In order to understand the changes and characteristics of microbial communities during early development of Protosalanx chinensis, samples were collected at five developmental periods, which are the embryonic period (heartbeat stage, XT), the endogenous nutrition period (the first day after hatching, H₁), the mixed nutrition period (the fourth day after hatching, H₄), the open feeding period (the seventh day after hatching, H₇), and the exogenous nutrition period (the tenth day after hatching, H₁₀). Microbial community succession during early development was observed by 16S rRNA gene sequencing technology, especially the characteristics and key microbial genera before and after feeding. Combined with the transcriptome data of the same batch of samples, the microbial genera related to immunity and metabolism were analyzed based on the association network method. The results showed that there were significant differences in β diversity among the different periods at the early developmental stage (P＜0.001). The dominant bacteria in the XT period were Flavobacterium and Chryseobacterium, and the dominant bacterium in the H₁ period was Pseudomonas. The main bacteria in the H₄ period were Flavobacterium and Pseudomonas. The main bacteria in H₇ and H₁₀ periods were Flectobacillus and Pseudomonas. The abundance of Pseudomonas was stable at all developmental periods. Node bacteria such as Flectobacillus were significantly associated with the expression of various immune and metabolic genes. This study has obtained the microbial succession information of the early developmental stage of P. chinensis for the first time, and screened out the dominant bacteria and node bacteria, which will provide references and ideas for the scaled cultivation of P. chinensis fry.

Apolipoprotein A-Ⅰ (Apo A-Ⅰ) plays important roles in the regulation of atherosclerosis, viral infections, lipid metabolism and other aspects. However, there are few studies on chicken Apo A-Ⅰ (chApo A-Ⅰ), and its biological function is not well understood. Based on the bioinformatics analysis of chApo A-Ⅰ, this study further performed the expression and purification of the recombinant protein of chApo A-Ⅰ via the pET-28a prokaryotic expression system. Mouse polyclonal antiserum was prepared by immunizing mice with purified recombinant protein. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titer of the polyclonal antiserum, and Western blot (WB) and indirect immunofluorescence assay (IFA) were used to determine its reactivity. Then, the polyclonal antiserum was further applied for subcellular localization analysis of chApo A-Ⅰ. Bioinformatic analysis revealed that the chApo A-Ⅰ protein contains signal peptide at 1-18 amino acids, which is composed of continuous alpha helix at the N-terminal. Homology analysis of amino acid sequences revealed that the chApo A-Ⅰ protein had the highest homology with turkey and the lowest homology with fish. The polyclonal antibody prepared using successfully expressed and purified recombinant protein His-chApo A-Ⅰ had an ELISA titer above 1×10⁵ and specifically reacted with the eukaryotic expressed chApo A-Ⅰ protein in WB and IFA. Particularly, the antibody can recognize the Apo A-Ⅰ protein in chicken serum, but cannot cross-react with Apo A-Ⅰ proteins in the serums of mice, rabbits, cattle or pigs. This polyclonal antibody was further applied for subcellular localization analysis of full-length chApo A-Ⅰ (chApo A-Ⅰ-FL) and chApo A-Ⅰ without signal peptide (chApo A-Ⅰ-NS). Observed by confocal microscope, it was found that chApo A-Ⅰ-FL protein was mainly localized near the cell membrane, but chApo A-Ⅰ-NS protein was localized in the cytoplasm, and most of them were diffusely distributed. The specific polyclonal antibody and the results of subcellular localization of chApo A-Ⅰ provide a basis for further research on the biological function of Apo A-Ⅰ.

The effects of aboveground parts of Tetrastigma hemsleyanum, T. hemsleyanum leaves (THL), on the growth performance, immune function and intestinal microflora of broilers were evaluated. A total of 240 broilers were randomly divided into four groups: the control group (fed with a basal diet) and low dose group, medium dose group and high dose group (fed with a basal diet supplemented with 1%, 3% and 5% THL powder, and denoted as THL-L, THL-M and THL-H, respectively). The body masses of broilers at 21 and 42 days of age in each group was measured, and the immune organ indexes and the contents of immunoglobulin A (IgA), IgG and IgM in serum and the relative abundance of intestinal microflora in cecum contents of broilers at 42 days of age were determined. The results showed that compared with the control group, the supplementation of THL powder significantly enhanced the average daily feed intake (P＜0.05) and average daily gain (P＜0.05) of broilers at 42 days of age, and the promoting effect of the THL-M group was the best. At the same time, compared with the control group, the thymus index and the bursa of fabricius index of broilers in THL-L and THL-M groups were significantly increased (P＜0.05). The contents of IgA and IgM in serum of broilers in THL-L, THL-M and THL-H groups were significantly higher than those in the control group (P＜0.05). However, IgG content in THL-M and THL-H groups was significantly higher than that in the control group (P＜0.05). The relative abundances of Lachnospiraceae and Clostridiaceae were significantly increased by the supplementation of appropriate amount of THL powder (3%) (P＜0.05). This study indicated that T. hemsleyanum leaf powder could significantly improve the immune function, the intestinal microflora composition and the growth performance of broilers, and the most significant effect was found with supplementation of 3% THL powder.

