Competing endogenous RNAs (ceRNA) refers to RNAs harboring the same microRNA response elements (MRE), competitively binding the miRNA to regulate gene expression post-transcriptionally. Eventually, the biological functions of cells are altered. The efficiency of ceRNA is affected by the cellular environment, miRNA activity, intermolecular affinity with RNAs, etc. Both circular RNA (circRNA) and long non-coding RNA (lncRNA) can act as ceRNA; circRNA is more effective based on its higher stability and conservation, enabling its transition into different tissues. This paper discussed the factors affecting the ceRNA mechanism, then reviewed the research progress of ceRNA-mediated regulation of circRNAs in muscle growth, fat deposition, mammary gland and follicular development in animals. In the hope of providing new insight for the in-depth study of the ceRNA regulatory network in the development of critical economic traits for animals.

Simple and accurate early pregnancy diagnosis is an important means to reduce open cows, improve female fecundity and improve farm economic benefits. Although traditional pregnancy diagnoses methods such as rectal palpation, B-ultrasound examination, and progesterone detection have provided important technical support for breeding production, they have not yet achieved the earliest detection of cows after mating, and some farms are still difficult to meet the high technical requirements or laboratory conditions. Pregnancy-associated proteins (PAGs) are pregnancy-specific proteins secreted by embryonic trophoblast cells, which play an important role in the pregnancy of dairy cows and are widely used in the diagnosis of early pregnancy in dairy cows. This article briefly summarizes the existing diagnostic methods for pregnancy, discusses the development process of PAGs diagnostic methods in detail, looks forward to the future development direction of pregnancy diagnostic technology, and makes important progress in "early", "accurate" and "simple", and provides relevant research information and important reference.

As an osseous organ of deer family, deer antler undergoes annual cyclic regeneration based on stem cells, and its unique biological properties have gradually made it an ideal model for biology, medicine and other fields. Antler ossification is closely related to changes in sex hormone levels in the body: Under the physiological environment of low sex hormone levels and rib bone loss in deer, antlers grow at a rate of up to 2.7 cm·d^-1, and ossifying as they grow; Then sex hormone levels rise and antlers enter a period of rapid ossification, forming bony tissues weighing more than 30 kg in just three months. This phenomenon that antler can achieve rapid osteogenesis under the endocrine conditions of massive body bone loss and low levels of sex hormones, which called antler reverse osteogenesis. This paper reviewed the current research status and occurrence mechanism of antler reverse osteogenesis from the perspectives of cell differentiation, hormone, osteogenesis, osteolysis and cytokines, with the aim of investigating the mechanisms of antler reverse osteogenesis and providing a reference for improving antler production and animal welfare and health.

Persistent infections with herpesviruses are detrimental to the health of farm animals and cause impairment of animal husbandry performance. Due to their robust capability of immune evasion, herpesviruses can effectively antagonize the recognition and consequent elimination by the immune systems of livestock hosts. During the process of replication, herpesviruses take advantage of the host cellular machineries to produce various types of molecules that disrupt the MHC-Ⅰ (major histocompatibility complex class Ⅰ) -mediated antigen presentation pathway, thereby evading the viral clearance by cytotoxic T lymphocytes (CTLs). For herpesviruses, one of the most active research focuses has centered on elucidating the molecular mechanisms by which viral proteins manipulate to induce reduction of MHC-Ⅰ expression. This review summarizes the research advances that have been made over the past decade concerning the modulation of MHC-Ⅰ molecules caused by infection with herpesviruses in livestock. Mechanistically, the involvements of viral proteins were illustrated based on the stages of MHC-Ⅰ antigen presentation. In addition, the domains and functions of the viral homologues were analyzed. By highlighting the current issues and future trends in this field, our perspectives shed novel insights into the therapeutic treatments as well as the development and optimization of vaccines for protecting domestic animals from herpesvirus infections.

Type VI secretion system (T6SS) is a widespread "secretion device" and "killing machine" in Gram-negative bacteria, which transmits bacterial toxins to prokaryotic or eukaryotic cells to inhibit or kill them. T6SS plays an important role in the pathogenic process of Salmonella, it can independently code five sets of T6SSs (T6SS-1-T6SS-5), which are respectively coded by Salmonella pathogenicity island 6 (SPI-6), SPI-19, SPI-20, SPI-21, and SPI-22. In addition, T6SS participates in the interspecific competition of Salmonella, biofilm formation, metal ion uptake and transportation, and interaction with host cells, playing an essential role in the life cycle of Salmonella. This article mainly reviews the assembly, genomic structural characteristics, and the latest research progress of secretion regulation network of T6SS in Salmonella, providing theoretical guidance for in-depth research on the pathogenic mechanism of Salmonella and the development of new antibacterial strategies.

Canine tumor disease is a common disease in veterinary clinic with high incidence and great harm. It is one of the important causes of canine death, especially for middle-aged and elderly canines. In order to better diagnose and treat canine tumor diseases, it is of great significance to identify potential biomarkers and therapeutic targets. Non-coding RNA(ncRNA)refers to a class of RNA molecules that do not encode proteins and have many functions. ncRNA can regulate the development of tumor cells by regulating gene transcription and it is expected to become a biomarker and potential therapeutic target for the diagnosis and treatment of canine tumor diseases. Therefore, this article reviews the diagnostic value and application prospects of non-coding RNA in canine mammary tumors, melanoma and osteosarcoma, in order to provide new ideas for the diagnosis, prevention and treatment of canine tumors.

Cats are one of the most popular pets in China, and the number of cat owners has been increasing in recent years. Cat allergens are one of the most common animal-derived allergens. The adverse effects to allergic population and society caused by feline allergens, such as rhinitis, conjunctivitis and urticaria, are gradually attracting extensive attention. This review aims to summarize the biological characteristics, sensitization mechanisms, detection methods, and control solutions for feline allergens, which will provide a theoretical basis for in-depth research and effective intervention of feline allergens.

Ferroptosis is a newly discovered programmed cell death pattern characterized by iron-dependent accumulation of lipid peroxidation. Iron metabolism disorder and oxidative stress play an important role in bacterial infection, and it has been proved that some bacteria could induce host cell death by ferroptosis. In this paper, the effects of bacterial infection on host iron metabolism and the mechanism of bacteria-induced ferroptosis were reviewed, in order to provide new research ideas for the prevention of bacterial infection in animals.

The purpose of this study was to evaluate the genetic parameters of lean meat percentage traits and the relationship between their main growth traits, and to analyze the effects of litter effect on lean meat percentage and main growth traits, so as to provide reference for the improvement of lean meat percentage traits. In this study, the corrected 115 kg lean meat percentage (LMP), corrected 115 kg daily gain (ADG), corrected 115 kg backfat thickness (BFT), and corrected 115 kg eye muscle area (LMA) traits were studied in a Duroc pig population (26 392 animals) at a core farm of Guangdong Zhongxin Breeding Technology Co.. The R software was used to analyze the fixed effects on LMP, ADG, BFT, and LMA in Duroc pigs. The genetic parameters of estimation with and without litter effect for lean meat percentage traits, and the genetic and phenotypic correlations between LMP trait and growth traits were evaluated using DMU software. The results showed that the number of phenotypic records for each of these traits reached more than 20 000, and the heritability of the LMP trait was 0.34±0.019 when the litter effect was taken into consideration, the heritability of the LMP trait was 0.45±0.017 when the litter effect was not considered. The genetic correlation coefficient between LMP and ADG traits was relatively small, but it was highly significantly positively correlated with the LMA trait. In addition, the weight of the litter effect in the overall variance for the LMP, ADG, BFT, and LMA traits in this Duroc population was 0.15, 0.21, 0.15, and 0.14, respectively, and the mean annual estimated breeding value (EBV) for LMP showed an increasing trend. The above results showed that the heritability of LMP, ADG, BFT, and LMA of this Duroc population belonged to medium heritability and was greatly affected by the litter effect. The fitting effect of the these traits was further enhanced and the accuracy of genetic evaluation was increased by treating the litter effect as a random variable in the genetic evaluation model. In addition, the effects of other traits , such as maternal genetic effects, are needed to be further discussed. The above results showed that the loin muscle area trait was cooperatively selected by lean meat percentage traits, and further explained the reason why loin muscle area was used as auxiliary traits in the current lean meat percentage prediction equation.

