Chromatin assembly factor-1 (CAF-1) is a histone chaperone composed of 3 subunits(p150, p60, and p48).CAF-1 mainly interacts with proliferating cell nuclear antigen (PCNA) during DNA replication and is responsible for recruiting histones H3 and H4 depositing on newly synthesized DNA to facilitate nucleosome assembly. More and more studies have analyzed the structure and biological function of the CAF-1 complex,and found that CAF-1 plays an important role in the process of somatic cell reprogramming. Therefore, this paper focuses on the structure, biological function and the role of CAF-1 in somatic cell reprogramming.

With the development of high-throughput sequencing and bioinformatics in recent years, as a newcomer, circular RNA (circRNA) has attracted much attention in the field of biology. circRNAs are endogenous non-coding RNAs (ncRNAs) that usually derived from back-splicing of pre-mRNA to form a covalent closed loop. circRNA has no freely expressed 3' and 5' ends in eukaryotic cells, this special structure makes it highly insensitive to exonucleases. This article reviews the brief research history, type, alternative splicing, biological function of circRNA and its latest research progress in poultry to provide new perspectives for genetic improvement of poultry.

Energy balance refers to the process of balance between energy intake and energy expenditure, and only when the body is in energy balance can it keep normal life activities. There are numbers of feeding related nucleus in the hypothalamus, which form a complex and sophisticated neural network, and hypothalamus acts as the key junction of neural and endocrine regulation on energy balance. This review will discuss the function of hypothalamus in food intake and energy balance from the nucleus, neuropeptide, neural projection and peripheral hormones that affect the energy balance of hypothalamus.

Innate immune cells release fibrous net substances which are composed of chromatin DNA and various intracellular protein components to the extracellular environment after being stimulated by exogenous substances. Those substances are called extracellular traps (ETs). ETs are new types of host defense mechanism that can capture or kill pathogens, control the spread of pathogens, and protect the body from infection. Diseases caused by foodborne zoonotic pathogens, such as Escherichia coli and Salmonella, are public health issues of widespread concern worldwide. This article reviews the research progress of the formation of ETs stimulated by foodborne pathogens, the biological activity of ETs, and the influence of some bacterial proteins on the formation of ETs. This article is intended to provide theoretical reference for the prevention and control of foodborne diseases.

This study aimed to identify the selected gene regions related to the apparent NDF digestibility during the breeding process of Suhuai pigs (a national new breed containing 25% Huai pig lineage and 75% Large White pig lineage) and provide an important foundation for analyzing the genetic mechanism of crude fiber tolerance in Suhuai pigs. Firstly, the apparent digestibility of neutral detergent fiber (NDF) of 331 160-day-old Suhuai pigs were measured. The estimated breeding value (EBV) of apparent NDF digestibility in Suhuai pigs were calculated. Suhuai pigs with extremely high 10% (n=33) and extremely low 10% (n=33) of EBV_NDF were selected to perform 80K chip genotyping. The fixation index (Fst) and the number of segregating sites by length (nSL) methods were adopted to analyze the selection signals distribution in Suhuai pig populations, and to detect the candidate genes related to fiber digestion located in selected signal regions. Finally, the common SNPs of selected sites in the Fst and nSL analyses were selected to genotype in the whole group of Suhuai pigs, and then the association analysis between SNPs and apparent NDF digestibility was carried out to further determine the candidate SNPs related to the apparent NDF digestibility in Suhuai pigs. A total of 51 367 effective SNPs sites were used for the further analysis through the quality control of chip data of Suhuai pig populations with high and low apparent NDF digestibility. Furthermore, 146 selected signals regions and 361 selected genes were identified via Fst and nSL selection signals analysis. Among them, many genes had been reported to be related to intestinal health and intestinal development, including MTHFD1L, PHLPP1, TRPM6, MCC, NEDD9, UVRAG and KLF5. Only 8 common SNPs sites were identified in the two analyses. Association analysis results of SNPs with apparent NDF digestibility showed that rs81363074, rs327393763, and rs81404927 were significantly associated with apparent NDF digestibility (P<0.05), and the rs81347101 and rs318870857 were extremely significantly associated with the apparent NDF digestibility in Suhuai pig populations (P<0.01). Among them, the rs81363074 was located on the second intron of the MCC gene. One hundred and forty-six selected signals regions were identified based on the methods of Fst and nSL selection signals in Suhuai pigs with high and low apparent NDF digestibility. The selected candidate genes MTHFD1L, PHLPP1, TRPM6, MCC, NEDD9, UVRAG and KLF5 were identified to affect the apparent NDF digestibility in Suhuai pigs. Besides, 5 SNPs sites were significantly related to the apparent NDF digestibility. This results of this study provided an important foundation for analyzing the genetic mechanism of fiber tolerance trait in Suhuai pigs.