The aim of this study was to systematically investigate the differences in the soluble expression level of the African swine fever virus (ASFV) CD2v protein by different prokaryotic expression vectors, and the immunoreactivities of the inclusion body and soluble CD2v proteins were compared using clinical anti-ASFV antibody-positive sera. Five prokaryotic expression vectors, namely, pCold-TF, pET28a, pMAL-C6T, pGEX-4T-1 and pET32a, were utilized to express the CD2v protein without the signal peptide and transmembrane region, respectively. The inclusion body CD2v protein expressed by the pET28a vector and the soluble CD2v protein expressed by the pCold-TF vector were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, and the immunoreactivity of the purified proteins was detected by indirect enzyme-linked immunosorbent assay (ELISA). The results showed that the CD2v protein expressed by the pCold-TF vector was soluble mainly, while the CD2v protein expressed by the pMAL-C6T vector was insoluble (inclusion body) and soluble, and the CD2v protein expressed by the other vectors was mainly as inclusion body. The indirect ELISA results for clinical anti-ASFV antibody-positive sera showed that the immunoreactivity of soluble protein was significantly better than that of the inclusion body protein (P＜0.05). The trigger factor (TF) tag of pCold-TF promoted the soluble expression of the CD2v protein, and the immunoreactivity of the expressed protein was greater than that of the inclusion body protein. This study lays the foundation for further immunogenicity research on the CD2v protein and provides a candidate strategy for the soluble expression of other important antigens.

This study aims to use microbial fermentation technology to improve the utilization of soybean residues and to develop a new type of fermented feed with superior quality and competitive price. In this experiment, fermented soybean residues were used to feed 1 day-of-age Xianju chickens, lasting for 42 d. Five groups were set up, including the control group T₁, fed with the basal diets; the antibiotic group T₂, fed with the basal diets supplemented with 40 mg/kg methylene salicylic acid bacitracin; the treatment groups T₃, T₄, and T₅, fed with the basal diets in which 2%, 4%, and 6% soybean meal were replaced by fermented soybean residues, respectively. The results were shown as follows. 1) Compared with the control group T₁, the 42 day-of-age body mass and average daily gain of Xianju chickens were significantly higher in each group, and the feed to gain ratio was significantly lower in T₄ group (P＜0.05). 2) Compared with the control group, the albumin content was extremely significantly higher in T₄ group (P＜0.01), and the superoxide dismutase activity was extremely significantly higher in T₂ and T₅ groups (P＜0.01), while the malondialdehyde content was extremely significantly lower in T₂ group (P＜0.01). 3) Compared with the control group, the apparent digestibility of crude protein and crude fiber was significantly increased in T₄ group (P＜0.05) and the apparent digestibility of crude protein was extremely significantly increased in T₅ group (P＜0.01); the duodenal amylase activity in T₄ group (P＜0.05), the duodenal protease activities in T₄ and T₅ groups (P＜0.01), and the chymotrypsin activities in T₂, T₃, and T₅ groups (P＜0.05) were significantly improved. In conclusion, replacing 4% soybean meal in the basal diets with an equal amount of fermented soybean residues has the best feeding effect, which could significantly promote the growth performance, apparent digestibility of crude protein and crude fiber, and digestive enzyme activities in vivo of Xianju chickens, improve their serum indexes, and has the potential to replace antibiotics.

The purpose of this study was to screen key mRNAs and long non-coding RNAs (lncRNAs) that affect gonadal development in duck embryos and to explain scientifically gonadal development defects in the mulard duck. Three male embryonic gonadal tissues of the mulard duck (BF₁, BF₂, BF₃) and the muscovy duck (F₁, F₂, F₃) were collected to extract RNA and perform high-throughput sequencing, and differential genes and lncRNAs were screened to predict target genes and perform functional annotations. Finally, the sequencing data were verified by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The results showed that a total of 1 109 differentially expressed genes were screened from the gonadal tissues of the mulard duck and the muscovy duck. Compared with the muscovy duck, 857 genes were up-regulated and 252 genes were down-regulated in the mulard duck. Among them, the aldo-keto reductase family 1 member D1 gene (AKR1D1), 17β-hydroxysteroid dehydrogenase 3 gene (17β-HSD3), and cholesterol side-chain cleavage enzyme gene (P450scc) may be related to gonadal differentiation and development in the mulard duck. Meanwhile, 733 significantly differentially expressed lncRNAs were obtained. Compared with the muscovy duck, a total of 660 lncRNAs were significantly up-regulated and 73 lncRNAs were significantly down-regulated in the mulard duck. Target gene prediction analysis showed that a total of 136 down-regulated lncRNAs and 893 up-regulated lncRNAs may be involved in differential gene expression and had potential regulatory relationships, among which, TCONS_00246198 targeted 17β-HSD3, and TCONS_00229529 targeted tetraspanin-2 gene (TSPAN2), suggesting that the above lncRNAs may participate in duck embryonic gonadal development by targeting key genes. The qRT-PCR results showed that the expression levels of differential genes and lncRNAs were consistent with the expression trends in transcriptome sequencing, indicating that the data obtained by high-throughput sequencing are relatively reliable. RNA binding protein immunoprecipitation (RIP) assay results revealed that compared with IgG, the enrichment level of TCONS_00246198 reached 71.51 times. The above results indicate that TCONS_00246198 interacts directly or indirectly with the 17β-HSD3 protein, which means that they may have a targeting relationship. In summary, this study obtains a batch of key mRNAs and lncRNAs that may affect duck embryonic gonadal development, and it is speculated that the differential lncRNAs can regulate the expression of differential genes. This study provides a scientific basis for understanding the differences in duck embryonic gonadal development and the mechanisms of avian gonadal development.