The study aimed to better understand the population structure and genetic diversity of Neijiang pigs, make a better protection and utilization of the genetic resources of Neijiang pig. The SNPs of 133 Neijiang pigs (12 boars, 121 sows) was detected based on low-coverage whole genome sequencing. Two sets of SNPs data, with different calling rates 90% or 95%, were obtained and named as NJ90 and NJ95. Population structure of Neijiang pigs was analyzed by population stratification, genetic distance, genetic relationship and boar genome family. The population genetic diversity was estimated by allele frequency, effective number of alleles, proportion of polymorphic and heterozygosity. Finally, the inbreeding coefficient of population was evaluated by different methods. The results showed as follows: 1) NJ90 contained 135 760 SNPs. The allele frequency, effective number of alleles, and proportion of polymorphic were 0.87, 1.27, and 0.76, respectively. The observed heterozygosity and expected heterozygosity were 0.15 and 0.21. NJ95 contained 32 266 SNPs. The allele frequency, effective allele number, and proportion of polymorphic were 0.79, 1.44, and 0.74, respectively. The observed heterozygosity and expected heterozygosity was 0.30 and 0.31. 2) The results of NJ90 showed that the average genetic distance of the population was 0.20, and the average kinship coefficient was 0.9%. The results of NJ95 showed that the average genetic distance of the population was 0.25, and the average kinship coefficient was 0.7%. 3) According to the boars of NJ90 and population clustering as well as kinship, the population could be divided into 6 families. 4) According to SNPs homozygosity, the average inbreeding coefficients of NJ90 and NJ95 were 0.27 and 0.01, respectively. According to the runs of homozygosity, the average inbreeding coefficients of NJ90 and NJ95 were 6.65% and 0.02%. In conclusion, the Neijiang pigs are relative abundant in genetic diversity, far in genetic distance and kinship, and low in inbreeding degree. The population can be divided into 6 families according to boars clustering and kinship analysis, which has a good basis for the protection, exploration and utilization of genetic resource. Moreover, low-coverage whole genome sequencing can cover genome information in a wider range, which is of more reference value for population genetic diversity and genetic structure analysis.

This experiment was conducted to study the genetic diversity, relationship and family structure of Yacha pigs conserved population. The single nucleotide polymorphism (SNP) in 166 Yacha pigs was detected by 50K SNP bead chip. The observed heterozygosity, expected heterozygosity, polymorphism information content and minor allele frequency were calculated by Plink software to analyze the genetic diversity of Yacha conserved population. The runs of homozygosity (ROH) were calculated and identity by state (IBS) distance matrix was constructed by Plink software. The genetic relationship was analyzed according to the G matrix result constructed by GCTA software. The phylogenetic tree constructed by Mega X software was used to analyze the family structure of Yacha conserved population. The results showed that, 45 211 SNPs were detected in 166 Yacha pigs, in which 36 243 SNPs passed quality control. The effective allele number, polymorphism information content, proportion of polymorphic markers, minor allele frequency were 1.529, 0.254, 0.875 and 0.233, respectively. The expected heterozygosity and observed heterozygosity were 0.329 and 0.344, respectively. The average IBS genetic distance of Yacha conserved population was 0.259 5, the result of IBS genetic distance and G matrix showed that most Yacha pigs had moderately genetic relationship. A total of 3 226 ROHs were detected, and the length of 40.96% of these ROHs was less than 100 Mb. The average inbreeding coefficient based on ROH was 0.069. The phylogenetic tree result showed that, there were 8 families in Yacha boars, which is the same as that by manual pedigrees, but with individual differences among families. In summary, the effective population size and the genetic diversity of Yacha conserved population is low, while the inbreeding degree is not serious. Therefore, it’s necessary to importing or creating new blood to expand the effective population content and improve the genetic diversity of the population.

This study aimed to screen the candidate genes affecting skin color of Black-bone chicken based on the combination of transcriptome and proteome analysis. Six white skin (WS) chickens and six black skin (BS) chickens at 150 days of age were selected from HW1 Black-feather Black-bone chicken population in this study. Total RNA and protein were extracted from breast skin tissue, high-throughput transcriptome sequencing (RNA-Seq) and proteomics quantification analysis were performed. Next, the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) associated with the skin color of Black-bone chicken were identified, and several DEGs were further verified by real-time fluorescent quantitative PCR (RT-qPCR). Finally, the candidate genes related to skin color trait of Black-bone chicken were further screened by combining functional enrichment analysis and protein-protein interaction network (PPI) analysis. The results showed that 293 DEGs were identified in the transcriptome and 4 DEPs were identified in the proteome, among which the overlapping genes were CRP, DCT and GPNMB. We then randomly validated 5 DEGs using RT-qPCR, which showed that RT-qPCR gene expression results were consistent with RNA-seq results. Enrichment analysis indicated that these DEGs and DEPs were significantly enriched in 71 GO terms and 4 KEGG pathways, including melanocyte differentiation, melanosome, melanosome membrane, and melanogenesis pathways. Subsequently, 10 Hub genes were captured by the cytoHubba plug-in of Cytoscape software from the PPI network constructed by DEGs and DEPs, and these genes were mainly enriched in immune-related GO terms and pathways. It was further found that Hub genes and melanin-related genes constituted two sub-networks, among which PTPRC, SOX10, GPNMB and CRP genes connected the two sub-networks. In all, 11 candidate genes related to skin color trait of Black-bone chicken were screened, including SOX10, EDN3, EDNRB2, MLANA, OCA2, DCT, GPNMB, ASIP, RAB38, PTPRC, CRP, which may play an important role in melanin synthesis process. In summary, this study provides a theoretical basis for the future exploration of the molecular regulation mechanism and the improvement of molecular breeding in Black-bone chicken skin color traits.

The present study aimed to evaluate whether the direct protein delivery of PhiC31 integrase through electroporation could induce the PhiC31-mediated recombination in chicken DF-1 cells. Here, prokaryotic expression and affinity chromatography purification was used to produce a small ubiquitin-related modifier (SUMO)-tagged His-PhiC31 fusion protein, and a SUMO proteinase was utilized to generate a natural PhiC31 protein by removing SUMO-His tag. The two types of PhiC31 protein were respectively incubated with pBCPB+ plasmid in tubes to induce intermolecular recombination. Subsequently, a landing pad (LP) plasmid harboring the attP^TT-DsRed2-attP^CT fragment was electroporated into the chicken DF-1 cells, followed by another round of electroporation of the natural PhiC31 protein along with a donor plasmid carrying the attB^TT-EGFP-HiBiT-attB^CT segment. The results showed that a prokaryotic expression vector (pET-28a-SUMO-PhiC31) was successfully constructed, and SUMO-His-PhiC31 fusion protein was expressed in a resolvable pattern in E.coli with induction of isopropyl-β-D-thiogalactoside (IPTG), and amount of the purified fusion protein reached 12 mg·L^-1; both SUMO-His-PhiC31 and natural SUMO-free PhiC31 were capable of triggering the intermolecular recombination between attP and attB sites of pBCPB+ plasmid, and the recombinant efficiency of SUMO-His-PhiC31 was comparable to that of PhiC31 proteins (50% vs. 52%); co-transfection of DF-1 cells with a PhiC31 expressing vector pCMVInt, LP and donor plasmids resulted in conversion of DsRed2 to EGFP, which was confirmed by fluorescent microscopy; the same results were observed when LP plasmid, the donor plasmid plus PhiC31 protein were successively electroporated to DF-1 cells, implying the occur of PhiC31-mediated recombination between attP^TT and attB^TT, attP^CT and attB^CT, respectively. The results indicated that the natural PhiC31 protein is biologically active. The protein is expected to serve as an important reagent in the Easi-CRISPR-TARGATT-mediated genome editing to generate transgenic chickens.

The study aimed to investigate the polymorphism of regulatory regions of several nutrition transport-related genes and screen the key variations affecting the feed conversion ratio of broilers. The blood DNA samples of 439 male yellow-feather broilers were collected in this study, and the polymorphic loci within 1 kb upstream of 3 monosaccharide transporter genes (GLUT2, GLUT5 and SGLT1), 8 amino acid transporter genes (B⁰AT1, B⁰⁺AT, CAT1, CAT2, LAT1, y⁺LAT1, y⁺LAT2 and rBAT) and 1 nutrient regulation gene (FGF1) were detected in the two-tail sample group with high and low feed conversion ratio by PCR sequencing. According to the difference in genotype frequency in the two-tail sample groups, the genetic variations with potential influence on feed conversion ratio were screened. The PCR-RFLP method was used to genotype the polymorphic loci in the whole sample and the association between these polymorphic loci with feed conversion ratio and other traits was analyzed. The results showed that a total of 111 SNPs sites were found within 1 kb upstream of 12 genes in this study, among which 8 SNPs sites had different genotype frequencies in the two-tail sample groups,including -379A>G of GLUT2, -490G>A of SGLT1, -639T>C of B⁰AT1, -46G>A, -90G>T, -180T>C, -235G>A of rBAT, and -884T>C of FGF1. Further whole population genotyping and association analysis showed that the -639T>C of B⁰AT1 was significantly associated with feed conversion ratio (P<0.05), individuals with TC genotype had significantly lower feed conversion ratio than those with TT and CC genotypes (P<0.05), showed a heterozygous advantage. The -884T>C of FGF1 was also significantly associated with feed conversion ratio (P<0.05), and the individuals with CC and TT genotypes had significantly lower feed conversion ratio than those with the TC genotype (P<0.05). In addition, the -379A>G of GLUT2 was not significantly associated with feed conversion or growth traits. In this study, the genetic variation of regulatory regions of several nutrient transport-related genes was analyzed, which provides the basis for using these variation sites as genetic markers to improve the feed conversion ratio of yellow-feather broilers.

