLIN3, KCNS3, ANGPTL2 and ALPL were associated with fat deposition process, and KCNS3 was considered as a candidate gene for intramuscular fat content; IGF-1, TMCO1, PRKAG3 and SHISA9 were associated with growth and development, and IGF-1 was reported to be central to the growth and development process; ASPH was involved in regulating carcass development and meat quality traits of beef cattle. In this study, the estimated parameters of the weight prediction model were used as phenotypes for GWAS, and some candidate genes associated with growth and development traits were identified, which provided a reference for other longitudinal data studies and new candidate molecular markers for regulating the growth and development of beef cattle to improve meat production in breeding.

The aim of this study was to analyze the regulatory mechanism of cathepsin D (CTSD) on the follicular granulosa cells of Qianbei Ma goat and to explore its effect mechanism on litter size traits.In this study, 36-week-old, healthy, polytocous female Qianbei Ma goat (n=5) were used as the research object, follicular granulosa cells from ovarian tissues collected after slaughter were isolated and cultured. The eukaryotic expression vector pEGFP-N3-CTSD was constructed, and the eukaryotic expression efficiency at the transcription and translation levels after introducing it into cells was examined; CCK-8 assay was used to detect the effect of recombinant plasmid on the proliferation of granulosa cells in different time periods. The effects of recombinant plasmid on the apoptosis and cycle of granulosa cells were detected by flow cytometry; Subsequently, RT-qPCR was used to detect the effect of recombinant plasmids on the expression levels of apoptosis-related genes Bcl-2, Bax, Caspase-3, cell cycle-related factors Cyclin A1, Cyclin D2, and Cyclin E mRNA at the cellular level. Finally, the prolificacy traits candidate genes BMPR-IB, FSHR, INHA were used as functional genes to verify the effect of recombinant plasmids on their mRNA and protein expression at the transcription and translation levels. The results of double enzyme digestion and sequencing confirmed that the eukaryotic expression vector pEGFP-N3-CTSD with CTSD gene of Qianbei Ma goat was successfully constructed, and the expression of CTSD in granulosa cells could be extremely significantly increased at the transcription and translation levels (P<0.01); The cell proliferation test results showed that the up-regulation of CTSD in granulosa cells could inhibit cell proliferation, and the inhibition efficiency on cell proliferation was extremely significant at 12, 24, 48 and 72 h (P<0.01); The results of apoptosis detection showed that the overexpression of CTSD could significantly promote the apoptosis of granulosa cells(P<0.01) and significantly down-regulate the expression of anti-apoptosis gene Bcl-2 (P<0.05), significantly up-regulate the expression of apoptosis-related genes Bax and Caspase-3 (P<0.01). In addition, the results of cell cycle detection showed that the recombinant plasmid pEGFP-N3-CTSD could significantly up-regulate the number of cells at G0/G1 and G2/M phases (P<0.01), and significantly down-regulate the number of cells at S phase(P<0.01), significantly increase the expression of cell cycle related factors Cyclin A1(P<0.01), and significantly decrease the expression of Cyclin D2 (P<0.01). The results of RT-qPCR and Western blot detection showed that the up-regulation of CTSD in granulosa cells could significantly down-regulate the expression of prolificacy trait candidate genes and proteins BMPR-IB, FSHR and INHA at transcriptional and translational levels (P<0.01).In this study, it was found that the high expression of CTSD could inhibit cell proliferation, promote cell apoptosis, change the cycle process of granulosa cells, change the expression of apoptosis-related factors Bcl-2, Bax, Caspase-3 and cell cycle-related factors Cyclin A1, Cyclin D2, Cyclin E, and significantly reduce the expression of prolificacy trait candidate genes and proteins BMPR-IB, FSHR and INHA in granulosa cells(P<0.01). It is suggested that CTSD may regulate the biological behavior of granulosa cells and affect the expression of candidate genes of prolific traits in goats by changing the expression of related factors at the cell level, and then indirectly become an important factor affecting litter size traits in goats. This study laid a foundation for further exploring the regulation mechanism of CTSD on litter size traits and the mechanism of its effect on granulosa cells in goats.