The non-structural protein 3 (Nsp3) of coronavirus, a component of the replication and transcription complex, is one of the potentially important antiviral targets. In this study, the transmembrane region, signal peptide, and epitope of Nsp3 were predicted, and then the region with better antigenicity (50-550 amino acids) of Nsp3 protein in a representative strain (WSU 79-1683) of type Ⅱ feline coronavirus (FCoV) was amplified by polymerase chain reaction. Subsequently, it was subcloned into pCOLD-TF prokaryotic expression vector. Under the low-temperature condition, the recombinant fusion protein His-Nsp3 with a molecular weight of about 130 kDa was successfully induced by isopropylthio-β-D-galactoside. The targeted recombinant protein His-Nsp3 was purified using a non-denaturing nickel affinity column, and the purified protein was used as an antigen to immunize BALB/c mice for preparing Nsp3 polyclonal antiserum. Western blotting (WB) and indirect immunofluorescence assay (IFA) results showed that Nsp3 polyclonal antiserum could specifically recognize Nsp3 protein in FCoV-infected cells. The subcellular localization of Nsp3 protein in FCoV-infected cells was studied by double-labeling IFA combined with laser confocal microscopy. The results showed that Nsp3 protein aggregated in FCoV-infected cells and co-localized with the endoplasmic reticulum. The specific antibody preparation and subcellular localization study of Nsp3 protein provided an important basis for further analysis of the biological function of Nsp3 protein.

This study aimed to investigate the changes in the composition and diversity of the rumen bacterial community in mid-lactation Holstein cows. In trial 1, a total of 20 healthy high-yielding Holstein cows at mid-lactation were continuously fed with the same basal diet for eight weeks and the rumen contents were collected on the 7th day at weeks zero, four, and seven. In trial 2, a total of 30 healthy high-yielding Holstein cows at mid-lactation were supplemented with 20 g/d rumen-protected methionine to the basal diet, and the rumen contents were collected on the 7th day at weeks zero and eight. The rumen contents collected at different time points were analyzed for changes in composition and diversity of the bacterial community, as well as differences in functional stability in both trials. The results showed that there were no significant differences in the alpha diversity, beta diversity, and functional stability of rumen bacteria at different time points (P＞0.05) in both trials. In trial 1, six and one differential bacteria, such as Actinobacteriota, Proteobacteria, Cyanobacteria, Chloroflexi, Synergistota, and Fibrobacterota, as well as Lachnospiraceae_NK3A20_group, were found in highly abundant bacteria at phylum and genus levels, respectively. In trial 2, three and one differential bacteria, such as Actinobacteriota, Spirochaetota, and Elusimicrobiota, as well as unclassified_f_Lachnospiraceae, were found in highly abundant bacteria at phylum and genus levels, respectively. It is indicated that the rumen bacterial community composition of Holstein cows at mid-lactation changed to a limited extent over time under the same feeding conditions, with no significant changes in their diversity and functional stability. Similar results were obtained when supplemented with rumen-protected methionine to the basal diet. In conclusion, the results suggested that the rumen microbial community composition and function of Holstein cows at mid-lactation were relatively stable, and there is no need to specifically consider the changes in rumen bacterial community structure and function along with lactation progress in the related experiments developed during the mid-lactation.

Krait software was used to analyze the distribution characteristics of perfect microsatellites in the whole genome of Protosalanx chinensis, which was published in 2020 with a higher degree of splicing, and to develop polymorphic microsatellite DNA (also known as simple sequence repeat) markers. The results showed that a total of 587 554 perfect microsatellite loci were obtained in the whole genome of P. chinensis, with a total sequence length of 11 803 017 bp, accounting for 2.53% of the whole genome length. Among six repeat types of microsatellites, the number of dinucleotide was the largest (401 585, accounting for 68.35%). In the 99 pairs of primers designed for microsatellite loci， 39 were polymorphic. Among them, 14 microsatellite markers with favorable polymorphism were selected to test one representative population selected from each of the migratory, landlocked, and introduced populations. The results indicated that 14 microsatellite markers with favorable polymorphism could achieve effective amplification in the three representative populations. The genetic diversity and genetic structure of the three populations were analyzed, and it was found that the migratory population (Chongming Island population) had abundant genetic variation (the mean expected heterozygosity is 0.614, and the mean polymorphism information content is 0.576), which could be clustered into a genetic group different from the freshwater populations [including Taihu Lake population (landlocked) and Lianhuan Lake population (introduced)], and there were large genetic distance and extremely high level of genetic differentiation level between them [the genetic differentiation index (F_st) is higher than 0.25, P＜0.05]. The genetic variations between the two freshwater populations (Taihu Lake and Lianhuan Lake populations) were relatively scarce and the genetic distance between them was small. Although there was significant genetic differentiation between them, the genetic differentiation level was relatively low (F_st=0.102, P＜0.05). These results indicate that the migratory population has potential conservation value of germplasm resource, which provide basis for the development of microsatellite markers and construction of genetic maps, and furthermore provide references for the subsequent evaluation of large-scale population germplasm resources of P. chinensis.