There are certain differences in the resistance to diseases of different pig breeds, one of the possible reasons for this difference is the diversity of swine leukocyte antigen (SLA) between different pig breeds. The study of SLA gene differences between Chinese indigenous and foreign pig breeds can provide important reference for clarifying the disease resistance mechanism of Chinese indigenous breeds. In this study, 8 healthy Min pigs and 4 Large White pigs were resequenced, and the obtained SNPs were then quality controlled for subsequent analysis. VCFtools and GEVALT softwares were used to analyze the SNP and haplotypes of SLA class I genes, and VEP tool in ENSEMBL was used to annotate SNPs, thus we can expound the diversity of SLA class I genes in two breeds at the global level. Mega software was applied to compare the sequences of nucleotide and coded amino acid of exon 2 and 3 of SLA class I classic genes of the two pig breeds. ProtParam tool and Protscale program on the Expasy server were performed to analyze the protein characteristics, and DnaSP software was used to calculate nucleotide diversity, the differences in antigen presentation ability of SLA class I classic genes between Chinese and foreign pig breeds were analyzed. The results showed that SLA class I genes of Min pigs had more SNPs and shorter haplotype blocks, and the number of missense mutations was more in Min pigs. Two alleles could be identified in exon 2 and 3 of SLA class I classic genes. The nucleotide diversity of alleles in Min pigs was higher than that in Large White pigs in the SLA class I classic genes. Moreover, Min pigs had more nucleotide and amino acid mutations at the antigen-binding site of SLA class I classic genes. The amino acids coded by SLA class Ⅰ classic genes of two pig breeds were hydrophilic, and it was more hydrophilic in Min pigs than that in Large White pigs. In summary, SLA class I genes have more polymorphisms in Min pigs. The number of base and amino acid mutations in the antigen-binding site of SLA class I classic genes of Min pigs are more than those of Large White pigs. The mutations in the ARSs of SLA class I classic gene of Min pigs detected in this study can provide a reference for the selection of pig disease resistance breeding markers.

The aim of this study was to analyze the relationship between mitochondrial DNA variation and litter size, and to find the molecular genetic markers affecting the reproductive perfor-mance of sheep, so as to provide a theoretical basis and method for the selection and breeding of sheep with high reproductive performance. Multiple Small tailed han sheep (XM), multiple Hu sheep (HM), twins Mongolian sheep (MT), singletons Mongolian sheep (MS), twins Oula sheep (OT), singletons Oula sheep (OS), twins Gansu alpine fine wool sheep (GT) and singletons Gansu alpine fine wool sheep (GS) all with a record of lambing were used as the research subjects. The mtDNA polymorphism was detected by PCR and sequencing, and the correlation analysis was conducted in combination with the litter size. The results showed that the genotype distribution between XM and HM was basically the similar at A1099T, T1112C, C13576T, T13837C and T13855C sites, and the genotype distribution between XM and MS, OT, OS, GT, GS were significantly different (P<0.05). There was significant difference in genotype distribution between MT and MS at T15668C site (P<0.05). There were significant differences in genotype distribution between OS and OT at T15445C, T15627C, T15657C, T15732C, G15977A, A16101G and C16429T sites (P<0.05). And there was significant difference in genotype distribution between GT and GS at A15923G site(P<0.05). The T15668C variable site significantly affected the litter size of Mongolian sheep (P<0.05). The variable sites of T15445C, T15627C, T15657C, T15732C, T15956C, G15977A and C16429T significantly affected the litter size of Oula sheep (P<0.05). Also, the A15923G site significantly affected the litter size of Gansu alpine fine wool sheep (P<0.05). What's more, Hap_2 (TTCTTACGC) and Hap_4 (TTTTTATGC) haplotypes also had certain effects on the litter size.

By metal-like toxic substance-arsenic exposure to the skin fibroblasts of Liaoning cashmere goats, the toxic effects of arsenic on cashmere goat skin fibroblasts and the mechanism of inducing cell apoptosis were explored, which aimed to provide a theoretical basis for further studying the growth effects of arsenic on cashmere goat skin cells. One 7-month-old male Liaoning new line cashmere goat with normal functions and the weight of about 25 kg was randomly selected, its abdominal skin was taken and the cells were cultured. The dimethyl arsenic acid was formulated into 11 different concentration groups (0, 0.1, 0.2, 0.4, 0.8, 1,5, 10, 25, 50, 100 mmol·L^-1), and 3×10⁴cells·mL^-1 skin cells were exposed to the drug for 24, 48, 72, and 96 h. After repeating the experiment by MTT method for 3 times, the proliferation and inhibition of cashmere goat skin fibroblasts were detected, the time points and 4 concentration groups(Control, Promotion, Critical, 50% inhibitory concentration (IC₅₀)) for the subsequent experiment were set up.The changes of cytoskeleton, cytoplasm and organelle were observed by immunofluorescence and TEM (transmission electron microscopy), respectively. The comet assay was used to detect DNA damage. Flow cytometry (FCM) was used to detect the changes of cell apoptosis. The mitochondrial transmembrane potential (ΔΨm) and the number of lysosomes were observed and analyzed by the staining intensity of the fluorescent dye Mito-Tracker Green. The OD values of different concentrations at 24 h could clearly reflect the proliferation and inhibition trend of dimethyl arsenic acid on cashmere goat skin fibroblasts, while the proliferation effect at 48, 72, and 96 h was gradually weakened or even disappeared, and the cell proliferation and inhibition effect was the best at 24 h.Therefore, the control group (without any treatment), the promotion group (0.8 mmol·L^-1), the critical group (1 mmol·L^-1), and the IC₅₀ group(38.68 mmol·L^-1) for 24 h were selected. Based on all results, when skin fibroblasts were treated with dimethyl arsenic acid(0.1-1 mmol·L^-1) for 24 h, the cytoskeleton was intact, the cell DNA was not tailed, and the apoptosis rate was low, the number of mitochondria increased, the membrane structure was clear, the cristae was dense, the morphology was complete, the membrane potential and the number of lysosomes increased. When the concentration of dimethyl arsenic acid was greater than 1 mmol·L^-1, it inhibited the growth of cashmere goat skin fibroblasts and caused obvious cytotoxicity (P<0.01), at the same time, the cytoskeleton morphology changed, the DNA was not tailed, the apoptotic rate significantly increased, the mitochondria swollen, the morphology was irregular shape, the cristae was broken and dissolved, the membrane potential reduced, and the number of lysosomes reduced. The cytoskeleton morphology of IC₅₀group was very different, and cometary tails were most obvious in the DNA of the cells(P<0.01), the number of apoptosis significantly increased, the membrane potential significantly decreased(P<0.01), and the number of lysosomes significantly decreased (P<0.01). In this study, the toxic effect of dimethyl arsenic acid on the skin fibroblasts of Liaoning cashmere goats and the mechanism of inducing cell apoptosis were explored, which indicated that a certain concentration of dimethyl arsenic acid had a toxic effect on the skin fibroblasts of Liaoning cashmere goats and further induced apoptosis.