The aim of this study was to explore and screen the differentially expressed long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs in caput epididymis between Taihang goat kids and adult Taihang goats, and to construct a competitive endogenous RNAs (ceRNAs) network associated with the regulatory mechanism of immune-related genes in caput epididymis of Taihang goats. Three 2-month-old healthy Taihang goat kids with similar body weight and three 2-year-old healthy adult Taihang goats with similar body weight were selected in this study. After castration, the caput epididymis were collected for whole transcriptome sequencing. The differentially expressed mRNAs, lncRNAs and miRNAs in caput epididymis were screened between goat kids and adult goats using DESeq2. Based on the principle of ceRNA-score, the differentially expressed ceRNAs was obtained by using miRanda software and R-package (reshape2, dplyr, tidyr). GO and KEGG enrichment analysis were used to study the function of mRNAs in the ceRNAs mechanism. The ceRNAs network related with immune function was drawn in caput epididymis of Taihang goats. Finally, 8 mRNAs, 8 miRNAs and 8 lncRNAs were randomly selected to verify the accuracy of the whole transcriptome sequencing results by qRT-PCR. According to the analysis results, compared with caput epididymis of goat kids, there were 6 461 differentially expressed mRNAs in caput epididymis of adult Taihang goats， including 2 997 up-regulated mRNAs and 3 464 down-regulated mRNAs; There were 1 147 differentially expressed lncRNAs, including 703 up-regulated lncRNAs and 444 down-regulated lncRNAs; There were 182 differentially expressed miRNAs, including 81 up-regulated miRNAs and 101 down-re-gulated miRNAs. Three hundred and sixty-six lncRNAs with ceRNAs regulatory relationship were obtained, including 213 up-regulated lncRNAs and 153 down-regulated lncRNAs; There were 3 131 mRNAs with ceRNAs regulatory relationship, including 1 253 up-regulated mRNAs and 1 878 down-regulated mRNAs; There were 140 miRNAs with ceRNAs regulatory relationship, including 48 up-regulated miRNAs and 92 down-regulated miRNAs. Among the genes in the ceRNAs mechanism, lymphocyte antigen 6 complex locus protein G5B (LY6G5B)，epididymal-specific lipocalin-9 (LCN9), adisintegrinand metalloproteinase 28 (ADAM28) and Mucin 15 (MUC15) were the significantly up-regulated genes related with immune function, which expression in adult goats were higher than those in goat kids (P<0.01). GO and KEGG analysis of the DEGs in the ceRNAs mechanism showed that of differentially expressed genes were involved in protein processing in endoplasmic reticulum, protein export, mucin type O-Glycan biosynthesis, ECM-receptor interaction and other pathways related to cell protein synthesis, secretion and transport. The results of qRT-PCR showed that the expression trends of the differentially expressed mRNAs, lncRNAs and miRNAs selected randomly were consistent with the whole transcriptome sequencing results, except for chi-miR-320-3p. In this study, the analysis results of the immune-related ceRNAs network in caput epididymis showed that lncRNA-MSTRG.22929.11, lncRNA-MSTRG.57822.5, lncRNA-MSTRG.26758.1, lncRNA-MSTRG.12113.3, lncRNA-MSTRG.59930.2 and other lncRNAs were related to immunity and sperm maturation in caput epididymis. In this study, the differentially expressed ceRNAs were screened in caput epididymais between Taihang goat kids and adult Taihang goats. The ceRNAs network related to immunity will provides a foundation for the study of lncRNAs as ceRNAs in the regulation mechanism of epididymal immune function in Taihang goats.