To prepare the specific antibodies against nucleocapsid (N) protein of feline coronavirus (FCoV), the full-length cDNA of N gene was amplified from type ⅠFCoV (ZJU1709) and subcloned into pCold TF prokaryotic expression vector. The recombinant N protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) at low temperature and affinity-purified using nickel column. The rabbit polyclonal antiserum was prepared using the purified recombinant N protein as antigen. Finally, the enzyme-linked immunosorbent assay (ELISA), Western blotting (WB) and indirect immunofluorescence assay (IFA) were applied to identify the reactivity of rabbit polyclonal antiserum. The results showed that the soluble recombinant N protein with a molecular weight of about 100 kDa was successfully induced and further purified under non-denaturing condition at a concentration of 1.4 mg/mL. The ELISA titer of the prepared rabbit polyclonal antiserum of N protein can reach 1×10⁶, and it can react with the recombinant Flag-N eukaryotic protein and N protein in FCoV-infected cells by WB and IFA. Cross-reactivity analysis showed that the polyclonal antibody can react with N proteins of alphacoronavirus, including various subtypes of FCoV, as well as canine coronavirus (CCoV), porcine transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV), but failed to react with N proteins of betacoronavirus of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and gammacoronavirus of infectious bronchitis virus (IBV). In summary, the soluble recombinant N protein expressed by using pCold TF prokaryotic system showed strong immunogenicity. The rabbit polyclonal antiserum against N protein of FCoV can recognize N proteins of multiple species from alphacoronavirus, but showed no cross-reactivity with N proteins of betacoronavirus and gammacoronavirus. This antibody facilitates to study the replication mechanism of FCoV and develop efficient methods for antigen or antibody detection.

In order to explore the pathogen of emulsification disease in Chinese mitten crab (Eriocheir sinensis) in Tianjin district, the epidemiological investigation, pathogen isolation, 18S rDNA gene sequence analysis, artificial infection test, and immunohistochemistry (IHC) analysis were carried out for identification of the pathogen, tissue distribution and temporal variation characteristics in this study. The results showed that the main clinical symptoms of naturally infected crabs included slow or almost no response to external stimuli, emulsify hemolymph accumulation in the cephalothorax cavity, opaque and whitish muscles at cephalothorax and joint membrane at the base of claw joints, and white semi-fluid emulsification of the hepatopancreas. The fungal strain P13, isolated from the diseased crab, was identified as Metschnikowia bicuspidata according to the phylogenetic analysis based on 18S rDNA gene sequences. Artificial infection test results showed that P13 could infect healthy Eriocheir sinensis under the laboratory condition, with cumulative mortality of (55.0±2.4)%, causing clinical symptoms similar to those found in the naturally infected crabs, and the Metschnikowia bicuspidata can be isolated from the experimental crabs again. IHC analysis results indicated that P13 infected various tissues of Eriocheir sinensis, including hepatopancreas, hindgut, gill, heart, and muscle, causing pathological damages in different degrees and temporal variation characteristics in the targeted tissues. In conclusion, Metschnikowia bicuspidata was the pathogen of the emulsification disease in Eriocheir sinensis. This study preliminarily revealed the main target tissues of Metschnikowia bicuspidata and the characteristics of tissue distribution and temporal variation, which provides a theoretical basis for clarifying the pathogenesis of Metschnikowia bicuspidata, and offers technical support for the control and prevention of emulsification disease in Eriocheir sinensis.

Developmental pluripotency-associated 2 and 4 (DPPA2/DPPA4) are important regulators of genome activation in 2-cell-like embryonic stem cells (2CL ESCs), meanwhile, they can regulate the proliferation of ESCs. However, the functions of DPPA2/DPPA4 during early bovine embryo development and their potential mechanisms remain unclear. In this study, RNA interference technology combined with embryo microinjection technology were used to knock down DPPA2/DPPA4 in bovine embryos. It was found that, compared with the negative control (NC) group, there was no significant difference in the cleavage rate from 8-cell to 16-cell stages and blastocyst rate at the blastocyst stage, but the total cell number, trophoblast cell number, and inner cell mass (ICM) cell number of blastocysts were significantly decreased in the knockdown (KD) group (P＜0.001, P＜0.05, and P＜0.01). RNA-sequencing analysis further showed that, when compared with the NC group, the core transcription factor RBPJ (recombination signal binding protein gene for immunoglobulin kappa J region) of NOTCH signaling pathway was significantly down-regulated (P＜0.05) in the late morula stage at the KD group. The previous studies demonstrated that RBPJ knockdown would lead to severe damage to blastocyst quality of early bovine embryos. Therefore, the results revealed that DPPA2/DPPA4 can affect cell proliferation by regulating the expression of RBPJ duringbovine preimplantation embryo development.

This study aimed to investigate the effects of FYN binding protein (FYB) gene mutation on lactation performance of Holstein cows in southern China. The lactation traits of 785 Holstein cows from 2014 to 2020 were collected, and the single nucleotide polymorphisms (SNPs) in FYB gene were detected by SNaPshot and the effects of the mutation on structural and physicochemical properties of FYB protein were analyzed. The associations between the mutations and lactation traits and serum biochemical indexes were analyzed by the least square method. The results showed that the allele frequencies of C and T at rs109262355 in FYB gene were 0.36 and 0.64, respectively, which were consistent with Hardy-Weinberg equilibrium (P＞0.05). An extra hydrogen bond with a length of 3.34 ? in the related region of FYB protein caused by the mutation from C to T at rs109262355 was formed, which reduced the hydrophobicity and flexibility of the protein in this region. 305 d milk yield, peak milk yield, and milk fat rate between CC and TT genotype individuals in rs109262355 was significantly different (P＜0.05), with the dominant allele T of milk yield and the dominant allele C of milk fat rate. The serum low-density lipoprotein (LDL) was significantly different between CC and TT genotype individuals (P＜0.05); alkaline phosphatase (ALP) and total cholesterol (TC) in serum were highly significantly different between CC and TT genotype individuals (P＜0.01). The rs109262355 mutation in FYB gene can affect the milk yield and milk fat percentage of dairy cows, which could be used as a candidate genetic marker in lactation traits of Holstein cows in southern China of marker-assisted selection breeding.