This study aimed to clone IGFBP4 and IGFBP5 genes of yak, and detect their expression level in different tissues and liver at different growth stages of yak. In this study, 9 healthy female Maiwa yaks with triplicates were selected from 1 day old, 15 months old and 5 years old with body weight of (12.35±1.85), (98.88±2.50) and (268.55±27.82) kg, respectively. The heart, liver, spleen, lung, kidney and intestine of 5 years old yak and the liver of yak at 3 growth ages were collected. The IGFBP4 and IGFBP5 genes were cloned and their sequences were analyzed by bioinformatics method. In addition, the expressions of IGFBP4 and IGFBP5 were detected by qRT-PCR, Western blot and immunohistochemistry in 6 tissues and liver at 3 growth stages of yak. The results indicated that the CDS sequence of IGFBP4 and IGFBP5 genes were 777 and 816 bp (GenBank accession number:MT012934 and MT003005), encoding a total of 258 and 271 amino acids, respectively. The homology of IGFBP4 and IGFBP5 genes of yak compared with the cattle reached 99.6% and 99.5%, respectively. At the same time, compared with the kidney, heart, intestine, spleen and lung, the expression of IGFBP4 and IGFBP5 genes were the highest in the liver of 5 years old yak (P<0.01). The expression of IGFBP4 gene were significantly higher than that of IGFBP5 gene in these tissues (P<0.01). With the increase of age, the mRNA and protein expression of IGFBP4 and IGFBP5 in yak liver were showed an upward trend in the liver of yak, which was the expression of 5 years old >15 months old >1 day old. Compared with 1 day old and 15 months old, the expression level of IGFBP4 and IGFBP5 in yak liver were the highest in 5 years old (P<0.01). Moreover, the expression of IGFBP4 was extremely significant higher than that of IGFBP5(P<0.01). The results suggest that IGFBP4 and IGFBP5 may co-regulate liver growth and development of yak. It will benefit further study of the role and expression regulation of IGFBP4 and IGFBP5 in the growth and development of yak liver.

The aim of this study was to analyze the activity region and transcription factors in the promoter of goose MyoG gene, and to elucidate the transcriptional regulation mechanism. Firstly, the 1 245 bp 5' flanking region of MyoG was amplified by PCR, followed by sequencing and bioinformatics analysis. Secondly, dual luciferase reporter vectors with 4 different deletion fragments of the promoter were constructed, and then transfected into C2C12 cell lines. Furthermore, the key transcription factor in the core promoter region was predicted using online software, site-directed mutagenesis of the transcription factor binding sites HNF4 (—521-—503 bp), USF (—379-—370 bp) and E2 (—296-—281 bp) were carried out, then 3 mutation reporter vectors were constructed, and the key transcription factors of MyoG gene was preliminary identified in C2C12 cell line. Finally, the expression profile of MyoG and key transcription factor in breast muscle, leg muscle, heart, liver, spleen, lung, kidney and hypothalamus of 70-day-old goose were determined through qPCR. The results showed that the amplified 5' flanking region of MyoG gene contained promoter elements. The key cis-regulatory elements was located at —624-—154 bp region using the dual luciferase reporter vector. Combined with site-directed mutation demonstrated that USF was the key transcription regulatory factor of goose MyoG gene. Tissue expression profile studies further revealed that MyoG and USF expressed in 8 tissues, and they were higher co-expressed in goose breast muscle, leg muscle and heart (P<0.01). The 5' flanking region of goose MyoG had promoter transcriptional activity, the core promoter region was detected at -624-+37 bp, and USF was the key transcription regulatory factor. The experimental results can provide a theoretical basis for exploring molecular regulation mechanism of MyoG gene in goose muscle development.

The aim of this study was to explore the effect mechanism of melatonin(MT) on porcine spermatogonial stem cells (SSCs) in vitro culture. The SSCs in the testicles of 3 7-day-old healthy Large White pigs were isolated by differential adhesion method. After morphological observation, alkaline phosphatase staining, marker gene detection and immunofluorescence staining identification, the SSCs were used as experimental materials, the MT concentration gradient (0, 50, 250, 500, 1 000 μmol·mL^-1) groups were set up to treat SSCs, each group had 3 replicates (n=3), and the blank control group was treated with 0.1% DMSO. The cell viability, reactive oxygen species (ROS), glutathione (GSH) content and apoptosis gene expression were detected after MT treatment. The results showed that:1)The isolated clonal colony cells had the growth characteristics of SSCs, which could be stained by alkaline phosphatase and the stem cell marker genes OCT4, SOX2 and SSCs marker genes NANOG, PLZF, UCHL1 were expressed; 2)After treated with more than 50 μmol·L^-1 MT for 48 h, the cell viability of SSCs were significantly improved (P<0.05); 3)MT could significantly reduce the level of ROS in pig SSCs (P<0.05), and extremely significantly increase the content of intracellular GSH (P<0.01); 4)MT could significantly inhibit the expression of apoptosis proteins Bax and Caspase3 in pig SSCs (P<0.05). These results indicate that MT has the functions of removing ROS in pig SSCs, increasing GSH content, inhibiting the expression of apoptotic genes and improving cell viability, which can provide a reference for enhancing the reproductive performance of boars during breeding.