This study aimed to establish a compound probe system for indentifying the sex of bovine early embryos, reduce the pollution caused by electrophoresis of conventional PCR method, improve the identification accuracy and reduce the identification cost. In this study, using SRY as the target gene for identifying the sex of bovine embryos, the imported YCD-PCR sex identification kit was used as the control group (n=9 543), and the single primer separate amplification system (FSPSA, n=6 570) and the double primer mixed amplification system (FDPMA, n=22 238) of fluorescence quantitative PCR were used as the experimental groups, respectively. The comparison among the groups was conducted in the aspects of the efficiency of sex identification, non-reaction rate, percent of female and male embryos, ratio of female and male embryos, pregnant rate of embryo transfer，female calf rate of female embryos, results of sex identification under the different number of sampled cells and cost of sex identification to optimize the fluorescence quantitative PCR technology for bovine early embryo sex identification. The results showed that， in FSPSA group, the percent of female embryos and the efficiency of sex identification were significantly higher than those in the other two groups (P<0.01), the non-reaction rate were significantly lower than those in the other two groups (P<0.01), and the ratio of female and male embryos was obviousty different from that of the other two groups (1.03∶1 vs 1∶1.03 and 1∶1.02), and the female calf rate of female embryos was significantly lower than that of the FDPMA group and control group (P<0.01); In the FDPMA group, the percent of female embryos, percent of male embryos and the female calf rate of female embryos were not significantly different from those in the control group(P>0.05), and the ratio of female and male embryos was similar to that in the control group (1∶1.03 and 1∶1.02), but the non-reaction rate was significantly lower than that in the control group (P<0.01), and the efficiency of sex identification was significantly higher than that of the control group (P<0.01). The efficiency of sex identification of the groups sampled separately 4-6 cells and 7-10 cells was significantly higher than that of the groups sampled 1-3 cells and separated or/and died cells(SD) (P<0.01), however, there were no significant differences between 1-3 cells group and SD group and between 4-6 cells group and 7-10 cells group(P>0.05). The pregnancy rate of embryo transfer was the highest in the 4-6 cells group (49.68%), but there was no significant difference among all the sampled groups(P>0.05). Compared with the control group, the identification cost of the FDPMA group was reduced by 46.76%. In conclusion, the double primer mixed amplification of fluorescent quantitative PCR technology is more feasible and conducive to the industrial promotion and application of sex identification of bovine embryos because of the highest calving accurate rate of female embryos, the highest pregnancy rate of embryo transfer when sampled 4-6 cells, and the lower cost of sex identification.

The objective of this study was to investigate the effects of different feeding levels on testicular development, testosterone (T) concentration, the expression of testosterone synthesis-related genes and androgen receptor (AR) in testis of sheep. Eighteen healthy 4-month-old Dorper×Jinzhong crossed F1 ram lambs with similar body weight of ((35±0.5) kg) were selected and randomly divided into 3 groups for a comparative slaughter trial. They were offered one type of feed at 100%(AL group), 65%(AL65 group) and 40%(AL40 group) of ad libitum intake, respectively. When any lamb in AL group reached a body weight of 50 kg, all animals were slaughtered. After the testicular circumference and length were examined, tissue samples were collected for analyzing testosterone concentration and histological parameters using ELISA and H-E staining method, respectively. The expression of testosterone synthesis-related genes and AR mRNA were detected using quantitative real-time PCR. Localization and quantification of AR in testis of lambs were determined by immunohistochemistry and Western blotting analysis. The results showed that the length and circumference of testis in the AL40 group were significantly lower than those in the AL group(P<0.05), no significant difference was observed between the AL40 and AL65 groups(P>0.05). The thickness of germinal epithelium of lambs in AL40 group was not significantly different from that in AL65 group, but both were significantly lower than that in AL group (P<0.05). The density of spermatogenic cells and leydig cells were remarkably increased with the increasing feeding levels(P<0.05). Testosterone concentration, the expression of STAR, 3β-HSD and AR genes and AR protein were also significantly increased(P<0.05). Immunopositive product of AR was detected in leydig cells,primary spermatocytes, spermatids, peritubular myoid cells and perivascular smooth muscle cells. In conclusion, the testicular development, T level, the expression of testosterone synthesis-related genes and AR were regulated by dietary nutrition level. Dietary nutrition could modulate the process of spermatogenesis by regulating T concentration and the sensitivity of spermatogenic cells to T, which could influence the sexual maturation and reproductive performance of male animals.