The effects of the combination of sea cucumber peptide and Cistanche deserticola (SCPCD) on the hormone regulation and testicular anti-oxidative damage of acute-exercising mice were preliminarily explored in this study. The mice were randomly divided into five groups, including the blank group, the control group, SCPCD-low dose group (SCPCD-L) (0.3 mg/g), SCPCD-medium dose group (SCPCD-M) (0.6 mg/g), and SCPCD-high dose group (SCPCD-H) (2.0 mg/g), and all the mice received continuously intragastric administration by different doses of SCPCD for 42 d. An acute exhaustive swimming (AES) model was conducted, and a mating test was carried out on the 45th day. The sexual behavior ability parameters, hormone levels, and anti-oxidative related indexes were finally measured. The results showed that the optimal mass ratio of sea cucumber peptide and Cistanche deserticola in combination of them was 2∶1. AES led to the energy expenditure and accumulation of metabolites in mice (P＜0.05), but the acute exhaustive swimming time was prolonged and the contents of lactic acid and blood urea nitrogen were reduced by SCPCD treatments of mice. Besides, AES caused decreases in testosterone and estrogen contents, but they were significantly increased in the SCPCD-M and SCPCD-H (P＜0.05). The levels of follicle-stimulating hormone and irisin were significantly reduced (P＜0.01) in SCPCD-M, while the adrenocorticotropic hormone content was increased (P＜0.05). In addition, the sperm malformation rate decreased in SCPCD treatment, while the sperm motility and total antioxidant capacity of testis were significantly improved (P＜0.05). In summary, the SCPCD presented the physiological activities on delaying fatigue, improving testosterone level, and reducing the oxidative damage of sperm and testicular tissue in AES model. These results provide a scientific basis for the development and application of functional foods related to hormone regulation.

In order to investigate the molecular epidemiology and genetic variation of porcine circovirus type 2 (PCV2) in Zhejiang Province, a total of 1 725 clinical samples suspected of PCV2 infection were collected from different regions of Zhejiang Province between 2016 and 2020. All these samples were subjected to pathogenic detection by polymerase chain reaction (PCR). The whole genomes of some positive samples were amplified, cloned and sequenced, and compared with sequences of 16 reference strains in the GenBank for genetic evolution analysis. The results showed that 359 samples were tested positive, with an average positive rate of 20.8%, and the positive rates from 2016 to 2020 were 38.1%, 23.2%, 24.1%, 12.5%, and 10.7%, respectively. The whole genomes of 36 isolates were sequenced. Multiple sequence alignments showed that the nucleotide sequence homologies among 36 PCV2 strains were 94.0%-99.9%, while the homologies were up to 94.7%-98.5% compared with the domestic vaccine strains, and 92.9%-99.8% compared with the reference strains. The phylogenetic analysis showed that these 36 isolates belong to three genotypes, including 11 strains of PCV2a genotype, eight strains of PCV2b genotype and 17 strains of PCV2d genotype. Among these, PCV2d was the predominant genotype. The length of PCV2 ORF2 gene was 705 bp in 16 isolates and 702 bp in 20 isolates. The nucleotide sequence homologies of ORF2 gene among these 36 isolates were 88.7%-100.0%, while the homologies were 90.2%-99.6% compared with the domestic vaccine strains, and 85.0%-100.0% compared with the reference strains. The amino acid sequence analysis of Cap protein indicated a large number of point mutations in addition to the only highly conserved glycosylation site. Moreover, different genotypes had characteristic mutation sites, and the specific mutation sites of dominant genotype PCV2d were mainly concentrated in I⁵³, N⁶⁸, L⁸⁹, T¹²¹, R¹⁶⁸, and I²¹⁵. This study provides a reference for the immune prevention of PCV2 in Zhejiang Province and accumulates effective materials for the development of new vaccines.

In order to explore the relationships between morphometric traits and body mass (BM) of sea cucumber (Apostichopus japonicus), correlation analysis and path analysis were used to obtain the main morphometric traits affecting body mass. Three morphometric traits including body length (BL), body width (BW) and body mass of 366 individuals of F₅ generation breeding population at one year old were measured, and one compound index that combined body length and body width to produce the square root of the length-width (SLW) index was calculated. The results showed that the correlation coefficients between morphometric traits and body mass were highly significant. The stepwise regression analysis showed that morphometric traits (SLW and body width) and body mass were significantly positive correlations (P＜0.001). Based on path coefficients, the multiple linear regression mode equation on the effects of body mass was established as BM=－16.14+6.70SLW+4.31BW. The morphometric traits that directly affected body mass were SLW＞BW, and SLW was the main morphometric trait that significantly affected body mass and its path coefficient was 0.62. Body width had an indirect effect on the body mass via SLW. The fitting relationships between morphometric traits and body mass were expressed as following BM=0.39SLW^2.66 (R²=0.86, P＜0.001), showing negativeallometry tendency. The results provide valuable information and theoretical guidance for A. japonicus breeding programs. Body width should be selected directly and body length simultaneously, when body mass is the breeding target.