As a binding protein of ATF/cAMP response element, Transcriptional activator 4 (ATF4) play a pivotal role in mammalian growth and development. The present study aimed to obtain the ATF4 gene sequence of Xinong Saanen dairy goats (Capra hircus), elucidate its structure and investigate its function on lipid metabolism and milk protein synthesis through siRNA interference of the ATF4 gene in goat mammary epithelial cells(GMECs). In order to clarify the expression differences of the ATF4 in different tissues of Xinong Saanen dairy goats, the breast tissues of Xinong Saanen dairy goats at different lactation stages were collected, and the CDS region of the ATF gene was cloned and analyzed by bioinformatics and tissue expression profile. The results showed that the complete CDS region of the ATF4 gene was 1 059 bp, encoding 352 amino acids, the ATF4 protein is acidic protein without transmembrane structure, with a molecular weight of 38.545 24 ku, a theoretical isoelectric point of 4.61, and 35 serine phosphorylation sites. The advanced structure of ATF4 protein was composed of α-helix, extension chain irregularly coiled and β-turn were 39.77%, 7.10%, 51.14% and 1.99%, respectively. ATF4 interacted with CEBPB, CEBPG, ATF3, FOSL2, ATF1, DDIT3, TRIB3, BTRC, CREB1 and DISC. Homology analysis showed that sequence homologies of Xinong Saanen dairy goat with sheep (Ovis aries) and cattle (Bos taurus) were 97.45% and 91.48% respectively. Tissue expression analysis revealed that the highest expression of ATF4 gene was in the rumen, followed by muscle. The expression level of the ATF4 gene was the highest in early lactation, followed by that in peak lactation. After interfering with ATF4 gene expression in mammary epithelial cells of dairy goats, the results showed that mRNA levels of FASN, FABP3 and BLG were down-regulated significantly. Moreover, the mRNA levels of PPARA, PPARG and LALBA were significantly up-regulated. The above results suggest that ATF4 might be involved in the regulation of lipid metabolism and milk protein synthesis in GMECs. The present study provides basic data for further investigation of the molecular mechanism of ATF4 on milk fat metabolism and milk protein synthesis in dairy goats.

The purpose of this study was to investigate the relationship between Sonic hedgehog (SHH) and wool bending formation and its regulatory mechanism. In this study, primary sheep keratinocytes were cultured, overexpression and silencing vectors were constructed, and cell transfection, immunocoprecipitation, quantitative real-time PCR and Western blot were used to explore the relationship between SHH and wool bending, and to analyze whether SHH regulates insulin-like growth factor binding proteins 5(IGFBP5) through early growth response 2(EGR2/Krox20) to affect wool bending. The results showed that sheep keratinocytes were successfully isolated and cultured in vitro, and were like "paving stone" under the microscope. Immunofluorescence identification results showed positive expression. CCK-8 results showed that the growth curve of sheep keratinocytes was "S" shape, which was in line with the trend of cell proliferation. After overexpression and silencing of SHH, the expressions of fleece-related genes KRT71, β-catenin and TCHH were significantly increased and decreased. After overexpression of Krox20, the expression of wool bending related genes was significantly increased. Co-immunoprecipitation assay showed that SHH could interact with Krox20 and positively regulate Krox20 in sheep keratinocytes. The results of real-time PCR and Western blot showed that overexpression of SHH significantly up-regulated the relative expression of IGFBP5, while silencing of SHH significantly down-regulated the relative expression of IGFBP5. The relative expression of IGFBP5 increased significantly after overexpression of Krox20. The results indicate that SHH affects wool bending through Krox20 regulation of IGFBP5 in sheep keratinocytes, which provides a theoretical basis for the related molecular regulation mechanism of hair bending traits.

The purpose of this study was to identify Guyuan cattle as relatively independent beef cattle genetic resources, further describe their growth and development rules, and provide certain theoretical basis for the identification and breeding of germplasm resources for meat of Guyuan cattle. The evolutionary history of Guyuan cattle was analyzed through historical documents and investigation. The whole genome genetic variation data of 337 individuals of 13 breeds including Guyuan cattle were detected through GGP Bovine 100K gene chip and downloaded from public databases, and the genetic background of Guyuan cattle was analyzed by multidimensional scaling analysis and adjacent phylogenetic tree. Taking the Red Angus×Guyuan crossbred cattle as the control population, the phenotypic data such as body weight, body size, backfat thickness and loin eye area of Guyuan cattle at different growth stages were measured and collected, with refe-rence to the phenotypic data of Qinchuan cattle and Mongolian cattle published in the Journal of Chinese Livestock and Poultry Genetic Resources·Cattle, the population rule of relevant indicators of Guyuan cattle was excavated from the phenotypic description. The results showed that: 1) Guyuan cattle was a unique population with the same shape and appearance after long-term breeding due to the mutual influence of Guyuan native cattle with neighboring Mongolian cattle and Qinchuan cattle; 2) The genetic structure of existing Guyuan cattle population was close to the neighboring Qinchuan cattle and Jinnan cattle, and it was a relatively independent resource population. 3) Compared with heifer of Guyuan cattle, somatic index of adult cow of Guyuan cattle was increased by 9.57%; 4) After crossbreeding with Red Angus cattle, brassiere index and somatic index of adult cow of crossbred Guyuan cattle were respectively increased by 11.59% and 12.70%, body length index and brassiere index of bull of crossbred Guyuan cattle were respectively increased by 15.15% and 19.26%; 5) Beef-purpose index of adult cow of Guyuan cattle was 2.65±0.46, beef-purpose index of bull of Guyuan cattle was 2.87±0.35, and Guyuan cattle was draft cattle; 6) The backfat thickness of bull of Guyuan cattle was (5.29±1.41)mm, higher than the backfat thickness of bull of Mongolian cattle (4.00±1.00)mm; 7) The loin eye area of bull of Guyuan cattle was (62.17±8.51)cm², between the loin eye area of of Qinchuan cattle (79.80±9.70)cm² and Mongolian cattle (50.40±9.80)cm². Guyuan cattle is a new genetic resource of beef cattle, which has good germplasm characteristics for meat. However, its body size indexes and beef-purpose index have not reached the standard of specialized beef cattle. In the later stage, strengthen breeding and the stock breeding and genetic improvement of Guyuan cattle are needed to further develop its germplasm resource characteristics.

The aim of this study was to investigate the population patterns of health events in Holstein lactating cows in Ningxia, and analyze the effect of risk factors on wellness traits. Calving and health records of 58 549 lactating cows calving between 2015 to 2021 were collected from 13 herds in Ningxia. The correlation between health events were analyzed using the UpSet program and the Spearman correlation analysis. In addition, Logistic regression models were used to analyze the effects of risk factors on health events, such as parity, calving season and calving ease score. The results showed that udder health and reproductive disorders accounted for a large proportion of the health events of lactating cows, at 29.97% and 28.95%, respectively. The first 60 days after calving was a critical period for health events in lactating cows. As the parities increased, the incidence of udder health, digestive disorders and metabolic disorders all gradually increased, while the incidence of reproductive disorders tended to first decrease and then increase, with the lowest incidence in 2-parturient cows. In the phenotypic correlation analysis, the correlations between the different health events were all weak. Logistic analysis of risk factors found significant effects of parity, calving season and calving ease score on selected wellness traits in lactating cows. The risk of metabolic disorders was higher in cows that calved in spring and summer, the risk of udder health and reproductive disorders was higher in cows that calved in autumn and winter. And the risk of metabolic and reproductive disorders was increased by 1.30-1.82 times, for assisted at calving comparing to cows unassisted. Based on large-scale herd data, this study identified reproductive disorders and udder health are key health events for lactating cows in Ningxia, the various health events have weak phenotypic correlations, parity, calving season and calving ease score were all risk factors for wellness traits. The results provided a reference for managing herd health events on large-scale farms, and provided theoretical support for research and selection of dairy cow wellness traits.