This study aimed to investigate the effects of dietary fish oil on estrous cycles and body heat production of mice fed high-fat diets (HFD). Thirty-six C57BL/6 J female mice at 4-week-old were randomly divided into 3 groups (n=12):control group, HFD group and HFD + fish oil group, respectively. The control group was fed with a standard rodent chow diet (AIN-93G). The HFD group and HFD + fish oil group were fed HFD (60% energy from fat) without or with 5% fish oil (equally replace lard energy), respectively. During the trial, the mice were used for various examinations, including body composition (12-week-old), body energy metabolism (16-week-old), brown adipose tissue(BAT) temperature (18-week-old), body core temperature (rectum temperature, 18-week-old) and estrous cycles (20-week-old). At the end of the experiment, the blood sample was collected from eye sockets, and the serum was isolated for examination of follicle-stimulating hormone (FSH) and estradiol (E2). In addition, the subcutaneous fat, abdominal fat and interscapular BAT were collected, weighed and used for detection of the protein expression of genes related to thermogenic program in adipose tissues (UCP1, Cyto C) by Western blot, and the mRNA expression of genes related to thermogenic program in brown adipose tissues (UCP1, PRDM16, PGC1α, Cidea, Elovl3) by real-time fluorescence quantitative PCR. The results showed that HFD induced a significant increase in body fat content (12-week-old), and subcutaneous fat and abdominal fat mass (21-week-old) compared with the control diet (P<0.05). Notably, the HFD-induced increase of fat content was significantly reduced by fish oil supplementation (P<0.05). In addition, HFD led irregular estrous cycles, with prolonged cycle duration and shortened estrus period, and decreased the level of FSH and E2 in serum (P<0.05). However, the HFD-induced estrous cycle irregularity of mice was alleviated by fish oil supplementation and the level of FSH and E2 in serum was increased (P<0.05). Meanwhile, fish oil supplementation increased BAT activation/thermogenesis and promoted white adipose tissue(WAT) browning in HFD-fed mice, with enhanced the expression of thermogenic marker genes in interscapular brown adipose tissue (iBAT) and inguinal white adipose tissue (iWAT) (P<0.05). These findings suggest that dietary fish oil can alleviate HFD-induced estrous cycle irregularity, possibly associate with enhanced body thermogenesis via BAT activation and WAT browning.

The study aimed to improve the efficiency and safety of spermatogonial stem cells (SSCs) transplantation recipients so that to decrease male sterility caused by busulfan toxicity, which is helpful in revealing the damage mechanism of busulfan on testis. Testicular biotin tracing experiments were performed on busulfan-treated mice to verify the integrity of the blood testis barrier (BTB); Liquid-mass spectrometry was used to target the content of busulfan in the semen of the epididymal tail to verify whether busulfan could directly pass through the testis BTB into the seminiferous tubules; ELISA determination of inflammatory factors was performed in mouse serum to verify whether its concentration had changed. The results showed that the earliest observable red fluorescence in the seminiferous tubules traced by biotin was detected at 24 h, suggesting the damage of blood testis barrier (BTB). And the highest proportion of busulfan in the epididymal semen reached a summit at 30 min post-treatment then gradually decreased. In serum, the concentration of TNF-α, IL-1β and IL-6 progressively increased by busulfan treatment(P<0.05), and at 36 h the amount of the three indexes was significantly higher than that in the control group. The values peaked at 21 d with TNF-α of (1 855.51±10.32) pg·mL^-1, IL-1β of (293.59±3.34) pg·mL^-1,and IL-6 of (340.30±12.55) pg·mL^-1, then gradually decreased. In summary, busulfan could freely cross over BTB and the biotracer could break through BTB within 24 h. The serum inflammatory factors TNF-α, IL-1β and IL-6 concentration gradually increased and reached significant levels, reaching the highest level at 21 d, then gradually decreased.

The aim of this study was to investigate the effects of excess lysine on weaned piglets in vivo and in vitro. A total of 144 weaned piglets (Duroc×Landrace×Yorkshire) were randomly divided into 4 groups, 6 replicates for each group, 6 piglets for each replicate (3 barrows and 3 sows). The pigs in different groups were fed with excessive standardized ileal digestibility lysine (SID Lys) of 1.3%, 2.6%, 3.9% and 5.2%, respectively in the diet to observe its effects on growth performance, visceral indexes and physiological and biochemical indicators. Using IPEC-J2 as an in vitro model, the proliferation of IPEC-J2 cells was measured by adding lysine and other amino acids according to the type and ratio of amino acids in the culture medium and nutritional requirements, and attempting to reduce the adverse effects of excessive addition of lysine by balancing other amino acids. The results showed that, with the increase of the amount of SID Lys in the diets, the growth performance of weaned piglets significantly decreased (P<0.05), the mean corpuscular hemoglobin (MCH), some serum free amino acids and intestinal morphology were affected. When lysine was 2.0 mmol·L^-1in culture medium, the viability of IPEC-J2 cells significantly decreased(P<0.05), with the original lysine concentration in the medium (0.5 mmol·L^-1) being the optimum concentration for IPEC-J2 growth. Cell viability was improved to some extent when other essential amino acids were supplemented according to the balanced ratio of amino acids in the medium; Cell viability varied with treatment time when the corresponding concentrations of arginine and histidine were supplemented according to the optimum ratio of porcine basic amino acids. The study demonstrated that the addition of excessive lysine has negative effect on the growth of weaned piglets. The balance of amino acids should be fully considered in practical applications.