The objective of this study was to investigate the effect of dietary supplementation of Zn-L-selenomethionine (Zn-L-SeMet) on lactation performance and metabolic status of dairy cows at peak lactation period. Sixty multiparous Chinese Holstein dairy cows at peak lactation were selected. The cows were divided into 15 blocks with four cows in each block based on the similarity principles of the lactation time, body mass, parity and milk yield. The four cows in the same block were randomly assigned to four treatments: the control group (fed with basal diet, without supplementing Zn-L-SeMet), and Zn-L-SeMet supplementation groups (supplementing Zn-L-SeMet with 0.1, 0.2, 0.3 mg/kg Se, respectively, which was calculated in dry matter mass). The prefeeding period was two weeks, and the experiment lasted for 12 weeks. The results showed that: 1) the energy corrected milk, milk, milk fat, milk protein, milk lactose yields and feed efficiency of dairy cows increased linearly (P0.01) as the supplementation of Zn-L-SeMet increased; 2) the milk fat and lactose contents, and Se concentration in milk were improved linearly or quadratically, and the milk protein content was decreased linearly (P0.05) by administrating increasing amount of Zn-L-SeMet; 3) the activities of glutathione peroxidase and catalase in plasma were improved linearly or quadratically (P0.05) and malondialdehyde content in plasma was decreased linearly or quadratically (P0.01) with the increase of Zn-L-SeMet supplementation. The above results demonstrate that dietary supplementation of Zn-L-SeMet can effectively improve the performance and antioxidant capacity of dairy cows during the peak lactation. It is suggested that Se from Zn-L-SeMet can be effectively used by lactating cows, and Zn-L-SeMet is an effective dietary Se source.

In this study, we analyzed the main chemical components of matcha made from four tea cultivars, established a high-fat diet-induced C57BL/6J obese mouse model, and selected ‘Maolü’ matcha as an experimental dietary supplement with three doses of 0.1%, 0.5% and 1.0%. The results showed that matcha could reduce body mass gain, blood glucose level rise and liver lipid accumulation induced by the high-fat diet without affecting food intake, and the effect was concentration-dependent. Furthermore, we detected the liver function, oxidative stress level and inflammatory response in mice, and the results showed that dietary supplementation of 1.0% matcha significantly inhibited the abnormal increase of activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) induced by a high-fat diet in liver, and increased the activity of antioxidant enzyme. The expression levels of inflammatory factors, Toll-like receptor 4 (TLR4) and MyD88 were also significantly reduced. In conclusion, matcha effectively improves obesity-related fatty liver lesions and inflammation, and its potential mechanism is to inhibit the activation of the TLR4/MyD88 signaling pathway.

The purpose of this study was to elucidate how iron-depleted and iron-saturated forms of lactoferrin (Lf) exert its antibacterial activity in vitro. Bacteria were divided into three groups by treated with different forms of porcine Lf: the control group (without addition of Lf), apolactoferrin (Apo-Lf, iron saturation is 6.9%) group and hololactoferrin (Holo-Lf, iron saturation is 100.0%) group. The results showed that: 1) Native Lf and Apo-Lf significantly inhibited the growth of Escherichia coli K88 and Staphylococcus aureus (P＜0.01), which was not observed in Holo-Lf group. 2) Apo-Lf exerted antibacterial effect by chelating iron, and iron supplementation could eliminate the inhibitory effect of Apo-Lf on E. coli K88. 3) Scanning electron microscope (SEM) results revealed that Apo-Lf damaged the surface membranes of E. coli K88 and S. Aureus. However, Holo-Lf showed no effect on all of tested bacteria. 4) Predicted three-dimensional structures showed that the structures of Apo-Lf and Holo-Lf were markedly different, and the active site of Apo-Lf was more likely to directly interact with bacteria. Taken together, Apo-Lf inhibited the growth of E. coli K88 and S. Aureus by chelating iron or destroying the surface of bacteria.

The aim of this study was to investigate the effect of intranasal adjuvant B subunit of heat-labile enterotoxin (LTB) of the recombinant Escherichia coli in combination with ginsenoside Rg1 in mice and evaluate its biological safety profile. Mice were intranasally immunized with saline, inactivated vaccine of porcine reproductive and respiratory syndrome virus (PRRSV), inactivated vaccine of PRRSV admixed with LTB or Rg1 or LTB-Rg1 for three times, respectively. Immunoglobulin M (IgM), IgG and IgA antibody levels in serum and bronchoalveolar mucus were measured by enzyme-linked immunosorbent assay; mRNA expression levels of cytokines from spleen and lung were detected by real-time fluorescent quantitative polymerase chain reation; body mass and serum biochemical indexes of mice were monitored regularly. The results showed that LTB-Rg1 quickly increased the levels of PRRSV-specific IgM, IgG and IgA antibodies in serum and bronchoalveolar mucosa, prolonged antibody functioning time, and remarkably upregulated the expression levels of Th1?- and Th2-type cytokines when compared with the vaccine alone group. LTB-Rg1 had no effects on body mass, hepatic or renal functions. Therefore, LTB-Rg1 is a safe and potential nasal mucosal immune adjuvant that is worthy of further study.

