circRNAs are a subclass of endogenous non-coding RNAs with a closed continuous loop. In recent years, lots of studies have found that circRNAs are ubiquitous in eukaryotes. They not only have certain conserved characteristics and cell specificity, but also play a regulatory role in gene transcription. Regulation of nutrient transport across the placenta is very important for the fetal development, which even determines whether the fetus could be successfully delivered and also affects its future growth and development. Studies in pigs and mice have shown that circRNAs play an important regulatory role in placental nutrient transport and fetal development. This paper reviews the sources, production mechanisms, classification, characteristics and functions of circRNAs. Combined with animal production, the regulation role of circRNAs on nutrient transport of glucose, amino acids and fatty acids in mammalian placenta and fetal development are also reviewed.

Intracellular endoplasmic reticulum stress (ERS) is ubiquitous in the widespread stress response of livestock, poultry and human beings. Unfolded protein response caused by chronic ERS triggers apoptotic signaling pathways. Glucose-regulated protein 94 (GRP94) is a marker protein of ERS, which is essential for the regulation of apoptosis via endoplasmic reticulum signaling. This paper reviews the regulation mechanism of GRP94 and its interaction proteins on hepatocyte apoptosis induced by endoplasmic reticulum stress, and how GRP94 plays a role in liver disease, which will provide a scientific reference for the control on the ERS-inducing production conditions in livestock and the treatment of ERS apoptosis-related liver diseases in clinical medicine.

The aim of this study was to explore the effect and mechanism of FGF10 overexpression on the differentiation of subcutaneous preadipocytes in goats, which was mediated by adenovirus transporters. In this study, the subcutaneous preadipocytes were separated from goat by collagenase digestion, pAdTrack-CMV-FGF10 was constructed successfully through AdEasy adenovirus packaging system, and then was used to infect cells. In morphology, Oil red O staining was used for investigating the effect of FGF10 on adipogenic differentiation. The mRNA levels of adipocyte differentiation marker genes, lipid metabolism related genes, fibroblast growth factor receptors (FGFRs) and Kruppel like factors (KLFs) were measured by quantitative real-time PCR (qPCR). The results showed that the lipid droplets accumulation increased after 2 days of FGF10 overexpression, compared with control group.The mRNA levels of C/EBPα, LPL, ACACA, FGFR1 and FGFR3 significantly increased(P<0.01). The relative expression of FASN and ATGL were significantly up-and down-regulated (P<0.05), respectively. Meanwhile, overexpression of FGF10 significantly up-regulated the mRNA expression of KLF8-10, 16 and 17 genes(P<0.05), while significantly down-regulated the mRNA expression of KLF1, 2, 4 and 15 genes(P<0.05). These results indicate that FGF10 overexpression promotes lipid droplets accumulate possibly by regulating the expression of C/EBPα, LPL, FASN, ACACA, ATGL and partial members of KLFs. This study provides an important data support for elucidating the molecular mechanism for FGF10 controlling fat deposition in different parts of goats.

The purpose of the work was to explore the athletics-related genes of Mongolian horse at genome level. Based on whole-genome sequencing technology, the blood of Mongolian horse was sampled and sequenced. The specific genes and positive selection genes of Mongolian horse were further detected combined with the mammalian genome databases of 7 species including human, bovine, mouse, etc.. The 19 475 annotated protein-coding genes were obtained via data analysis, while 38 specific genes and 45 positive selection genes were screened in Mongolian horse. By GO enrichment, KEGG pathway enrichment analyses and previous studies, the 17 protein-coding genes of positive selection including MAPK13, CCN2, FSCN3, BTG1 and TF, etc. were considered to be probably involved in athletic performance of Mongolian horse. The biological functions of the above 17 protein-coding genes contained actin filament organization, positive regulation of cardiac muscle contraction, calcium ion binding, regulation of intracellular pH, angiogenesis, positive regulation of collagen biosynthetic process, etc.. The mRNA 3'UTR of MAPK13, LOC100057755, EHD4, MICAL3, TMBIM1 and F11R were targeted by 7 miRNAs (such as eca-miR-302c, eca-miR-8974, eca-miR-9077, etc.). The above athletics-related protein-coding genes of positive selection can make people comprehend athletics character of Mongolian horse, while also provide molecular theoretical basis for targeting competitive livestock breeding.