This study aimed to explore the genetic diversity and the ancestral types of Tahe red deer at the molecular level. DNA was extracted from fresh blood which was collected at the sawn antler stage, PCR amplification and direct sequencing were performed to analyze AMELY2, DBY, SRY genes on Y chromosome and ND1, COX1, ATP6, ND5, Cytb genes of mtDNA in 38 stud Tahe red deer species. Base composition, nucleotide diversity (Pi), mean nucleotide difference (K), Tajima’D value, haplotype number (H) and haplotype diversity (Hd) were calculated to evaluate the genetic diversity of stud Tahe red deer. The haplotype network diagram was constructed and the genetic distance between haplotypes was calculated. The phylogenetic tree was constructed and white-lipped deer was used as the outgroup to analyze the parental lines of stud Tahe red deer. The results showed that the length of the Y chromosome spliced by comparing the AMELY2, DBY and SRY genes was 3 577 bp, and a total of 17 SNPs polymorphic sites were detected, and 4 haplotypes were defined. The dominant haplotype was Hap-1, accounting for 65.79% of the frequency. The nucleotide diversity and haplotype diversity were 0.001 95 and 0.495 0, respectively, showing a low level of genetic diversity. The phylogenetic tree constructed based on the Y chromosome genes showed the existence of two branches, A and B. After comparison of ND1, COX1, ATP6, ND5 and Cytb genes of mtDNA, the splicing length was 6 160 bp, and a total of 41 SNPs polymorphic sites were detected, and 8 haplotypes were defined. The dominant haplotype was Hap-1, accounting for 47.37% of the frequency. The nucleotide diversity and haplotype diversity were 0.001 54 and 0.699 9, respectively, showing an imbalance of high haplotype diversity and low nucleotide diversity, and the genetic diversity was at a low level. The phylogenetic tree constructed based on mtDNA showed the existence of two branches, I and II. The genetic diversity of stud Tahe red deer is at a low level, and there are two ancestral types in the population of Tahe red deer.

This study was conducted to investigate the relationship between the occurrence of intrauterine growth retardation (IUGR) and the development pattern of insulin-like growth factor (IGF). Twenty pregnant sows with similar body condition were selected. The largest piglet selected was defined as normal birth weight (NBW) piglet and the smallest piglet was defined as IUGR piglet from each litter after parturition. They were divided into NBW group and IUGR group with 20 piglets per group. At 0, 7, 14, and 21 days of age, 5 piglets were randomly selected from each group, respectively. The blood was collected from the anterior vena cava and the plasma was collected to detect the IGF-1 concentration, and the samples of liver and longissimus dorsi muscle were collected to detect the gene expression levels of IGF-1, IGF-1R,IGFBP-3, and IGFBP-5. The results showed that the body weight and plasma IGF-1 content of IUGR piglets were significantly lower (P＜0.05) during 0 to 21 days of age, compared with the NBW piglets. The body weight of IUGR piglets during 0 to 14 days of age was increased (P＜0.05), but there were no significant difference (P＞0.05) in the body weight of IUGR piglets at 14 and 21 days of age. At 0 day of age, the IUGR piglets had significantly higher (P＜0.05) mRNA expressions of IGF-1R, IGFBP-3 and IGFBP-5 in muscle and IGFBP-5 in liver than NBW piglets; At 7 days of age, the IUGR piglets had significantly lower (P＜0.05) mRNA expression levels of IGF-1 in muscle or liver than NBW piglets; At 21 days of age, the IUGR piglets had significantly higher (P＜0.05) mRNA expression levels of IGF-1R in muscle than NBW piglets. The expression of IGF-1 in liver and muscle of NBW piglets and liver of IUGR piglets at 7 days of age was significantly higher (P＜0.05) than that at other days of age; The mRNA expression level of IGF-1 in muscle of IUGR piglets at 0 and 14 days of age was significantly higher (P＜0.05) than that of IUGR piglets at 7 and 21 days of age. The expression level of IGFBP-5 in liver and muscle of NBW piglets at 7 days of age was significantly higher (P＜0.05) than that at other days of age, and the expression levels of IGFBP-3 at 14 days of age and IGFBP-5 at 0 day of age were significantly up-regulated (P＜0.05) in liver of IUGR piglets. Collectively, these findings suggested that the IUGR may reduce the expression of IGF-1 and regulate the expression of IGF-1 receptor and binding protein in suckling piglets of Huanjiang mini-pig, which may lead to the decrease of IGF-1 content, retard growth and development of suckling piglets.

The study aimed to investigate the effect of miR-144-5p targeting WNT5a on proliferation and apoptosis of goat granulosa cells. Ovarian granulosa cells from 2-year-old healthy Leizhou female goats were selected to overexpress and repress miR-144-5p and WNT5a, and the effects of miR-144-5p and WNT5a on granulosa cell proliferation and apoptosis were investigated by fluorescent quantitative PCR (real-time qPCR, RT-qPCR), Western blot, dual luciferase reporter gene, CCK-8 and other techniques. The results showed that the expression of miR-144-5p was significantly higher in large follicular granulosa cells than that in small follicular granulosa cells (P<0.01), and the expression of WNT5a was significantly lower in large follicular granulosa cells than that in small follicular granulosa cells (P<0.01); CCK-8 assay revealed that miR-144-5p inhibited the proliferation of GCs, and overexpression of miR-144-5p resulted in significant downregulation of mRNA and protein expression of the apoptosis suppressor gene BCL-2 (P<0.01) and significant upregulation of mRNA and protein expression of the pro-apoptotic gene BAX (P<0.01); Wnt/β-Catenin pathway gene CTNNB1 mRNA and β-Catenin protein levels were highly significantly reduced (P<0.01); In addition, miR-144-5p could target WNT5a to inhibit its expression. Meanwhile, CCK-8 assay results showed that WNT5a promoted the proliferation of GCs, and overexpression of WNT5a significantly upregulated the mRNA and protein levels of the BCL-2 (P<0.01), significantly downregulated the mRNA level (P<0.05) and protein level (P<0.01) of BAX, and the Wnt/β-Catenin pathway gene CTNNB1 mRNA and β-Catenin protein levels were significantly increased (P<0.01). In summary, in GCs, miR-144-5p targets and negatively regulates WNT5a expression, regulates the Wnt/β-Catenin pathway, and then upregulates the BAX/BCL-2 ratio to inhibit granulosa cell proliferation and promote apoptosis, which may have an effect on ovulation and lambing numbers in goats.

The purpose of this study was to clone the microtubule associated protein 1 light chain 3B (LC3B) gene of yak and prepare the polyclonal antibody of yak LC3B, so as to lay a foundation for exploring the role of autophagy in the reproductive physiology of yak. The sequence of yak LC3B gene was obtained by gene cloning. The recombinant protein of yak LC3B was purified by prokaryotic expression and affinity chromatography. The purified protein was used as antigen to immunize 10-month-old Japanese big-ear white rabbits. The polyclonal antibody against LC3B was purified and prepared by Sepharose 4B column chromatography, and the antibody titer was determined by enzyme linked immunosorbent assay (ELISA). Western blot (WB), immunohistochemistry (IHC) and immunofluorescence (IF) were used to detect the expressions of LC3B protein in yak reproductive organs. The results showed that the yak LC3B gene (accession number:OP572227) was successfully cloned. The constructed recombinant plasmid pET-32a-LC3B induced the production of recombinant protein and expressed in inclusion bodies and supernatant. The size of purified yak LC3B recombinant protein was 34 ku (containing Trx and His tags). The rabbit anti-yak LC3B protein serum obtained by immunizing Japanese big-ear white rabbits had antibody titers of 1∶640 000 and 1∶320 000, respectively, with good specificity, which showed that the polyclonal antibody of yak LC3B protein was successfully prepared. The purified antibody was used to detect the expressions of LC3B in the reproductive organs of yaks. It showed that LC3B was expressed in the ovaries, oviducts and uterus of the yak, and recognized LC3B-Ⅰ and LC3B-Ⅱ after lipidation, it was located mainly in the cytoplasm and perinuclear. In conclusion, LC3B polyclonal antibody was prepared successfully by cloning LC3B gene in this study, and the expression of LC3B protein was detected in the reproductive organs. It is stated that the antibody prepared has good specificity and it can be applied to detect LC3B protein in yaks and further study on the levels of autophagy.