In this study, we aimed to prepare anti-ASFV pig-derived single-chain antibody (ScFv), and identify its biological activity, screen out the pig-derived ScFv with ASFV reactivity, and provide a new diagnosis for ASFV material. the peripheral blood of ASFV-infected and recovered pigs were collected to separate lymphocytes, then lymphocyte total RNAs were extracted and used as a template to obtain pig-derived IgG heavy chain variable region and light chain variable region genes by PCR amplification, using SOE-PCR technology to expand. The pig-derived ScFv gene was obtained by splicing; the pET-30a-ScFv expression vector was constructed, and the protein was expressed and purified. The reactivity of the ScFv antibody was identified by ELISA and IFA. The results showed that the pig-derived ScFv (VH-VLκ, VH-VLλ) genes were successfully amplified, and an scFv (VH-VLλ₁₁) antibody that was reactive with ASFV was identified. The results showed that an ScFv (VH-VLλ₁₁) antibody that was reactive with ASFV was successfully screened, providing new raw material for ASFV diagnosis, prevention and control.

Bluetongue virus serotype 7 (BTV-7) was firstly isolated from sentinel cattle in Guangdong Province in 2014; however, the epidemic situation of this serotype was still not clear in China. The objective of the present study is to isolate and analyze the genomic characterization of BTV-7 epideictic in China. Sentinel cattle were placed in Jinghong County, Yunnan Province, and blood samples were collected weekly for arbovirus isolation by inoculating cells. Complete genome sequence of the isolated virus was obtained by high-throughput sequencing. The infection feature of the isolated virus on cells were analyzed by virus plaque assay and proliferation curve analysis. The dynamics of viral nucleic acids and antibodies in the blood of infected sentinel cattle were monitored by serum neutralization test (SNT) and qRT-PCR. In May 2020, a virus strain (V303/YNJH/2020) causing cytopathic effects (CPE) on BHK-21 cells was isolated from the blood sample collected from sentinel cattle, which was serotyped as BTV-7. The genome of V303/YNJH/2020 was 19 154 bp in length (GenBank accession numbers: MW046280 to MW046289), which showed the closest relationship to the BTV-7 GDST008 strain isolated in Guangdong Province in 2014, with nucleic acid (nt) and encoding protein amino acid (aa) sequence identities of segment 1 to 6, segment 9 and 10 higher than 98% and 99%. However, the segment 7 and 8 of V303/YNJH/2020 belonged to the Western topotype, shared nt/aa sequence identities of 71.5%/81.6% and 79.6%/84.4% with the corresponding segment of GSDT008. Results of virus plaque assay and proliferation curve analysis showed that the proliferation ability of V303/YNJH/2020 in BHK-21 cells was significantly robust than that of GDST008. Sentinel cattle infected with V303/YNJH/2020 did not show observed clinical symptoms, but viral nucleic acids in the blood persisted up to 12 weeks, and neutralization antibodies in blood remained at titers of 1:256 during 4-9 weeks after virus infection. The BTV-7 strain V303/YNJH/2020 isolated in Yunnan Province in 2020 has the closest relationship with BTV-7 strain of GDST008, and possessed robust proliferation ability on cells than GDST008. The infection of V303/YNJH/2020 did not cause clinical symptoms in cattle, but viral nucleic acids and neutralization antibodies existed in the blood of infected cattle for a long time. The results of this study laid the foundation for the research on evolution, viral mutation caused by reassortment and pathogenicity of BTV-7 strains.