The objective of this research was to clone the histone deacetylases 1 (HDAC1) gene of yak and identify its expression patterns in various tissues and oocytes during meiosis process, respectively, which would provide theoretical basis for studying the role mechanism of HDAC1 in reproductive development of yak. The samples of yak heart, liver, spleen, lung, kidney, small intestine, brain, muscle, ovary and uterus were collected after slaughtering. Total RNAs in different samples were extracted. The coding sequence of HDAC1 gene was cloned by RT-PCR. Its structure and function were analyzed by bioinformatics softwares. The mRNA expression of HDAC1 in different tissues and oocyte during meiosis process was detected by quantitative real-time PCR (qRT-PCR). The results showed that the open reading frame HDAC1 was 1 302 bp, encoding 433 amino acids. It had high homology with cattle, sheep and goat. HDAC1 gene of yak was widely expressed in various tissues, the expression level of which was significantly higher in spleen, kidney and small intestine(P<0.01). The expression level of HDAC1 in MI and MⅡ stages was significantly higher than that in GV stage in oocytes of yak(P<0.01). This study suggested that HDAC1 was involved in meiosis process of oocytes in yak, which provided basic data for studying the role of HDAC1 in reproductive development of yak under the harsh natural environment of the plateau.

The purpose of this study was to explore the protein component information of sika deer antler in different growth stages, and provide a theoretical basis for the exploration of effective active substances in antler. In this study, the proteome characteristic and alterations at 6 time points(10, 20, 40, 60, 130 and 360 d after casting) throughout antler growth cycle were analyzed based on label-free and bioinformatics methods. The result showed that sika deer antler was rich in protein, and the protein content in antler on 10, 20, 40, 60, 130 and 360 d were 65.35, 70.90, 74.00, 82.25, 56.00, 28.02 mg·g^-1, respectively. A total of 636 proteins were successfully identified based on label-free, of which 218 proteins involved in protein synthesis, developmental process, cytoskeleton, transport were found to be significantly differentially expressed in different growth stages of antler. The protein expression in velvet horns in different growth stages has their own characteristics, which lays a theoretical foundation for the screening of pharmacological active components in sika antler horn and the development of velvet-related products.

The aim of this study was to explore the population variation of sustaining fertilization ability of different laying hen breeds, the variation degree in population and the correlations among the related traits, which would provide a theoretical basis for setting up reasonable insemination interval for different breeds and breeding for sustaining fertilization ability in laying hens. Each 158 healthy hens from White Leghorn, Columbia Rock, Barred Rock and Rhode Island Red laying hen breeds of 27 weeks of age were used in this study. They were inseminated for two consecutive days. The eggs were collected for each laying hen for incubating from the 3rd day for 4 weeks. The egg production, daily/weekly fertility rate, average fertility rate in week 1-4, maximum days of fertilization and maximum number of consecutive fertilized eggs were calculated. The correlation coefficients among these traits were analyzed. The results showed that the daily fertilization rate decreased with days, but the extents were different in 4 breeds. The maximum days with the fertility rate greater than 90% were 10, 9, 8 and 11 days for White Leghorn, Columbia Rock, Barred Rock and Rhode Island Red, respectively. Except for day 4 (P=0.01), the daily fertility rate for the first 9 days were not significantly different among breeds (P>0.05); Except for day 13 (P=0.08) and 16 (P=0.19), daily fertility rate from day 10 to 18 was significantly different among breeds (P<0.05); Daily fertility rate from day 19 to 28 was significantly not different among breeds (P>0.05); There was no significant difference among breeds in weekly fertility rate in the first (P>0.05) and 4th week (P>0.05), but in the 2nd (P=0.005) and 3rd week (P=0.04) the difference was significant; There were significant differences in egg production, fertilized eggs number in week 1-4, average fertility rate in week 1-4, maximum days of fertilization, maximum number of consecutive fertilized eggs, fertilized eggs in week 1, 2, 3 and 2-3 among breeds (P<0.05), and all the above traits was the highest in Rhode Island Red hens; These traits also showed great variation among individuals within the breed, and the variation was small in Rhode Island Red and high in Columbia Rock and Barred Rock; The correlations among these traits were positive, and the correlation coefficients were high in fertilized eggs number between week 2 and week 1-4 (r=0.86, P<0.000 1) and between week 2-3 and week 1-4 (r=0.92, P<0.000 1). The results indicated that there were differences in the sustaining fertility ability among different breeds, and the breed-specific artificial insemination interval should be established; There was great variation in these traits within the breed,which indicated that further efforts can be made in breeding; The number of fertilized eggs in week 2 is an ideal trait for evaluating and selection for the sustaining fertilization ability of hens by reducing the labor in trait estimating without losing the accuracy.