The objective of this study was to investigate the effects of supplemental essential oils on gut lesions, CAZy spectrum and eggNOG pathways of intestinal flora in broilers challenged with Clostridium perfringens. A total of 112 1-day-old male Arbor Acre broiler chicks were randomly divided into two groups: Control group(Ctrl) and Essential Oil group(EO). Each group had 7 replicates and each replicate had 8 chicks. The broilers in group Ctrl were fed the basal diet, while the broilers in group EO were fed diet supplemented with 120 mg·kg^-1 of essential oils (comprised of carvacrol and thymol). All broilers were challenged with C. perfringens during d 14 to 20 and were sacrificed on d 21. The overall intestinal injury degree was observed for intestinal lesion score, and the ileal content was collected for metagenomic next-generation sequencing analysis. The results showed that the supplementation of EO significantly alleviated the gut lesions (P<0.05).The abundance of most ileal bacterial genes were increased significantly at the second level and the EC level of the carbohydrate active enzymes (CAZy) database(q<0.05) in which the proportion of glycosyltransferases was the highest. The addition of EO remarkably influenced the composition of homologous protein genomes of ileal bacteria, and the relative abundance of genes were generally reduced (q<0.05) at the second level and the orthologous group(OG) level of the eggNOG database (evolutionary genetics of genes: Non superior Orthologous Groups).In addition, most of the reduced genes were bacterial virulence related genes, such as Catalyzes_ the synthesis of GMP from XMP, DsbA and dTDP-4-dehydrorhamnose 3,5-epimerase. The above results indicated that, dietary supplementation of EO alleviated the gut lesions caused by Clostridium perfringens, which could be related to the regulation of the abundance of carbohydrate active enzymes and virulence-related genes of the intestinal flora.

The present study was aimed to determine the effects of maternal addition with probiotics and synbiotics on muscular fatty acid composition and related gene expression of offspring. A total of 64 pregnant Bama mini-pigs were selected and randomly divided into control (antibiotic-free basal diet), antibiotic (50 g·t^-1 virginiamycin), probiotics (200 mL·d^-1 probiotic mixture), and synbiotics (500 g·t^-1 xylo-oligosaccharide + 200 mL·d^-1 probiotics mixture) groups. The sows were fed with their corresponding experimental diets during pregnancy and lactation. After weaning, two piglets per litter (32 piglets per group) were selected and fed with a basal diet. At 125 days of age, 8 pigs per group were selected and their biceps femoris (BF) and psoas major (PM) muscles were sampled to determine medium- and long-chain fatty acid composition and related gene expressions. Results showed that, compared with the control group, the C20:2 content in BF muscle was significantly increased (P<0.05) and the expression of stearyl coenzyme A desaturase (SCD) and sterol-regulatory element binding protein-1 (SREBP-1) in BF muscle were significantly up-regulated (P<0.05) in the antibiotic group. In the probiotics group, the C17:0 content in BF and PM muscles were significantly decreased (P<0.05), and the expression of hormone sensitive lipase (HSL) in BF muscle and peroxisome proliferator-activated receptor γ (PPARγ) in PM muscle were up-regulated (P<0.05). In the synbiotics group, the content of C18:2n6c, C20:1, and n-6 PUFA in BF muscle were significantly decreased (P<0.05), and the expression of PPARα in PM muscle was significantly up-regulated (P<0.05). In the antibiotic and probiotics groups, the C20:0 content of BF muscle was significantly decreased (P<0.05), while the expression of PPARα in BF muscle, triglyceride lipase (ATGL) and fatty acid binding protein 4 (FABP4) in PM muscle were significantly up-regulated (P<0.05). The expression of carnitine palmityl transferase 1 (CPT-1) and lipoprotein lipase (LPL) in PM muscle were significantly up-regulated (P<0.05) in the probiotics and synbiotics groups. In the antibiotic, probiotics and synbiotics groups, the C18:1n9t content in BF muscle was significantly decreased (P<0.05), and the expression of ATGL, FABP4, and LPL in BF muscle were up-regulated (P<0.05). In conclusion, maternal addition with probiotics and synbiotics could alter offspring’s muscular fatty acid composition by regulating the expression of genes related to lipid metabolism, which are beneficial to improve the nutrition value and flavor of meat. In addition, maternal antibiotic addition could improve offspring’s muscular fatty acid composition.

The A33 and H3L proteins of orthopoxviruse are the two major targets of neutralizing antibodies. In order to detect the immunogenicity and neutralizing activity of A33 and H3L homologous proteins of ectromelia virus (ECTV), we amplified target sequence of EVM135 and EVM085 from the genome of ECTV-Moscow strain, and constructed the pET30a-EVM135 and pET30a-EVM085 recombinant prokaryotic expression plasmids, which were then transformed into Rosetta competent cells and induced by IPTG, the recombinant proteins were purified by Ni-chelating affinity chromatography and renatured by stepwise dialysis, and antiserum were prepared by immunized rabbits. The indirect ELISA detection showed that the antibody titers of EVM135 and EVM085 reached 1∶120 000 and 1∶360 000, respectively. Western blot and immunofluorescence tests revealed that the two polyclonal antibodies had great specificity and reactivity. The neutralization assay of ECTV showed that the titer of anti-EVM135 polyclonal antibody was 1∶16, while the EVM085 was 1∶128. Through the high expression of EVM135 and EVM085 proteins, and subsequent analysis of their immunogenicity and neutralizing activity, a thorough investigation of the pathogenic and immune mechanisms of orthopoxviruses can be conducted. This research can pave the way for the establishment of a diagnostic method, which can contribute to the development of effective prevention and control measures against monkeypox.

The purpose of this study is to investigate the changes of virus populations in the diarrheal feces of piglets in intensive pig farms in Sichuan Province. In 2019, 156 pig diarrheal fecal samples were collected from 15 pig farms in Sichuan Province. Three samples from each farm were mixed into a pool sample, and their total RNA was extracted and subjected to high-throughput sequencing. Sequence assembly was performed using SOAP denovo software, and the virus species and molecular characteristics were studied. The results revealed that the virus population in the fecal samples of diarrheal piglets included 20 viruses from 12 families, mainly Coronaviridae. The main pathogens associated with diarrhea are porcine epidemic diarrhea virus (PEDV), porcine infectious gastroenteritis virus (TGEV) and porcine rotavirus (PoRV). By SOAP denovo software, we assembled and obtained the complete sequences of the PEDV N gene and the partial genomes of TGEV, porcine astrovirus (PAstV-2), Posavirus 1 and PPV-4. The phylogenetic analysis results revealed that PEDV, TGEV, PAstV-2, Posavirus 1 and PPV-4 were more closely related to the reference strains. The above results indicated that the virus species in the feces of piglet diarrhea in Sichuan Province in 2019 were complex and diverse, and that the abundance of Coronaviridae increased, which provided a foundation for the prevention and control of piglet diarrhea in Sichuan Province.

There is no commercially diagnostic kits for swine acute diarrhea syndrome coronavirus (SADS-CoV). To fill the gap, in this study, monoclonal antibodies (MAbs) against the SADS-CoV nucleocapsid (N) protein were prepared and the sequences of MAbs were analyzed. The N protein was expressed using prokaryotic expression system. The purified N protein was used as an antigen to immunize Balb/C mice. After cell fusion, screening, and subcloning, five stable secreting MAbs against N protein were obtained, and designated as 1C10, 4B10, 6G1, 6F3 and 6E8,respectively. The variable region gene sequences of MAbs were obtained by nested PCR. Indirect immunofluorescence assay (IFA) results showed that all MAbs could specifically recognize Huh7 infected with SADS-CoV. ELISA results showed that the five MAbs could react with the purified N protein, while only the MAb 6E8 could react with SADS-CoV. The results of western blot showed that the five MAbs specifically recognized with the purified N protein, as well as the native N protein in cells infected with SADS-CoV. Isotyping revealed that the MAbs 1C10, 4B10, 6G1, and 6F3 were of the IgG1 class, and 6E8 was of the IgG2a class, and which have a kappa light chain. Truncated expression analysis showed that the recognition region of N protein of the five MAbs was 1-142aa. Overall, the MAbs against SADS-CoV N protein were promising a useful tool for development of new immunodiagnostic methods and research N protein function and structure.

The purpose of the study was to establish a rapid clinical diagnostic method with high sensitivity and specificity for porcine epidemic diarrhea virus. Using the cDNA of porcine epidemic diarrhea virus as the template, the target gene was amplified by the combination of recombinant enzyme and polymerase. In this study, primers were designed for the amplification of recombinant enzyme polymerase, the reaction system of RPA was optimized, the specificity and sensitivity of the reaction were tested, and the optimum temperature and time were determined, analysis and identification were carried out by agarose gel electrophoresis and side-stream chromatography. The experimental results showed that the optimal temperature and time for the reaction were 30 ℃, 20 min, and the minimum number of plasmid copies that could be detected was 10² copies·μL^-1 also showed good specificity in the detection of porcine delta coronavirus, transmissible gastroenteritis virus, porcine rotavirus and porcine circovirus. The above results show that by exploring and optimizing the reaction conditions of RPA, a polymerase chain reaction combined with agarose gel electrophoresis (Basic RPA) and lateral flow chromatography (LF-RPA) were developed for the detection of porcine epidemic diarrhea virus, it has shorter detection time, higher sensitivity and lower requirement for instrument, site and detection environment, which provides more choice and more effective technical support for clinical diagnosis and rapid detection of PEDV in the future.