Caprine kobuvirus (CKoV) is an emerging virus in goats in China, this study aimed to establish a real-time fluorescent quantitative PCR assay for detecting CKoV. Through designing primers targeting the 3D gene of CKoV and optimizing the reaction conditions and system, a TB Green Fluorescent Quantitative PCR assay was successfully established. The assay had a good linear relationship with CKoV in the range of 2.23×10²-2.23×10⁸copies·μL^-1 with a correlation coefficient (R²) of 0. 999 2, and its amplification efficiency was 110%. Moreover, the assay has good specificity and stability, and the detection limit was 2.23×10¹copies·μL^-1. Furthermore, out of 79 diarrhea samples of lambs collected from Sichuan, Chongqing, and Yunnan regions from January 2020 to April 2020, 25.3%samples (20/79) were detected as CKoV positive, and the farms’ positive rate was 53.3% (8/15). In addition, 13 complete 3D genes of CKoV were obtained in this study, and evolution analysis showed that these 13 3D genes had unique evolutionary trends. In conclusion, this study provides a new molecular method for detecting CKoV and basic epidemiological data of domestic CKoV.

This experiment was conducted to investigate the impact of H9N2 subtype avian influenza virus (H9N2 AIV) infection on the gut microbiota in mice. Twenty-four SPF BALB/c male mice were selected and randomly divided into the control group and infection group. The control-group mice were intranasally inoculated with normal allantoic fluid, and the infection-group mice were intranasally challenged with allantoic fluid containing 1.2×10⁵ plaque forming units of H9N2 AIV. The feces of control-group mice at day 0 and 33 and infection-group mice at day 4, 8, 21, and 33 post-infection (4, 8, 21, and 33 dpi) were collected. 16S rRNA sequence method was used to analyze the fecal bacteria after extracting DNA from the feces. The results showed that no significant differences were found in the alpha diversity or relative abundance of fecal bacteria in control-group mice between day 0 and day 33 (P>0.05); Comparatively, there were significant differences in the relative abundance of fecal bacteria between control-group mice and infection-group mice (P<0.05), though no significant differences were found in the alpha diversity of fecal bacteria (P>0.05). Of note, the relative abundance of the phylum Firmicutes decreased, while that of the phylum Proteobacteria increased in the fecal bacteria of the infection-group mice at 4 dpi compared to the control-group mice. Thereafter, the relative abundance of the phylum Firmicutes gradually increased, while that of the phylum Proteobacteria gradually decreased in the fecal bacteria during H9N2 AIV infection. In line with this finding, we observed a separation of over-all fecal bacterial community beta diversity from control-group mice and infection-group mice at 4, 8, 21, and 33 dpi by principal coordinates analysis (PCoA) at the OTU level. These results indicated that the intestinal bacterial community in mice is stable during homeostasis, while the composition of intestinal bacteria significantly changes after pulmonary H9N2 AIV infection and does not recover at 33 dpi.

This study was conducted to explore the universality and homology of the sequence encoding Staphylococcus aureus surface protein A (SasA) in S. aureus strains existing in raw milk. Combined with the investigation into the structure and composition, we can elucidate its role in adhesion to dairy cattle mammary epithelial cells. Seventy-three S. aureus strains were isolated and purified from five dairy cattle farms in the north of China using aseptic techniques. After DNA extraction, we used PCR to amplify and identify SasA gene, then compared the sequence with reference sequences to analyze its conservation. SRR1(serine-rich repeat region 1) and NRR(non-repeat region)were reconstructed using prokaryotic expression system and purified. The differences in adhesion effect of NRR, BSA and SRR1 on dairy cattle mammary epithelial cells (Mac-T) were detected by flow cytometer. Compared with BSA and SRR1, NRR exhibits greater adhesion to cells. To find out the main adhesion domain in NRR, different lengths of NRR fragments were used to inhibit the intact NRR fragment adhesion. PCR combined with the homologous analysis of sequences showed 86.3% of S. aureus carried SasA gene and the gene similarity was over 95% with the adhesion effect of NRR1-2 (230-540 aa) being most obvious. The above results have indicated that SasA gene is ubiquitous and highly conserved in bovine S. aureus. NRR1-2 module plays a major role in adhesion to dairy cattle mammary epithelial cells and is roughly located at the 230-540th residues of the protein domain. It suggests that specific cell receptors, which can bind to this module, might be sites where SasA interacts with the host cells as a kind of adhesion.