The objective of this study was to investigate the regulation of miR-125b-2 on spermatogenesis and sperm maturation in animals and find the target genes of miR-125b-2 and related signaling pathways. In this study, the miR-125b-2 knockout mouse model was established by using CRISPR/Cas9. Three wild type (WT) and three miR-125b-2 knockout (KO) mice were selected and dissected, followed by testis tissues collection. Their total RNA were extracted and mixed into sample pool, respectively. The transcriptomes were analyzed by Illumina HiSeq^TM 4000 RNA-Seq technology. All of Unigene sequences were annotated using the Blast search both in Nr and KEGG database. The differentially expressed genes (DEGs) in testis were conducted into cluster analysis. The results showed that 324 DEGs were identified, and 180 DEGs were annotated. GO analysis results showed that the 180 DEGs were significantly enriched in 80 biological process items including sperm chromatin condensation, 22 cellular component items including mitochondrial inner membrane presequence translocase complex, and 41 molecular function terms including structural constituent of ribosome. KEGG analysis results indicated all annotated DEGs were significantly enriched in 74 signaling pathways, in which the top 3 pathways were RNA transport, mRNA surveillance pathway and selenocompound metabolism. In conclusion, all DEGs identified between WT and KO mice testes were mainly involved in sperm chromatin and mitochondria function. Especially, the expression levels of 3 spermatogenesis related genes, (Papolb, Tssk1 and Slc22a14) were significantly increased. The results provide a basis for further study on the function of miR-125b-2 in regulation of spermatogenesis process.

This study was conducted to investigate the correlation between general nutritive components(GNC), metabolizable energy(ME) and enzyme hydrolysate gross energy(EHGE) of different corn DDGS for ducks. The GNC and EHGE of 11 kinds of corn DDGS with different origins were determined. Based on the EHGE results, five corn DDGS with different EHGE and the energy gradient were selected, and they were combined with corn starch to formulate 5 mixed diets with crude protein(CP) 20%. Eighty-four healthy adult male Pekin ducks were randomly divided into 7 groups, 12 ducks for each group and 2 ducks were reserved. Ducks in the first 5 groups were fed the 5 mixed diets with CP 20%, respectively, ducks in the sixth group was fed corn starch, and ducks in the seventh group was kept starving to determine endogenous energy in order to measure the apparent metabolizable energy(AME), true metabolizable energy(TME) and EHGE of different corn DDGS. The results showed that:1) There was remarkable variation in GNC among different corn DDGS, especially ether extract(EE), ash(Ash) and crude fiber(CF), and their coefficient of variation(CV) were all greater than 15%. Furthermore, the significantly positive correlation was observed between gross energy, EE and EHGE(r=0.651, P<0.05; r=0.769, P<0.01); 2) There was significantly linear positive correlation between EHGE and AME or TME of 5 mixed diets(r=0.998, P<0.000 1; r=0.999, P<0.000 1), and the regression equations were AME_mix=0.780×EHGE_mix+3.096(R²=0.997, P<0.000 1), TME_mix=0.778×EHGE_mix+4.556(R²=0.997, P<0.000 1); 3) The EHGE of 5 corn DDGS were 11.98, 12.73, 13.17, 14.49, 15.24 MJ·kg^-1, AME were 12.41, 12.93, 13.20, 14.37, 14.68 MJ·kg^-1,and TME were 13.77, 14.27, 14.57, 15.73,16.05 MJ·kg^-1, respectively. And there was significantly linear positive correlation between EHGE and AME, TME(r=0.995,P=0.000 4;r=0.996,P=0.000 3), and the regression equations were AME_DDGS=0.728×EHGE_DDGS+3.677(R²=0.991, P=0.000 4), TME_DDGS=0.732×EHGE_DDGS+4.980(R²=0.992, P=0.000 3). There is large variation in GNC among corn DDGS with different origins, especially EE, Ash and CF; There is significantly linear positive correlation between EHGE and ME of corn DDGS and mixed diets for ducks, and ME can be predicted based on EHGE by linear regression model.