The aim of this study was to investigate the effect of dietary chitosan oligosaccharide (COS) as an alternative to antibiotic on the growth performance, slaughter performance, serum biochemical parameters, intestinal barrier function, and meat quality of Cherry Valley ducks. A total of 540 ducks (one day old) were randomly assigned to 3 groups with 6 replicates per group and 30 ducks per replicate. Ducks in the 3 treatments were respectively fed a basal diet (control group), a basal diet added with either 40 mg·kg^-1 zinc bacitracin (antibiotic group) or 50 mg·kg^-1 COS (COS group) for 42 days. At the end of the experiment, one male duck (close to the average body weight) from each replicate was randomly selected and weighted. Blood, intestinal and muscle samples were collected for determination of serum biochemical indices, intestinal barrier function and muscle quality, respectively. The results showed that:1) Compared with control and antibiotic groups, dietary COS supplementation markedly increased the average daily feed intake (ADFI), average daily gain (ADG) of ducks, and the spleen and thymus indices (P<0.05), whereas no significant effects were found in the slaughter performance (P>0.05). 2) Compared with the control group, the supplementation of COS or antibiotics significantly increased the serum globulin (GLB) content, the activity of superoxide dismutase (SOD) and the total antioxidant capacity (T-AOC) of leg muscle (P<0.05), the secretory immunoglobulin A (sIgA), and the immunoglobulin M (IgM) in the duodenal mucosa (P<0.05), signifcantly decreased the serum urea nitrogen (BUN) content and the A/G value, the malondialdehyde (MDA) content and the drip loss of leg muscle(P<0.05). 3) Compared with control and antibiotic groups, dietary COS supplementation significantly increased IgM content in jejunal and ileal mucosa (P<0.05), signifcantly decreased diamine oxidase (DAO) and lipopolysaccharide (LPS) levels in serum (P<0.05). Compared to the control group, dietary COS supplementation markedly increased the mRNA expression of occludin (OCLN) in the duodenal mucosa (P<0.05) and the redness value (a^*) of leg muscle (P<0.05), significantly decreased the serum D-lactic content (P<0.05). In addition, the mRNA expression of OCLN in the jejunal mucosa in antibiotic group was higher than that in control group (P<0.05). The results suggest that the supplementation of COS could increase growth performance, enhance the immunity ability, improve the intestinal barrier function, and meat quality in Cherry Valley ducks. Therefore, COS could be used as an alternative to antibiotics in Cherry Valley ducks production.

The multiple real-time RT-PCR method was used to improve the speed and sensitivity of multiple pathogen detection, which can promote rapid diagnosis and prompt treatment of calf diarrhea. Four pairs of effective primers and probes were designed,synthesized and experimentally screened targeting the conserved region of ORF2 Gene from bovine astroviruses (BAstV), 5'-UTR from bovine viral diarrhea virus 1 (BVDV-1), N pro gene from bovine coronavirus (BCV) and VP6 gene from bovine rotavirus (BRV), respectively. The concentrations of the primers, probes and the reaction parameters and conditions of the real-time RT-PCR reaction system were further optimized with four recombinant plasmids containing the target fragments of BAstV, BVDV-1, BCV and BRV respectively. The standard curves were drawn to assess the specificity, sensitivity and reproducibility of the real-time RT-PCR reaction system. The results showed that the optimum concentrations of the primers were 300, 300, 400 and 500 nmol·L^-1, and the optimum concentrations of the probes were 250, 150, 100 and 300 nmol·L^-1 for BAstV, BVDV-1, BCV and BRV respectively, and the annealing temperature and time were 50.0℃ and 45 s. The minimum detectable limits of BAstV, BVDV-1, BCV and BRV were 1 000, 100, 100 and 100 copies·μL^-1 respectively, and the quadruple real-time RT-PCR has good specificity and repeatability and the positive detection rate of clinical collection of fecal samples were higher than that of the PCR method. In conclusion, the results showed that the quadruple real-time RT-PCR method established in this paper can be used for the rapid differential diagnosis of BAstV, BVDV-1, BCV and BRV which are common causes of diarrhea in calves.

The relationships between the structure, subcellular localization and function of D1133L gene of African swine fever virus (ASFV) were explored by predictive analysis and subcellular localization. Mega6.0 software was used to make phylogenetic trees of five helicase genes encoded by ASFV, and Swiss-Model software was used to predict the secondary and tertiary structures of GenBank D1133L gene; according to the ASFV (LR743116.1) sequence published in GenBank (LR743116.1), the D1133L gene was synthesized and its recombinant eukaryotic expression plasmid pCMV-D1133L was constructed. After expression, the gene was verified by Western blot (WB), the recombinant plasmid was transfected into PK-15 cells, and the subcellular localization of the protein was observed with indirect immunofluorescence assay (IFA). The results showed that the 5 ASFV helicases were relatively conservative. The total length of D1133L gene was 3 402 bp, G+C content was 6.1%, and A+T content was 10.6%; the expressed protein was 124.36 ku, and the secondary structure indicated that helix accounted for 48.90%, the extended chain accounted for 13.50%, random curl accounted for 33.27%; the tertiary structure indicated that QMQE was 0.20, and the coverage rate was 84%, it has a helix-dominated advanced structure; using LOCSVMPSI analysis and prediction, the probability of D1133L protein localization in the nucleus is the same as that in the cytoplasm. The WB results verified the expression of pCMV-D1133L, and the result of IFA proves that the helicase D1133L encoded by ASFV was localized in the nucleus and cytoplasm at the same time. These results of this study have accumulated data for further study of D1133L protein inhibiting immune response and clarifying the pathogenic mechanism of helicase proteins of ASFV.