At present, the addition of antibiotics in animal feed is completely banned in China, thus particular attention has been focused on finding novel antibiotic substitutes in the field. Hence, we aimed to isolate the bacteriocin with antibacterial activity produced by Bacillus subtilis SXAU18, and obtain its antibacterial recombinant protein using prokaryotic expression system. In this study, we isolated the target protein by (NH₄)₂SO₄precipitation, chloroform extraction, ultrafiltration and tested its antibacterial activity by SDS-PAGE analysis and Oxford cup diffusion method. Predicted protein was expressed and purified by prokaryotic expression system and AKTA system. The antibacterial activity of the recombinant protein was determined by the Oxford cup diffusion method. We identified a 15 ku protein containing a unique peptide sequence GSSIFGLAPGK from the antibacterial substance produced by Bacillus subtilis SXAU18, which exerts pronounced antibacterial activity against S. aureus, S. epidermidis, M. luteus and L. monocytogenes. The mass spectrometry and bioinformatics analysis indicated that the 15 ku protein is DarA. Recombinant expression of DarA in prokaryotic expression system and its purification revealed that the DarA was mainly expressed in a soluble form and could be purified to homogeneity, which showed as a single band. The antibacterial test showed that the recombinant DarA has antibacterial activity. The results indicate that DarA from Bacillus subtilis SXAU18 have antibacterial activity against S. aureus, S. epidermidis, M. luteus and L. monocytogenes, and this protein can be obtained through recombinant expression.

The multidrug efflux transporter AcrB is an important cause and biological basis of multidrug resistance (MDR) in E. coli. To find an AcrB inhibitor to solve the problem of multidrug resistance, AutoDock Vina was used for screening with AcrB as the target in this experiment, and glycyrrhizic acid was obtained. DS Visualizer was employed to analyze interactions, and the combined drug susceptibility test was used to verify the bacteriostasis of glycyrrhizic acid combined with antibiotics. The inhibitory effect of glycyrrhizic acid on AcrB was verified by Nile red efflux assay, test on proton gradient across the inner membrane and test on outer membrane permeability. Finally, the effect of glycyrrhizic acid on AcrB expression-related genes was tested by real-time fluorescence-based quantitative (RFQ)-PCR. The results showed that glycyrrhizic acid formed hydrophobic force with multiple amino acid residues on AcrB protein, and formed multiple hydrogen bonds with the docking site, the binding force is 47.720 4 kJ·mol^-1；3.125 mg·mL^-1 glycyrrhizic acid, cefotaxime sodium and fosfomycin sodium had synergistic effect on E. coli E320 with fractional inhibitory concentration index (FICI)≤0.5; glycyrrhizic acid could block the efflux of Nile red and has the same tendency as known inhibitor PAβN; 3.125 mg·mL^-1 glycyrrhizic acid had no significant effect on outer membrane permeability and proton gradient across the inner membrane; glycyrrhizic acid could significantly increase the expression level of mRNA in negative regulators AcrR and MarR. This experiment proved that glycyrrhizic acid had the potential to become an efflux pump inhibitor to reduce multidrug resistance in E. coli.