African swine fever (ASF) is a highly infectious disease of pigs, which seriously threatens the development of the global pig industry. The purpose of this study is to screen specific nanobodies against the NP419L protein of ASF virus (ASFV) and evaluate its application of anti-ASFV antibody detection. We prepared the recombinant NP419L protein by E.coli expression system and Ni-NTA affinity chromatography purification. The purified recombinant protein was used to immunize the Bactrian camels. After the fifth immunization, peripheral blood lymphocytes were collected and total RNA was extracted. A phage display library against NP419L protein was constructed by nested PCR, enzyme digestion, ligation and electroporation. Nanobodies against NP419L were obtained through three rounds of screening using phage display screening technology. The eukaryotic expression vectors of nanobodies fused with horseradish peroxidase (HRP) proteins were constructed and transfected into HEK293T cells. The secretory expression of the fusion protein and its binding to NP419L protein were identified by ELISA and indirect immunofluorescence (IFA), and preliminary evaluation of the obtained nanobody fused with HRP proteins in ASF detection using competitive ELISA. The results showed that the NP419L recombinant protein with the expected size of 48 ku was successfully prepared with high purity; a phage display library with a positive rate of 89.5% and a library capacity of 6×10⁸ was constructed; six strains of anti-NP419L protein nanobodies were obtained by screening; the fusion proteins of these six nanobodies with HRP were secretively expressed, the results of ELISA showed that the six fusion proteins specifically bind to the recombinant NP419L protein, one fusion protein was found to bind to NP419L protein expressed with eukaryotic system by IFA, and this fusion protein can be used as a probe for the detection of ASFV antibodies. In the study, six nanobodies against ASFV NP419L protein were successfully screened. The preliminary characterization of its binding protein and its application of antibody detection were also carried out, which provided the basis for the subsequent development of ASF diagnostic technology and the functional study of NP419L protein.

The aim of this study was to establish a specific, sensitive and rapid real-time fluorescence quantitative PCR (FQ-PCR) method for the detection of ASFV-G-ΔI177L. Specific primers/probes were designed for the conserved region of ASFV I117L gene to improve the sensitivity of the detection method and verify the repeatability and specificity of the detection method by optimizing the reaction conditions and procedures. A total of 170 clinical samples were tested and compared with OIE recommended testing methods. The results showed that the specific primer/probe pair based on ASFV I117L gene could amplify 2.2×10¹-2.2×10¹⁰ copies·μL^-1 pMD18-I177L standard plasmid, and the established standard curve R² reached 0.995 7. The minimum detection template concentration was 2.2×10¹copies·μL^-1. The amplification reaction was performed by one-step method, which took 35 min, and the coefficient of variation within and between batches was less than 1.8%. Specific amplification of ASFV I177L gene was detected, but no positive amplification was observed for nucleic acid samples of other 5 common porcine viruses and enzyme-free water. This method was used to detect 170 clinical samples, and it was in good agreement with the method recommended by OIE. This study successfully established a specific, sensitive and rapid ASFV-G-ΔI177L detection method, which provides a good technical support for the clinical monitoring and diagnosis of African swine fever virus.

Swine erysipelas is an infectious disease caused by Erysipelothrix rhusiopathiae which endangers breeding industry. Understanding the spatial and temporal distribution pattern and influencing factors of swine erysipelas in China can provide reference for early warning and prevention and control measures of swine erysipelas in the future. In this study, 31 provincial administrative regions of China were selected as the study area, and the spatial and temporal distribution characteristics of the incidence of erysipelas in China from 2010 to 2020 were analyzed by combining H-P filtering, spatial autocorrelation analysis and spatio-temporal scanning statistics. Meanwhile, MGWR, GWR and OLS model were used to explore the spatial impact of social and climatic factors on the incidence of erysipelas in China. The results show that: In terms of time, the epidemic showed a trend of increasing first and then decreasing, the seasonal index of case numbers was greater than 120% in June-September and less than 90% in January-April. Spatially, there was a significantly spatial positive correlation in the number of cases per year (Moran's I value ranged from 0.127 to 0.295). The first-level agglomerations detected by spatio-temporal scanning during 2010-2014 and 2015-2020 were 5 and 4, respectively. The agglomerations occurred from June to August, and the agglomerations tended to move southward. The results of comparing multiple models show that the MGWR model has the best fitting effect (R² ranged from 0.43 to 0.84). Wind speed, temperature, road density, number of live pigs, and the proportion of rural population can significantly affect swine erysipelas cases to a certain extent, and the influencing factors in different regions have different fluctuation directions and intensities. Our results indicated that the epidemic distribution has obvious aggregation in time and space. The outbreak mainly occurred in the southeastern part of China. Wind speed and rural population ratio are the main factors affecting swine erysipelas cases.

The aim of this study was to explore the rules of the genetic variation and spatio-temporal transmission of the Chinese IBV isolates, all the sequences of N gene from GenBank database were downloaded. Bioinformatic software including MEGA 6.0, RDP 4.95, SimPlot 3.5.1, BEAST v1.10.4, jmodeltest 2.1.7, Tracer v1.7.1, TempEst v1.5.1, FigTree v1.4.3 and SpreaD3 v0.9.7 were respectively used to analyze the characteristics of phylogenetic tree, recombination events, origin,population dynamics and spatio-temporal transmission of the IBV isolates. The phylogenetic tree analysis showed that a total of six genotypes were identified, and the LX4-type was the predominant genotype. The recombination analysis showed that recombination event was found in N gene in one of isolate from Sichuan province. The maximum clade credibi-lity tree showed that the Chinese IBVs were most likely to be originated in Liaoning province in the early 1930s. Bayesian phylogeographic analyses indicated that multiple transmission routes and three epicenters in China were found, including the Northeastern Region (Heilongjiang, Liaoning, and Jilin), the Northern and Eastern Region (Hebei, Shandong and Jiangsu) and the Southern Region (Guangdong and Guangxi). Shandong has been the source of spreads in China. Our study revealed that the IBVs' origin, transmission routes and a recombination event occurred in N gene in China, suggesting that it is necessary to continue to carry out the molecular epidemiology study and strengthen the analysis of the N gene of IBV.

The aim of this study was to explore the changes of different metabolites in duck ileum infected with Clostridium perfringens type A (CpA) by non-targeted metabonomics. The 37-day-old healthy ducklings infected with CpA were selected for the experiment. After the CpA infection was successful, pathological observation and PCR detection were adopted, the metabonomics of duck ileum was detected by liquid chromatography-mass spectrometry (LC-MS) at 66, 90 and 114 h in the control group and CpA-infected group, and the potential differential metabolites and related metabolic pathways were screened and analyzed. Finally, the differential metabolites in the control group and CpA-infected 114 h group were selected for verification. The duck CpA infection model was successfully established by autopsy, pathological observation and pathogenic nucleic acid detection.There are some overlapping ileum samples in PCA, which suggests that there may be the same ionic compounds in the ileum of infected ducks and control ducks. There are 14, 16 and 24 kinds of total differential metabolites in ducks infected at 66, 90 and 114 h, respectively, and there are 13, 14 and 28 enriched metabolic pathways, mainly focusing on arachidonic acid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, tyrosine metabolism, etc. At the same time, the results of differential metabolites verification showed that the change trend of valine and alanine content in ileum after 114 h infection was consistent with the results of non-targeted metabolomics. To sum up, the pathogenic mechanism of CpA infection in ducks may regulate the synthesis of related metabolites by affecting the metabolic pathways of related amino acids.