Mastitis is a frequent disease of dairy cows and results in significant economic losses for dairy producers. In this study, Establishment of inflammation was performed by using different concentrations of lipopolysaccharide (LPS). After that, mammary alveolar cell-T (MAC-T) were randomly cultured in the standard medium (CK), standard medium with 100 μg/mL LPS (LPS₁₀₀), standard medium with 10 mmol/L rapamycin (RAP₁₀), and standard medium with 100 μg/mL LPS＋10 mmol/L RAP (LPS₁₀₀＋RAP₁₀) for 24 h. Cells and culture supernatant were collected at the end of the treatment. The results showed that incubation with LPS for 24 h significantly increased the concentrations of interleukin-8 (IL-8), interleukin-1β (IL-1β) and tumor necrosis factor- α (TNF- α) in MAC-T cells (P＜0.05) without affecting the cell viability and apoptosis. Rapamycin addition individually had no significant effects on baseline concentrations of IL-1β and TNF-α (P＞0.05), but abolished the increased production of inflammatory cytokines stimulated by LPS (P＜0.05). Culturing with LPS increased the phosphorylation and translocation of p65 protein in nuclear factor-κB (NF-κB) signaling pathway, while this elevation was disappeared with the rapamycin supplementation (P＜0.05). Phosphorylation levels of c-Jun N-terminal kinase (JNK) and p38 proteins in mitogen-activated protein kinase (MAPK) signaling pathway were lower in LPS₁₀₀＋RAP₁₀ group than that in LPS₁₀₀ group (P＜0.05). These results indicate that rapamycin alleviates LPS-induces inflammatory response in MAC-T cells through the NF-κB/MAPK pathway, which gives a reference for the therapeutic potential of rapamycin in mastitis.

This experiment aimed to construct a cDNA library of Haemonchus contortus, which could provide the basis for further research on the protein interaction mechanism and screening out the interactive proteins of H. contortus. The L3 stage larvae of H. contortus were used to extract the total RNA by the TriZol method. Besides, the kit was adopted to construct a cDNA library of H. contortus and the cDNA was normalized. Then, the purified cDNA were transformed into yeast Y187 cells together with the linear pGADT7-Rec, and a yeast two-hybrid cDNA library of H. contortus was constructed by homologous recombination. The results showed that a homogenized yeast activation domain (AD) library with the recombinant rate of 100%, average inserted fragment length of 1 000 bp, and working fluid cell density of more than 3.5×10⁷ CFU/mL was constructed. The constructed expression library met the requirements of yeast two-hybrid screening. This library lays a foundation for the molecular mechanism study and vaccine development of H. contortus.

As one of the important members of the NOD-like receptor (NLR) family, NLRC5 plays an important role in the recognition of invading microorganisms, immune signal transmission and innate immune response regulation in vivo, but there are relatively few researches on NLRC5 in poultry. In this study, the Yangzhou goose was selected, and the sequence characteristics of NLRC5 was analyzed, and the expression profile of NLRC5 gene was detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The expression levels of NLRC5 in the liver, spleen and kidney of goslings infected with Salmonella enteritidis at 0, 1, 2 and 4 d post-infection were examined. The results showed that the amino acid sequence of goose NLRC5 was highly conserved in vertebrates, and shared high homology with duck (89.12%). The NLRC5 mRNA was widely expressed in the heart, liver, spleen, lung, kidney, duodenum, gizzard, brain and muscle, and its expression level was the highest in the spleen. In addition, the expressions of NLRC5 in the liver, spleen and kidney of goslings infected with S.enteritidis were significantly upregulated during 1-2 d, and then returned to normal at 4 d post-infection. The above results are helpful to understand the role of NLRC5 in the immune response of goose infected with S. enteritidis.

The effects of Tieguanyin extracts from different fragrance types (Qingxiang, Tgy-Q; Nongxiang, Tgy-N; and Chenxiang, Tgy-C) on the behavior of APP/PS1 double transgenic Alzheimer’s disease (AD) mice were studied, and the mechanisms of oxidative stress and inflammation were analyzed. Forty six-month-old female APP/PS1 mice were equally divided into five groups, which named as model group, donepezil group, Tgy-Q, Tgy-N and Tgy-C groups, and the control group consisted of 10 C57BL/6J mice. The drug was given by oral gavage for 70 consecutive days, and Y-maze test and open field test were performed during administration. At the end of the behavioral study, they were executed, and their plasma and brain tissue were collected, and then the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) content in plasma of mice were determined, and the morphological changes of glial cells were observed by immunohistochemical assay. The spontaneous alternation behavior rates of Tgy-Q, Tgy-N, Tgy-C extract treatments in the Y-maze experiment of mice groups were increased by 17.3%, 17.6% and 19.8% compared with the model group, respectively (P＜0.001), and the learning and memory abilities of mice in each group were significantly improved as compared with the model group. The results of open field test showed that the movement distances and residence time in the central area of mice fed with Tieguanyin extracts were significantly increased. The effective movement distances in the central area of mice was increased by 93.0% in the Tgy-C group (P＜0.001), followed by 82.0% in the Tgy-N group (P＜0.001) and 49.2% in the Tgy-Q group (P＜0.001). The optimal retention time in the central area of mice was 105.0% in the Tgy-C group (P＜0.01), followed by 84.1% in the Tgy-N group (P＜0.01), and 66.8% in the Tgy-Q group (P＜0.05). In the open field test, the spatial exploration ability and anxiety behavior of Tgy-C group were the best in all the treatments. In this experiment, oxidative stress levels of three Tieguanyin treatments were decreased, which are the main mechanisms of Tieguanyin against Alzheimer’s disease of APP/PS1 mice. Compared with the model group, the MDA contents of plasma in the Tgy-Q, Tgy-N, Tgy-C treatments were significantly reduced by 18.44%, 12.97% and 15.11%, respectively, and the SOD activities in the three groups were increased by 15.31%, 13.69% and 18.80%, respectively. Tieguanyin extracts also improved the morphological recovery of microglia and astrocytes in the brain of AD mice. The above research results indicate that three Tieguanyin extracts had a certain alleviating or protective effect on AD mice.