The purpose of this study was to investigate the effects of Bacillus Subtilis supplementation into milk replacer on the development of gastrointestinal tract of 7-28 day-old Hu lambs. Thirty 7 day-old Hu male lambs(double lambs) were chosen and divided into 2 groups randomly, fifteen lambs in each group and each lamb as a repeat. Lambs in the 2 groups were fed milk replacer with 0 and 0.2% Bacillus Subtilis, respectively. The test lasted for 21 days. Eight lambs were selected from each group randomly and slaughtered at 28 day-old. The weight and net weight of content in the each stomach chamber and each intestinal segment, the length of the intestinal tracts were measured, and the relative weight, relative length and content distribution were calculated. The tissue samples from fundus gland region of the abomasum, the middle part of duodenum, jejunum and ileum were fixed in paraformaldehyde to analyse the histomorphology, and the apoptotic rate of small intestinal epithelial cells was detected. And the mRNA expression of occludin, ZO-1 and claudin 1 in duodenum, jejunum and ileum mucosa were measured. The results showed that:1) Bacillus Subtilis significantly decreased the duodenum index (percentage to body weight, P=0.037), relative length of jejunum (percentage to intestinal tract length, P=0.009) and villus width of duodenum of 7-28 day-old Hu lambs (P=0.001), and increased relative length of colon (percentage to intestinal tract length, P=0.012), relative weight of jejunum content (percentage to intestinal tract content weight, P=0.003), villus height and crypt depth of ileum (P=0.010, P=0.034); 2) Bacillus Subtilis significantly increased the mRNA expression of ZO-1 protein in duodenum mucosa of lambs (P=0.012), in addition, there was a tendency that Bacillus Subtilis up-regulated mRNA expression of oocludin protein in ileum mucosa (P=0.053) and reduced the apoptotic rate of ileum epithelial cells of lambs (P=0.079); 3) Bacillus Subtilis did not affect the relative weights of stomach,intestine segments (percentage to body weight, percentage to stomach weight, percentage to intestinal tract weight and percentage to gastrointestinal tract weight), relative weights of content of stomach and intestinal tract (percentage to body weight, percentage to stomach content weight, percentage to intestinal tract content weight and percentage to gastrointestinal tract content weight), gut relative lengths (percentage to intestinal tract length), histomorphology of abomasum and intestinal tract, mRNA expression of occludin, ZO-1 and claudin 1 proteins in intestine mucosa, and epithelial cells apoptosis rate of other parts of small intestine (P>0.05). It indicates that Bacillus Subtilis almost does not significantly influence the gastrointestinal tract developments of 7-28 day-old Hu lambs, but improves the barrier function of small intestine and decreases the apoptotic rate of small intestinal epithelial cells, beneficial for maintaining normal structure and function of intestinal tract.

This study aimed to investigate the effect of transient receptor potential melastatin 2 (TRPM2) ion channel on pulmonary microvascular endothelial cell (PMVEC) mitochondrial damage induced by H9N2 influenza virus infection. Basing on the established TRPM2 shRNA PMVEC, in this study, TRPM2 shRNA PMVEC and shRNA PMVEC (control) were infected by the dose of five multiplicity of infection (MOI) H9N2 influenza virus. At 24 and 48 h post-infection, the mitochondria of PMVECs mentioned above was extracted and the protein level was measured by BCA protein assay kit; The SOD activity, GSH level, NOS activity, Na⁺-K⁺-ATPase and mitochondrial complex Ⅳ were measured according to the manufacturer's instructions;The mitochondrial membrane potential change (using mitochondrial membrane potential assay kit with JC-1) and apoptosis (using annexin V-FITC/PI apoptosis detection kit) of PMVEC were observed by laser scanning confocal microscope. The results showed that the activity of SOD, Na⁺-K⁺-ATPase and mitochondria complex Ⅳ and the level of GSH in TRPM2-shRNA PMVEC were significantly higher than those of shRNA PMVEC (control) after H9N2 influenza virus infection (P<0.05 or P<0.01); However, the NOS activity was dramatically lowered than that of control PMVEC (P<0.01). Moreover, the observation of electron microscope showed that the damage of organelles of TRPM2-shRNA PMVEC was not as serious as that of control shRNA PMVEC; Furthermore, the mitochondrial membrane potential level also was higher than that of control shRNA PMVEC. The apoptosis of TRPM2 shRNA PMVEC detected by annexin V-FITC/PI staining showed that there were less PMVECs stained by green fluorescence in cytomembrane and red fluorescence in nucleus than that of control shRNA PMVEC. The results demonstrated that gene silencing of TRPM2 by shRNA alleviated effectively SOD, GSH, Na⁺-K⁺-ATPase, mitochondrial complex Ⅳ in PMVEC infected by H9N2 virus, and further to prevent the mitochondrial damage and apoptosis of PMVEC.