Heat shock protein 70-b2 (HSP70-b2) of Haemaphysalis flava is a member of the HSP70 family. The research was to evaluate the anticoagulant activity and immunogenicity of HSP70-b2, and to investigate the role of 14-Mer-like peptide of HSP70-b2 from H. flava. The gene of H. flava HSP70-b2 was amplified in vitro by RACE technology. The coding sequence of 14-Mer-like peptide of HSP70-b2 was site-directed mutated to amplify the HSP70-b2^M gene. Prokaryotic expression plasmids were constructed. The soluble recombinant proteins were expressed by prokaryotic expression system and were purified by nickel column chromatography. The anticoagulant activities of rHSP70-b2 and rHSP70-b2^M were evaluated using four clotting assays in vitro. Emulsifying recombinant proteins with complete and incomplete Freund’s adjuvants were subcutaneously immunized in Sprague Dawley (SD) rats. The serum antibody titers were detected by indirect enzyme linked immunosorbent assay (ELISA) to assess the immunogenicity of rHSP70-b2 and rHSP70-b2^M. Results revealed that the length of HSP70-b2 was 2 413 bp, the open reading frame (ORF) of HSP70-b2 was 1 905 bp, and the encoded protein had typical HSP70 protein family tags. The HSP70-b2 gene sequence encoding the 14-Mer-like peptide was successfully mutated into 14-Mer gene sequence encoding the 14-Mer peptide. Compared with the control group, rHSP70-b2 and rHSP70-b2^M had no significant effect on the prothrombin time (PT) and activated partial thromboplastin time (APTT) (P>0.05). However, rHSP70-b2 and rHSP70-b2^M prolonged the thrombin time (TT) and fibrinogen (FIB) coagulation time (P<0.05), which had similar anticoagulant activities (P>0.05). The antibody titers in rat serum increased with the increase of immunization times, and the antibody titers induced rHSP70-b2 was significantly higher than that of rHSP70-b2^M (P<0.01). The results suggested that rHSP70-b2 and rHSP70-b2^M exhibited anticoagulant activities by prolonging the TT and reducing the FIB content in vitro. Both rHSP70-b2 and rHSP70-b2^M had nice immunogenicity and could induce humoral immunity.

The aim of this study was to evaluate the immunoprotective effect of Es-GAPDH, a recombinant protein of Eimeria stiedae, on rabbits and to lay the foundation for the development of a recombinant subunit immunization vaccine for rabbits.The transcript levels of Es-GAPDH at different developmental stages of E.stiedae were analyzed by relative fluorescence quantitative PCR, and Es-GAPDH protein was prokaryotically expressed and purified. Then 42 45-day-old coccidia-free rabbits were randomly divided into 5 groups (unimmunized and unchallenged group, unimmunized and challenged group, Trx-His-S tag-challenged group, Quil-A saponin-challenged group and rEs-GAPDH immunization group). 1 mL PBS, 1 mL PBS, 1 mL PBS containing 100 μg pET-32a and 1 mg Quil-A, 1 mL PBS containing 1 mg Quil-A, 1 mL 100 μg rEs-GAPDH and 1 mg Quil-A were respectively injected subcutaneously via the neck to the rabbits, and were booster immunized with the same dose 14 d after the first immunization. After the secondary immunization, except for the unimmunized and unchallenged group, each rabbit in the other groups was orally infected with 1×10⁴ sporulated oocysts of E.stiedae. The clinical manifestations of each group were observed after the attack. Blood samples were collected and the animals were weighed at regular intervals every week. Twenty-one days after infection, the liver pathological changes were observed by autopsy, and the relative weight gain, feed to gain ratio, liver index, oocyst excretion, biochemical indexes, specific IgG antibodies and cytokines of each experimental group were measured and counted. The results showed that Es-GAPDH was transcribed at all developmental stages of E.stiedae, and there were differences in transcript levels, with the highest transcript level in the sporulating oocyst stage. The immunoprotection test showed that typical symptoms of rabbit hepatic coccidiosis appeared in the unimmunized and challenged group after infection, while rEs-GAPDH immunized group showed no significant symptoms. rEs-GAPDH immunized group showed 87.09% oocyst reduction, and significantly greater relative weight gain than the other three challenged groups (P<0.05), specific IgG antibody levels, cytokine (IFN-γ, IL-2, IL-4, IL-10, TGF-β) levels were significantly different from those of the unimmunized and challenged group (P<0.05), and histopathological sections also showed that the liver tissue was less destroyed and the number of parasites was lower in the immunized group compared with the unimmunized and challenged group. It can be concluded that the recombinant protein rEs-GAPDH of E.stiedae can reduce weight loss and oocyst excretion, trigger cellular and humoral immune responses in the host, have certain immunoprotective effects, and can be used as a candidate antigen for E.stiedae recombinant subunit vaccine.

The aim of this study was to prepare monoclonal antibody specific to chicken CTLA-4, and provide useful tools for research on the poultry immune checkpoint and vaccine development. Baculovirus expression system was used to express chicken CTLA-4 protein in vitro. The monoclonal antibodies against chicken CTLA-4 protein were prepared by fusion between SP2/0 cells and spleen cells from the 8-week-old Balb/c mice immunized with the expressed chicken CTLA-4 protein. The charcterization of monoclonal antibodies were analyzed by indirect immunofluorescence, Western blot and flow cytometry. The recombinant baculovirus expressing CTLA-4 protein in Sf9 insect cells was constructed. Three monoclonal antibodies against chicken CTLA-4 protein were successfully obtained, named as mAb-CTLA4-3D7, mAb-CTLA4-5A4 and mAb-CTLA4-6A12. The subclass of mAb-CTLA4-3D7 was IgG2a and type Lambda; The subclass of mAb-CTLA4-5A4 was IgG3 and type Kappa; The subclass of mAb-CTLA4-6A12 was IgG2a and type Kappa. All three monoclonal antibodies could react with DF-1 cells transfected with pCAGGS-CTLA-4-Flag and Sf9 insect cells infected with rBac-CTLA-4. Among them, mAb-CTLA4-3D7 had a good binding activity with chicken PBMC and could react to CD3⁺T lymphocytes, with the binding rate of about 16%. In this study, monoclonal antibodies against chicken CTLA-4 protein were successfully developed, and their reactivity and biological characteristics were analyzed, which provided a reference for immune checkpoints in the pathogenesis, signaling pathways, vaccine development and other research fields of avian diseases.

To further explore the gene function and construct the dominant epitope vaccine, sequences identification, bioinformatics and transcription analysis of ubiquitin-conjugating enzyme gene family of Echinococcus granulosus sensu lato (EgE2s) were conducted. Based on the genomic data of Echinococcus granulosus published before, the EgE2s gene family was cloned and analyzed by bioinformatics. In addition, the transcriptional levels of EgE2s in protoscolex (PSCs) and 28-day strobilated worms were detected by qRT-PCR. The results showed that, 19 EgE2s genes were cloned and sequenced successfully. CDS sequences among two of them were originally wrong had been corrected and submitted to NCBI to obtain accession number (EgE2L3: MZ277618; EgE2J1: MZ277619). The prediction of tertiary structures and conservative motifs indicated that EgE2s were highly conserved. qRT-PCR showed that the transcriptional levels of EgE2s in PSCs differed from 28-day strobilated worms, EgE2A (P<0.001) and EgE2J1 (P<0.01) were highly expressed in PSCs. The prediction of B cell epitopes showed that the 139-157 region of EgE2N was located outside the conserved motifs of EgE2s. Meanwhile, the T cell epitopes prediction showed that EgE2N had two strong binding sites with human and canine MHC I molecules, respectively, including a common epitope region 104-113. In conclusion, EgE2A and EgE2J1 may play important roles in the growth and development of PSCs. The 139-157 and 104-113 regions of EgE2N may be the target sequences for the development of drugs and vaccines.

The study aimed to explore the succession of intestinal flora diversity, structure and function of giant pandas at different ages. Fresh feces of 16 giant pandas of different ages were collected in Research Center for Qinling Giant Panda, divided into 5 groups and analyze based on 16SrDNA high-throughput sequencing technology and hierarchical clustering method. The results showed that the main bacterial phyla in giant pandas at different ages consisted of Firmicutes (60.44%) and Proteobactera (38.69%), and the relative abundance of Firmicutes increased while Proteobacteria decreased with age; at the genus level, the bacteria consisted mainly of Escherichia Shigella (57.35%) and Clostridium_sensu_stricto_1 (15.17%), while the gender difference has little effect on the intestinal flora structure of giant pandas. There was a significant difference in the Alpha diversity of intestinal flora (P<0.05), showing a trend of first increasing and then decreasing with age, reaching the highest in 18-22 years old. Beta diversity results show that the difference between groups is greater than the difference within groups, and the interpretation of sample differences by different groups has credibility. The dominant bacteria unique to adult giant pandas aged 13-14 years-Weissella (9.975%) is intestinal probiotics. The relative abundance in amino acid transport and metabolism, signal transduction, defense mechanisms and other pathways of 34 year old giant pandas is different from other age groups. Its unique dominant bacteria-Sarcina (53.29%) is conditioned pathogen. To sum up, this study believes that the intestinal flora diversity of giant pandas first increases and then decreases with age, and the abundance and function of common and unique dominant bacteria in different age groups are different, showing a certain succession law with age, suggesting that the corresponding lack of nutrients can be supplemented in the feeding process of giant pandas at different ages to maintain the intestinal microecology balance.