Duck hepatitis A (DHA), an acute and highly lethal infection in ducklings, is one of the main diseases threatening the duck industry. The purpose of this study was to develop a monoclonal antibody (McAb) that has cross-neutralization activity against two most prevalent serotypes of DHAV in China, DHAV-1 and DHAV-3. Twelve epitope peptides shared by VP1 proteins of DHAV-1 and DHAV-3 were synthesized artificially and respectively conjugated with keyhole limpet hemocyanin (KLH) protein. BALB/c mice were then immunized with those antigens and McAbs were prepared. The obtained McAbs were screened by detecting the cross-reactivity with DHAV-1 and DHAV-3, the inhibitory efficiency on virus proliferation, the neutralization titer, and the protection rate against virus infection. A total of 12 hybridomas were obtained, the McAbs secreted by 6 hybridomas (C1, C4, C7, C12, C13, C16) in those had specific cross-reactions with DHAV-1 and DHAV-3. The McAbs secreted by hybridomas (C1, C4, C7 and C16) had higher inhibitory efficiency on DHAV-1 and DHAV-3 proliferation in duck embryos, ranging from 75.34% to 100.00%. The neutralization titer of McAbs secreted by hybridomas (C1, C4, C7 and C16) against DHAV-1 and DHAV-3 were 1:3 & 1:5, 1:3 & 1:3, 1:10 & 1:11, 1:9 & 1:9, respectively. The protection rate of McAbs secreted by hybridomas (C7 and C16) against DHAV-1 and DHAV-3 infection were 70% & 80%, 100% & 60%, respectively. In this study, four McAbs with cross-neutralizing activity against DHAV-1 and DHAV-3 were developed, two of those had a higher protection rate against virus infection, providing new materials and ideas for the prevention and control of DHA.

In order to detect Ehrlichia ruminantium rapidly and accurately, a set of specific primer and TaqMan probe were designed for TaqMan real-time PCR assay and three sets of specific primers were designed for Eva Green real-time PCR assay according to the pCS20 gene to establish real-time PCR assays for detection of Ehrlichia ruminantium. The specificity, sensitivity and repeatability were tested, respectively. Then the TaqMan and Eva Green real-time PCR were used to detect Amblyomma samples and compared with OIE nested PCR. Results showed that the Ehrlichia ruminantium could be identified specifically and without any cross reaction with B.bovis, B.bigemina, Theileria annulata, E. Canis, E. bovis, E.equi and E. risticii. Besides, the TaqMan and Eva Green real-time PCR detection limits were 17.4 and 1.74 copies·μL^-1, respectively. The coefficients of variations were less than 1.5% for both intra-assay and inter-assay. Total 420 Amblyomma samples were detected by the established assays, positive rate was 25.48% in TaqMan real-time PCR and 29.29% in Eva Green real-time PCR. The sensitivity of TaqMan and Eva Green real-time PCR was significantly higher than that of OIE nested PCR. The results indicated that the detection assays could be applied for rapid and high through-put detection of Ehrlichia ruminantium.

To investigate the effect of paeonol on oxidative damage in mice with acute pancreatitis(AP), 50 SPF male mice were selected and randomly divided into 5 groups with 10 each, namely the blank control group, AP model group and paeonol high-, medium- and low- dose groups. The mice in the paeonol groups were given 100, 50, and 25 mg·kg^-1 paeonol intragastrically, and the mice in the blank control group and the AP model group were given equal volumes of saline. After continuously intragastric administration for 5 days, mice in the AP model group and different doses of paeonol groups were injected intraperitoneally with 20% L-arginine. After 6 hours of injection, serum samples of mice were taken to measure the oxidation indexes (MDA, SOD), and pancreatic tissues of mice were taken to observe the pathological changes. At the same time, myeloperoxidase(MPO) immunohistochemical sections of pancreatic tissue were made to observe the histopathological changes and the distribution and expression of MPO. The mRNA expression levels of HO-1,Keap1 and Nrf2 in pancreatic tissues of each group were detected by fluorescence quantitative PCR, and the protein expression levels of HO-1,Keap1 and Nrf2 were detected by Western blot. The results showed that different doses of paeonol significantly reduced the MDA content and increased the activity of SOD in mice with acute pancreatitis (P<0.01). Immunohistochemical results showed that high and medium dose of paeonol effectively reduced the expression of MPO. Compared with the model group, medium and high dose of paeonol significantly increased the mRNA and protein expression of HO-1 and Nrf2 (P<0.01), low dose of paeonol significantly increased the mRNA and protein expression of Nrf2(P<0.01). Different doses of paeonol significantly decreased the expression of Keap1 mRNA and protein(P<0.01). In conclusion, paeonol may alleviate oxidative damage induced by L-arginine in mice with acute pancreatitis by activating Nrf2/HO-1 signaling pathway. This study provides theoretical reference for the pathogenesis of acute pancreatitis and the development of related drugs.

This study aimed to construct rant gene-deleted mutant and complemented strain to study the effect of MFS efflux pump Rant on the resistance of R. anatipestifer. R. anatipestifer RA-LZ01 strain was used as the parental strain. The deletion mutant Δrant was obtained by the suicide plasmid pRE112-mediated homologous recombination, and rant gene was replaced with erythromycin resistance cassette. The E. coli-R. anatipestifer shuttle plasmid pCPRA was used as the vector to construct the complemented strain cΔrant. The minimal inhibitory concentrations (MICs) of 11 classes of clinically common antibiotics against wild strain, deletion mutant and complemented strain were determined with micro broth dilution assay. The growth ability and virulence of the above 3 strains were also determined. The results showed that compared with the wild strain, the MIC value of tetracycline antibiotics to the deletion mutant was significantly reduced, and the MIC values of other antibiotics did not change. The growth rate of the deletion mutant was significantly reduced by comparison with that of the parental strain. Furthermore, the deletion of the rant gene significantly reduced the virulence of R. anatipestifer. These results indicate that rant gene of R. anatipestifer mediates the efflux of tetracycline antibiotics and can significantly affect the growth and virulence of R. anatipestifer.