This study aims to elucidate the target genes of microRNA (miRNA) involved in the oxidative stress reaction in the dorsal and ventral hippocampus of piglets and its possible regulatory mechanism. In the study, based on the data of miRNAs and transcriptome packets previously put into the gene expression database (GEO), the target genes of differentially expressed miRNAs in the dorsal and ventral regions of the hippocampus of Rongchang piglets under oxidative stress were predicted by bioinformatics analysis. The predicted results and transcriptome data were overlapped and screened to get the target pairs, which were used to construct a network map of miRNA-mRNA interaction. GO classification and KEGG analysis were performed to explore the molecular mechanisms involved in the regulation of oxidative stress in the dorsal and ventral regions of the hippocampus of piglets. The results showed that the oxidative stress-induced differential expression of 309 pairs of miRNAs-mRNAs in the dorsal hippocampus and 247 pairs in the ventral hippocampus of piglets. Among those, there were 67 pairs of negatively regulated miRNA-mRNAs in the dorsal hippocampus (2 pairs of up-regulated miRNA-down-regulated mRNAs, 65 pairs of down-regulated miRNA-up-regulated mRNAs), and 41 pairs in the ventral hippocampus (3 pairs of up-regulated miRNA-down-regulated mRNAs, 38 pairs of down-regulated miRNA-up-regulated mRNAs). GO classification and KEGG analysis results displayed that targeted up-regulated genes played a major role in regulating oxidative stress damage and maintaining body homeostasis, while targeted down-regulated genes in the dorsal hippocampus mainly regulated cell apoptosis and those in the ventral hippocampus mainly regulated cell proliferation and migration. And some differentially expressed miRNAs participated in the regulation of oxidative stress-related neurological diseases in a coordinated or divergent manner through targeting regulating genes. In conclusion, the study firstly described the interaction network and signaling pathway of oxidative stress-related miRNA-mRNAs in the hippocampus of piglets, and analyzed the differences and consistency in the regulation of oxidative stress in the dorsal and ventral hippocampus, which laid a foundation for the study of the pathological mechanism of oxidative stress-mediated neurological diseases.

This study aims to provide the best immune markers for the diagnosis of canine hair follicle tumors, improve the accuracy and shorten the time of tumor diagnosis, and be helpful for the clinical precise treatment. In this study, 26 cases of clinical canine hair follicle tumors were collected, and the tumor cases were labeled with immunohistochemistry (IHC) using immune markers CK5/6, CK8/18, CK19, P63, CD34, CD10, and Vimentin. The results of IHC staining of 26 tumors showed that CD34 was positive in 86.9% follicular tumors; Tumor cells of trichoepitheliomas were specifically positive for CD10 and CK8/18; CK19 was strongly positive in benign trichoepitheliomas and moderately positive in malignant tumors, and CD34 was moderately positive in benign trichoepitheliomas and weakly positive in malignant tumors; CD10 was specifically positive in the peripheral stroma of piloblastoma; Tumor cells in all cases were negative for Vimentin, while mesenchymal cells were positive. The results showed that CK5/6 and P63 can be used as positive markers of all follicular tumors in this study. CD34 can be used as a specific positive marker of hair follicular tumors except pilomatricomas and trichofolliculomas. CD10 and CK8/18 can be used as positive markers of trichoepitheliomas. CK19 and CD34 can be used to distinguish the benign and malignant trichoepitheliomas. CD10 can be used as a specific positive marker of the surrounding stroma of trichoblastomas. Vimentin can be used as a negative marker of hair follicle tumors.

The aim of this study was to investigate the characteristics of computer tomography images after Mycoplasma ovipneumoniae infection in goats. Twenty 1-year-old goats were randomly divided into the control group (n=5) and the experimental group (n=15). In the experimental group, mycoplasma pneumonia was artificially induced by intratracheal injection with 5 mL Mycoplasma ovipneumoniae standard strain IK3-3 (1×10⁷ CFU·mL^-1), the control group was injected with equal volume physiological saline. After inoculation, the clinical symptoms of the two groups were observed. At the same time, CT scan was performed on the chest at day 0, 7, 14, 21 and 28 after infection to analyze the characteristics of images. The goats were killed at day 29 after infection to observe the pathological and histopathological changes between the two groups. As a result, after successful infection, the experimental goats expressed obvious clinical symptoms of respiratory diseases, such as high temperatures, cough, serous and purculent nasal discharge. In the experimental group, CT images were mainly characterized by ground glass density shadow and mesh shadow, mostly in bilateral lobar lesions, preferably in the right anterior lobe. Interstitial pneumonia with bronchopneumonia was the main characteristic. In addition, air bronchogram was observed in severe group’s cases and thickened pleural was found in some cases. There was no significant difference before and after infection in normal group goats. It was suggested that CT technology was conducive for the early diagnosis of mycoplasma pneumoniae in goats and helpful to judge the outcomes.