This study aims at investigating the alleviating effect of diazene aceturate (DIZE) on the development of mouse pulmonary fibrosis by endogenously activating Angiotensin-converting enzyme 2 (ACE2). Bleomycin induced pulmonary fibrosis injury model in mice was established for this aim. Twenty-four male ICR mice were randomly divided into control group (CON group), model group (MOD group) and DIZE group (DIZE group). Mice in the two groups other than CON group were injected with bleomycin (5 mg·kg ^-1) intratracheal only once to establish pulmonary fibrosis injury model. One day after establishing the model, mice in DIZE group were given DIZE (15 mg·(kg·d) ^-1) gavage, and mice in CON group and MOD group were given gavage with normal saline at the same dose. After continuous intragastric administration for 28 days, all mice were executed, serum and lung tissue were taken for the following tests: 1) Lung tissue was weighed and lung coefficient was calculated; HE and Masson staining were used to observe the pulmonary histological changes; the content of hydroxyproline in lung tissue was determined by alkaline hydrolysis; 2) The contents of Ang Ⅱ and Ang 1-7 in serum were determined by indirect ELISA; 3) The expression levels of fibrosis related factor genes and proteins in lung tissues were detected by qPCR and Western blot. Results showed that: 1) Compared with CON group, lung coefficient and hydroxyproline content in MOD group increased significantly (P<0.01 & P<0.05). The lung tissue showed obvious pathological changes, mainly manifested as alveolar collapse, inflammatory cell infiltration, alveolar septum congestion and thickening, and large amount of collagen fiber deposition. After DIZE treatment, the lung coefficient and hydroxyproline content decreased significantly (P<0.01 & P<0.05); The degree of pulmonary fibrosis reduced significantly; 2) Compared with CON group, the expression of ACE2 protein in lung tissue of MOD group was down-regulated, and the content of Ang Ⅱ in serum increased significantly (P<0.01), the content of Ang 1-7 decreased significantly (P<0.01); DIZE group showed a reverse on the changes happened in MOD group ; 3) Compared with CON group, the expressions of Col1a1 and Col3a1 genes in the lung tissues of MOD group were significantly up-regulated (P<0.01), α-SMA and TGF-β1 gene and protein expressions were significantly up-regulated (P<0.01), the expression of the characteristic protein E-cadherin in epithelial cells was significantly down-regulated (P<0.01); Compared with the MOD group, the DIZE group mitigated the above changes. After 28 days of one-time intratracheal injection of bleomycin, pulmonary fibrosis injury was successfully induced in mice. The production of Ang Ⅱ and the increased expression of TGF-β1 are the major factors of bleomycin-induced pulmonary fibrosis in mice. DIZE may reduce the high level of Ang Ⅱ by activating the expression of ACE2 and thus has a substantial relieving effect on pulmonary fibrosis in mice.

Environmental cadmium (Cd) is easily enriched in animals and plants, and enter various organisms through the food chain, which in turn, endangers the health of human and animals. The aim of this study is to evaluate the chelation effect of sheep bone collagen peptide (SBCP) with cadmium and determine the protective effects of SBCP against Cd-induced liver injury in chickens. All Hy-line brown chicks were randomly assigned into four groups including control group (basal diet, n=10), Cd group (basal diet with 140 mg·kg^-1 Cd, n=10), SBCP group (basal diet with 3 g·kg^-1 SBCP, n=10) and Cd + SBCP group (basal diet with 140 mg·kg^-1 Cd and 3 g·kg^-1 SBCP, n=10). After 42 days, the cadmium residue level, oxidative stress status, histological changes and mRNA expression levels of mitochondrial apoptosis-related genes in liver were detected. The results showed that no Cd were detected in liver and chest muscle of control group and SBCP group by inductively coupled plasma-mass spectrometry (ICP-MS). Cd in liver and chest muscle of Cd group were 412.57 and 129.47 μg·kg^-1, respectively. Cd in liver and chest muscle of Cd + SBCP group were 216.06 and 49.68 μg·kg^-1, respectively. Meanwhile, compared to control group, the significantly reduced T-SOD and GSH-Px activities, and significantly increased levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and inflammatory cytokines such as IL-2, IL-8, TNF-α, IFN-γ were detected in Cd group. The histopathological results demonstrated that Cd exposure caused disordered arrangement of liver cords, nuclei concentrated necrosis and interstitial lymphocytic infiltration. In addition, Cd exposure significantly increased pro-apoptotic genes bax, cyt C, caspase-3 and caspase-9 expression levels, and significantly reduced the mRNA expression of anti-apoptotic gene bcl-2. However, compared to Cd group, SBCP treatment significantly ameliorated Cd-caused liver injury, reflected by the significantly increased T-SOD and GSH-Px activities, and the significantly decreased levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and inflammatory cytokines. In addition, SBCP treatment improved morphological changes of liver and restored the mRNA expression levels of apoptosis-related genes. Importantly, the results of infrared spectroscopy showed that SBCP chelated with Cd, and produced a new substance, the chelation rate of Cd and SBCP reached 47%. SBCP can reduce cadmium residue by its chelation effect and thus alleviated Cd induced oxidative stress, inflammatory injury and hepatocyte apoptosis. All these results provide theoretical basis that SBCP could be a promising candidate for the prevention of animals poisoning caused by heavy metal.

This study aimed to explore the function of Foxq1, a differentially expressed gene in the skin tissues at hair follicle induction stage (E 66) and cytodifferentiation stage (E 120), in hair follicle stem cells (HFSCs) of Shanbei white cashmere goat. In this study, 6 healthy pregnant Shanbei white cashmere goats with the same age and body weight were selected. And the back skin tissues were collected at E 66 (n=3) and E 120 (n=3) from the fetuses, respectively. The pAd-Foxq1 and pAd-Blank overexpression adenovirus vectors were transfected into HFSCs. EdU, MTT and flow cytometry were used to detect the effects of Foxq1 on the proliferation of HFSCs. qPCR and immunofluorescence assay were used to detect the expression of CTNNB1, a marker of activating Wnt signaling pathway, and the downstream genes of Wnt signaling pathway. The results of qPCR showed that Foxq1 was differentially expressed in the skin tissues of cashmere goats at E 66 and E 120 stages (P<0.001), which was consistent with the sequencing data. Comet tail fluorescence was observed under fluorescence microscope, indicating that Foxq1 adenovirus was successfully packed, and qPCR results showed that the overexpression efficiency of Foxq1 was more than 40 000 times (P<0.000 1). EdU and MTT results showed that the cell viability of hair follicle stem cells was significantly increased with the overexpression of Foxq1 (P<0.05), and the number of EdU positive cells was significantly increased (P<0.01). Flow cytometry results showed that the percentage of S-phase cells was significantly increased with the overexpression of Foxq1. qPCR results showed that the mRNA expressions of CTNNB1, TCF3 and CYCLIND1 genes were significantly increased with the overexpression of Foxq1 (P<0.05). Immunofluorescence result further demonstrated that the protein expression levels of CTNNB1, TCF3 and CYCLIND1 were significantly increased with the overexpression of Foxq1. This study revealed that Foxq1 could activate the Wnt/β-catenin signaling pathway to promote the proliferation of HFSCs, which illustrated the function and molecular mechanism of Foxq1 in the proliferation of HFSCs.

The aim of this study was to construct a PK 15 cell line stably expressing the E165R protein of African swine fever virus (ASFV). ASFV E165R gene was ligated to p3×FLAG-CMV-10 vector by PCR amplification, and then ligated to lentivirus vector by homologous recombination to obtain recombinant plasmid V2-FLAG-E165R. Lentivirus particles were obtained by lentivirus packaging in HEK 293T cells and transduced into PK 15 cells. The polyclonal cells obtained after primary screening with puromycin were then screened by endpoint dilution method to obtain a monoclonal PK 15 cell line stably expressing E165R protein. The constructed PK 15 cell line was identified by PCR amplification, Western blot and indirect immunofluorescence, and the mRNA expression levels of NF-κB, inflammation-related genes IL-6, IL-8 and TNF-α were further detected by RT-qPCR. The results showed that a PK 15 stable cell line overexpressing ASFV E165R protein was successfully constructed. Compared with the control cell line, E165R protein significantly enhanced the gene expression of NF-κB as well as inflammation-related genes P65、IKBa、IL-6, IL-8 and TNF-ɑ. These results will provide biological materials for further research on the effects of ASFV E165R on innate immune signaling pathway.