To prepare monoclonal antibodies specific to FAdV-4 Fiber2 protein, this study used NusA-Fiber2, a soluble recombinant protein expressed in prokaryotes, as an immunogen to immunize BALB/c female mice. Three hybridomas (2G5, 2G8 and 4C2) secreting monoclonal antibodies (MAb) against FAdV-4 Fiber2 protein were selected. Ascites were prepared from cell line 2G5 and purified. The specificity of the monoclonal antibody was determined by indirect immunofluorescence assay and Western blot. The prepared monoclonal antibody was used to coat the enzyme plate. Through a series of optimization, the detection method of FAdV-4 Fiber2 double antibody sandwich ELISA was established. The epitopes recognized by monoclonal antibodies were identified by progressively truncating Fiber2 protein. Results were as follows:Three hybridomas 2G5, 2G8 and 4C2 were successfully obtained. IFA and Western blot results showed that MAb 2G5 could react specifically with purified Fiber2 protein and FAdV-4 virus. The results showed that the sandwich ELISA was specific, sensitive and reproducible. Our results revealed that the antigenic epitope required for reactivity with the 2G5 was N-terminal aa1-33. In this study, monoclonal antibodies with good reactivity of Western blot and IFA were successfully prepared. This provides experimental data and theoretical basis for the development of rapid serological diagnosis and commercial vaccine against FAdV-4.

The coronaviruses of livestock, such as porcine epidemic diarrhea virus-PEDV, have genetic sequences that are highly similar to those of coronaviruses in bats, which can cause intestinal infections in pigs and cause a large number of deaths of piglets, which is extremely harmful to the health of animals. This study is based on the data mining of the epidemiological prevalence of PEDV strains in the United States as a priori information, combined with antibody screening of US swine in 2019 at entry port as the knowledge update, Bayesian statistical inference were employed to estimate the true prevalence of PEDV in US swine populations. The results show that the median true prevalence (median) of PEDV in the US swine population is 0.005 22 (95% CI=0.000 424-0.022 5), and the prevalence distribution of the 95% upper confident limit (UCL) is 3%. The probability density statistical properties of the posterior distribution prevalence are highly right-skewed to infer that the true prevalence of PEDV in the U.S. pig herd is mainly concentrated between in the 2.5% percentile (P_2.5%=0.0002) and the median (P_50%=0.005 22), but due to the variability and difference among different pig herds in the United States, the PEDV true prevalence distribution value of a random sampling may have a small probability overflowing the distribution greater than 0.03 (P ≤ 2.5%). In addition, in terms of assay selection for port screening, it is recommended that N protein-based ELISA can be used as the primary screening method, and then use S1 recombinant protein ELISA as the re-screening method to improve the port's ability of risk perception, control and identification capabilities to mitigate the entry risk of coronavirus into the country. Bayesian statistical inference and surveillance technology based on prior distribution and optimization of port laboratory testing results can provide scientific evidence-based risk decision technology for blocking the invasion of foreign pathogenic microorganisms to the greatest extent on the basis of existing knowledge and cognition, which can be of great significance to port inspection and quarantine, sampling monitoring and risk decision-making.

The exact distribution and expression of CTGF and FGF-2 in the lungs of newborn, young, adult and old yaks were studied by immunohistochemistry (IHC), Western blot and real-time fluorescence quantitative (qRT-PCR). The results of IHC showed that CTGF was mainly distributed in terminal bronchiole epithelium Clara cells and smooth muscle cells, pulmonary artery endothelial cells and medium membrane smooth muscle cells; The expression levels were the highest in the newborn and elderly groups; FGF-2 was mainly distributed in the smooth muscle of the terminal bronchiole and the smooth muscle of the pulmonary artery, and maintained a high expression level in all age groups. The results of WB showed that the expression of CTGF and FGF-2 could be detected in the lungs of yaks in different age groups, the expression of CTGF in newborn group and elderly group was significantly higher than that in young group and adult group (P<0.05), and the expression of FGF-2 in the newborn group and young group was significantly higher than that in adult group and old group (P<0.05). The results of qRT-PCR showed that the transcriptional level of CTGF was the highest in the newborn group, and there were significant differences among different age groups, while the transcriptional level of FGF-2 in the elderly group was the highest, followed by the adult group, and there were significant differences compared with other groups (P<0.05). CTGF and FGF-2 are related to the formation of adaptive structure and adaptive process of yak lung, and play a significant role in newborn and old age.

To study the effect of Fas/FasL pathway on cadmium-activated MAPK pathway in PC12 cells, PC12 cells with non-specific sequences inserted and Fas gene silenced were treated with 10 μmol·L^-1 CdAc₂ respectively for 12 h, PC12 cells were treated with 10 μmol·L^-1CdAc₂ and 40 μmol·L^-1 Z-IETD-FMK (caspase-8 specific inhibitor) alone or in combination for 12 h. The expression of Fas/FasL pathway-related proteins Fas, FasL, Fas-associated death domain protein (FADD), Cleaved caspase-8, Death domain associated protein (Daxx), Apptosis signal-regulating kinase 1 (ASK1) and MAPK pathway-related proteins ERK1/2, JNK1/2, p-ERK1/2, p-JNK1/2 were detected by Western blot. The morphological changes of apoptosis were detected by Hoechst33258 fluorescence staining, and the apoptosis rate was detected by flow cytometry. The results showed that Fas shRNA lentivirus significantly inhibited the increase of Fas/FasL pathway related-proteins expression and apoptosis rate in PC12 cells induced by cadmium (P<0.01), and alleviated the morphological changes of cell apoptosis caused by cadmium in PC12 cells. In addition, Fas shRNA and Z-IETD-FMK could significantly inhibit the increase of cadmium-induced phosphorylation levels of MAPK pathway-related proteins ERK1/2 and JNK1/2 in PC12 cells (P<0.01). Our results indicate that the regulation of Fas/FasL pathway on the MAPK pathway is involved in cadmium-induced apoptosis in PC12 cells.