In order to observe the effects of bioactive peptides from black-bone chicken on diabetic mice, the peptide was extracted from the breast muscles of black-bone chicken, through the steps of enzymolysis, centrifugation, ultrafiltration and freeze-drying, whose molecular mass is less than 5 kDa. Then the diabetic mice induced by streptozotocin (STZ) were randomly divided into low-dose, high-dose, drug and positive control groups (n=10), fed with 100 mg/kg bioactive peptide, 400 mg/kg bioactive peptide, 30 mg/kg acarbose and saline placebo, respectively. Besides, the normal control group was set and fed with saline. The initial body mass, fasting blood glucose level, and the final body mass, biochemical parameters after 30 d of continuous feeding were measured. Meanwhile, the pathological changes were observed. The results showed that at the end of the experiment, the body mass of positive control group decreased significantly (P＜0.05), whereas those of the other four groups increased. Compared with the positive control group, the low-dose, high-dose and drug groups had lower final fasting blood glucose levels (high-dose and drug groups, P＜0.05), lower total cholesterol level and low density lipoprotein cholesterol level, but higher high density lipoprotein cholesterol level (high-dose group, P＜0.05); higher superoxide dismutase activity, but lower malondialdehyde content (high-dose group, P＜0.05). In the low-dose, high-dose and drug groups, insulin level and islet area were significantly higher than those in the positive control group (P＜0.05); their liver and renal indexes were higher than those in the normal control group, but the pathological changes such as cell swelling and vacuolation were less than those in the positive control group. In conclusion, bioactive peptides from black-bone chicken have certain effects on hypoglycemia, hypolipidemia and tissue-protection of the pancreas, liver and kidney. In addition, the feeding dose of 400 mg/kg has better effect than that of 100 mg/kg.

There are sporadic immature individuals in the natural migration population of Chinese mitten crab (Eriocheir sinensis). The low temperature environment with high latitude and high altitude can significantly increase the proportion of immature individuals and facilitate collection. In this study, experiments were carried out to verify whether it can grow or reproduce normally in the subsequent third-year-old stage. In the spring of 2018, the two-year-old immature individuals from the Koluke Lake in Qinghai Province were collected as crab larvae and moved back to the ponds in the Yangtze River Basin for breeding, and the third-year-old crab was obtained for the first time. The experimental results showed that: 1) After domestication, the Chinese mitten crab population could successfully molt and grow in the ponds; the growth performance was similar to that of the conventional cultured crab, and the cultivated size was larger than that of the adult crab in the Koluke Lake area. 2) The third-year-old male and female individuals could be sexually mature, and the fecundity was similar to that of conventional crabs, and their offspring could be obtained by raising seedlings in earth ponds. 3) The nutritional compositions of third-year-old crabs cultured in ponds were intact, and some indexes were significantly different from those of conventional crabs, which might be related to the life history of plateaus and lakes. The results also showed that the size of the two-year-old immature crab was smaller than that of the conventional crab of the same age, and the size of the pond-cultured crab in the third-year-old stage was significantly larger than that of the Koluke Lake crab in the same period, indicating the existence of low temperature diapause and compensatory growth in the life history of E. sinensis. To sum up, the life cycle of the third-year-old crab population is more complex, and its nutrient deposition characteristics, mineral element contents, phased growth and accumulated temperature differences can provide new references for exploring the mechanism of low temperature diapause, life cycle prolongation and compensatory growth of the invertebrates.

In order to achieve high-efficiency expression of exogenous protein in sheep lung fibroblasts OAR-L1, and to explore a suitable transfection method for the cell line, the transfection efficiencies of polyethyleneimine (PEI), Lipofectamine^TM 2000 transfection reagent (Lipo 2000), Cytofect^TM fibroblast transfection kit (CF2) and lentivirus mediated cell transfection in the OAR-L1 cells were compared. The results showed that when OAR-L1 cells were transfected with fluorescent plasmids pLentiCMV-EGFP-Puro or pLentiCMV-mCherry-Puro, PEI-, Lipo 2000- and CF2-mediated transfection could be affected by the cell density and the amount of transfection reagents, besides the best transfection efficiency of each method was less than 30%. While the number of fluorescent cells obtained by lentivirus-mediated cell infection was not limited by these two factors, and was significantly higher than the former three methods. The recombinant virus solution could be stored at 4 or －80 ℃ for at least 15 d without decline of the infection efficiency. To co-express two exogenous proteins in the OAR-L1 cells, mixing two packaged lentivirus in equal proportions followed by infection could achieve a higher co-transformation rate. The above results show that lentivirus infection is a cell transfection method that could achieve high expression of exogenous proteins in the OAR-L1 cells, and provide certain references for the selection of transfection methods for other difficult-to-transfect cells.