In order to explore the effect of nano-selenium on the apoptosis of hepatocytes induced by fluoride, A total of 40 Kunming mice were divided into 4 groups (each group contents 10 mices) after 7 days of adaptive feeding, which were administered with normal saline, 24 mg·kg^-1 NaF, 1 mg·kg^-1 nano-selenium and the same dose of NaF (24 mg·kg^-1 NaF) + nano-selenium (1 mg·kg^-1). The entire experiment lasted for 28 days, during which all the mice had free drinking and eating. The pathological damage of hepatocyte was evaluated by HE staining, while Tunel staining was used to evaluate the apoptosis level of hepatocytes, and the expression level of apoptosis-related genes and proteins were detected by immunofluorescence, Western blot, and RT-qPCR. The results showed that nano-selenium effectively alleviates the swelling of liver cells, vacuolar degeneration and nuclear fragmentation caused by fluoride treatment, and down-regulates the expression of genes related to apoptosis, such as Bax, Caspase3/9 and protein P53, Caspase3, elevated Bcl-2/Bax expression level. Tunel staining results showed that fluoride treatment significantly increased the rate of cell apoptosis (P<0.01), while nano-selenium can effectively reduce the incidence of cell apoptosis(P<0.05). The expression level of Cyt-C in the fluoride group was significantly higher than that in the control group (P<0.05), while the expression in the nano-selenium treatment group showed a downward trend. In conclusion, nano-selenium can effectively alleviate fluoride induced hepatocyte apoptosis by regulating the expression of apoptosis-related factors.

The aim of this study was to evaluate the effect of conjugated linoleic acid (CLA) on the size and distribution of milk fat globule(MFG) of dairy cows. Twenty-four Holstein cows with similar body condition in mid-lactation(body weight (583±34.6) kg，milk yield (27.2±2.4) kg·d^-1) were randomly assigned to 4 groups, 6 replicate in each group, and one cow in each replicate. The individuals in control group (C group) were fed with basal diet, the individuals in low dose CLA group (L group) were fed with basal diet + 150 g·d^-1 CLA, individuals in medium dose CLA group (M group) were fed with basal diet + 300 g·d^-1 CLA, individuals in high dose CLA group (H group) were fed with basal diet + 400 g·d^-1 CLA. Through 5 d treatment, dry matter intake(DMI) and milk yield were recorded and milk components were analyzed. The average diameter and particle size distribution of MFG were measured by Mastersizer 3000 laser particle size analyzer. The results showed that CLA supplement did not significantly affect the DMI, milk yield, content of milk protein and milk lactose(P>0.05), but extremely significantly reduced milk fat content (P<0.01). The mean particle size D_[4,3]and D_[3,2] of MFG in CLA group cows were significantly lower than those in the control group cows(P<0.05). Furthermore, CLA supplementation decreased the distribution percentage of large globule particles, and increased the distribution percentage of small globule particles. In this experiment, it can be concluded that dietary CLA levels affect size and distribution of MFG, which provides a basis for clarifying the mechanism of milk fat depression caused by CLA.

Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi. Yet the biosynthetic pathway and key catalytic enzyme genes are not quite clear. Studies have demonstrated that the biosythesis of SW requires for the presence of a variety of SWN cluster genes. In order to determine the role of the swnR gene encoding NADB Rossmann-fold reductase in this gene cluster, we used PEG-mediated homologous recombination (HR) to transform a wild-type strain. The concentration of SW was measured in the fermentation broth of M. anisopliae wild-type strain (WT), mutant-type strain (MT) and a complemented-type (CT) strain using a Q Exactive Mass Spectrometer. The content of swainsonine in the fermentation broth of the obtained WT, MT and CT were (82.91±15.92), (5.71±2.23), (56.42±10.82) μg·mg^-1, respectively. The results showed that the content of SW decreased in the fermentation broth of the MT strain, and returned to the original level in the CT strain. These results indicate that the swnR gene plays a crucial role in the SW biosynthesis pathway of M. anisopliae, this will lay a theoretical foundation for the follow-up study of the catalytic enzyme gene screening and biosynthetic pathway research of swainsonine synthesis of endophytic fungi in locoweed.