This study aimed to investigate the expression of thermogenic genes and beige fat marker genes during the differentiation of Min pig adipocytes and to provide a basis for further study on the molecular mechanism of differentiation of Min pig adipocytes into beige adipocytes. Adipose tissue of 1-month-old Min pig was collected, preadipocytes were isolated, cultured and induced to adipogenesis, cell morphology was observed during differentiation and identified by Oil red O staining; The expression levels of thermogenic genes UCP3, PGC-1α, PPARα, and beige fat marker genes EBF2, CD81, PDGFRα were detected on day 0, 2, 4, 6 and 8 after differentiation. The results showed that the number of lipid droplets increased with the time of differentiation increased. The result of Oil red O staining showed that most of the cells reached the stage of mature adipocytes. The expression levels of PGC-1α, EBF2 and PDGFRα were significantly increased on day 2 compared with those on day 0 (P<0.01) and reached the highest level, and decreased on day 4, 6, and 8. The expression of UCP3 was significantly increased at day 4, 6, and 8(P<0.01), and decreased at day 8. The expression levels of PPARα and CD81 were significantly increased on day 2 (P<0.01), and there was no significant change on day 4, 6 compared with day 2. In conclusion, this study successfully conducted adipogenic differentiation of Min pig preadipocytes, revealed the expression of thermogenic and beige fat marker genes during differentiation, and laid a foundation for further study on the molecular mechanism of differentiation of Min pig preadipocytes into beige adipocytes and cold-resistance of Min pig.

The experiment was conducted to investigate the effects of early heat stress on the growth and development of broilers and gene expression in skeletal muscle. One hundred and ninety-two (male and female half) healthy newborn AA broiler chickens with similar body weight were randomly divided into 2 groups (4 repetition per group, 24 chickens per repetition):heat stress treatment (heat stress group, 36℃ for 24 h) and normal temperature (control group,33℃ for 24 h) at 3 days of age. At 42 days of age, 8 chickens from each treatment were selected and slaughtered, the plasma was collected, and carcass performance was measured. Pectoralis samples were separated to determine the gene expression related to skeletal muscle development. The results showed that early heat stress significantly decreased the body weight gain of chickens at 3 days of age, but significantly increased the average daily weight gain of broilers during the whole feeding period (P<0.05). Compared with the control group, the average daily intake of broilers during the whole feeding period was increased after early heat stress (0<P<0.1). The early heat stress had no significant effect on the carcass rate, half-clean chamber rate, full-clean chamber rate, liver proportion, heart proportion, pectoral muscle rate, leg muscle rate and abdominal fat rate of broilers (P>0.05). Early heat stress significantly increased the plasma ketone body content of 42-day-old broilers (P<0.05). In pectorals, early heat stress significantly increased the expression of insulin-like growth factor (IGF-1) and its receptor (IGF-1R) genes (P<0.05). The results suggest that early heat stress improves the growth and development of broilers in the later stage, and IGF-1 signals in skeletal muscle may be involved in this regulatory process.

To understand the prevalence and genetic diversity of Escherichia coli (E. coli) in the dairy farm environment and explore the genetic relationships of isolates from different samples, we conducted this experiment. In this study, E. coli was isolated from 209 samples which collected from a dairy farm environment in 2017-2019 and identified by 16S rRNA gene. The ERIC-PCR genotyping and phylogenetic grouping were performed respectively with non-duplicate E. coli. Three hundred and thirty-eight E. coli strains were isolated and the isolation rate was 67.46%. The E. coli was clustered into 14 types by ERIC-PCR genotyping. Type Ⅳ was the dominant, including 196 strains, followed by type Ⅰ (59 strains), type Ⅴ (31 strains) and type Ⅹ (11 strains). The remaining 41 strains were distributed in the other 10 types. The phylogenetic grouping results showed that 2 strains were not classified. The rest were classified into 6 types and the most strains were in group B1 (75.45%). The proportion of group A,C,E,D and F were 18.34%, 2.96%, 1.18%, 1.18% and 0.30%, respectively. E. coli had a wide range of DNA diversity in the dairy farm environment, and there was a close relationship between the strains from samples of different sources at different time. The phylogenetic grouping was mainly B1 group.

The purpose of this study was to characterize the molecular characteristics of the HE genes of bovine torovirus (BToV). An RT-PCR assay was used to detect BToV from 94 diarrheic fecal samples collected in Liaoning, Henan, and Sichuan provinces and full-length hemagglutinin-esterase (HE) genes were further amplified from the positive samples. Approximate 10.64% (10/94) samples were detected as BToV positive, and 15 full-length HE genes were sequenced from 10 positive samples, including 9 genotype Ⅱ strains and 6 genotype Ⅲ strains. Among the 10 positive samples, 5 samples were co-infected with genotype Ⅱ and Ⅲ strains, 1 sample was infected with genotype Ⅲ only and 4 samples were infected with genotype Ⅱ strains only. Compared with other available genotype Ⅲ BToV HE genes in the GenBank database, 4 unique amino acid variations were found in the HE gene, including 2 variations in the lectin domain which may affect HE receptor binding. Recombination analysis showed that BToV genotype Ⅲ strains may be derived from the recombination of genotype Ⅱand genotype Ⅰstrains in the HE gene. This study is the first confirmation of genotype Ⅲ BToV in China and the co-infection of genotype Ⅱ and Ⅲ strains. These findings contributed to the diagnosis and control of bovine diarrhea in China and enhance the current understanding of the genetic evolution of BToV.