To investigate the molecular mechanisms of apoptosis in goat bronchial epithelial cells infected with CPIV3 in vitro, the cells were inoculated with CPIV3 JS2013 strain that isolated from the previous study. The cultural supernatants of the cells were harvested at 12,24,36,48,72 and 96 h post inoculation(hpi), then qRT-PCR and TCID₅₀ assays were conducted to determine CPIV3 proliferation levels. The apoptosis was examined by Annein V-FITC/PI double staining cell apoptosis detection kit; and relevant indicators were detected by Caspase-3, Caspase-8 and Caspase-9 activity detection kit, qRT-PCR assay, and Western blot. The results showed that virus titers increased from 10^1.80 to 10^4.50 TCID₅₀·mL^-1 at 96 hpi. Observation with inverted microscope showed that CPIV3-infected cells had typical CPE, including shrink and disrupted. Flow cytometry analysis of apoptosis showed that the number of apoptotic cells increased to 19.66% at 48 hpi. Caspases activity assay demonstrated that CPIV3 infection increased the activation of Caspase-3, Caspase-8 and Caspase-9. Meanwhile, the expression levels of intrinsic pathway and extrinsic apoptotic pathway mRNA were increased. In addition, cleaved Caspase-3 was activated in CPIV3-infected cells. These findings indicate that CPIV3 infection could induce apoptosis and the activation of Caspase played important role in apoptosis.

In order to illuminate the cause for neurological sign and death in suckling piglets at a Bartha-K61-vaccinated pig farm in Hebei province, one wild stain of pseudorabies virus (PRV), named HBXT-2018, was isolated from the brain tissues of sick piglets and identified by PCR, Immunoperoxidase monolayer assay (IPMA), animal experiment and gene sequencing in this study. Experimental rabbits were subcutaneously injected with HBXT-2018 strain of 10⁵TCID₅₀, and they started to bite the inoculated sites at 24 h and all of rabbits died within 44-68 h. Neutralizing antibody titer of anti-PRV strain Bartha-K61 serum was lower than the dilution of 1:8, whereas that of anti-PRV variant serum was 1:58.9. Compared with Bartha strain, gB and gC nucleotide and amino acid sequences of HBXT-2018 had various mutations including single or continuous several base-pair or amino acid residue substitutions, insertion and deletion, and predicted antigenic epitopes changes happened in the amino acid sequences of gB and gC. Phylogenetic analysis based on gB, gC and TK showed that HBXT-2018 clustered in the same branch with those strains prevalent in China especially since 2011 whereas it was located at a different branch from Bartha strain and other PRVs in Europe and America. The results indicate that HBXT-2018 isolated in this study is a virulent strain with the same genetic characteristics as the PRV variants currently prevalent in China while it has a remarkably distant evolutionary relationship with Bartha strain. Anti-Bartha-K61 strain serum did not neutralize this isolate well, which suggests that the mutations in gB and gC might be related to the viral immune escape causing piglet pseudorabies in this pig farm.

To quantitatively detect the positive and negative strand RNAs of bovine viral diarrhea virus (BVDV) during intracellular replication, the specific reverse transcription primers with non-viral nucleotide sequences for distinguishing the positive and negative strand RNAs of BVDV from each other and primers for real-time PCR were designed using DNAStar and Primer express 3.0 software. Primer pairs composed of real-time PCR primers and non-viral nucleotide sequences were used for the establishment of strand specific reverse transcription real-time quantitative PCR (ssRT-qPCR). The sensitivity, reproducibility and specificity of the ssRT-qPCR were evaluated. The dynamic changes of the positive and negative strand RNAs of BVDV during intracellular replication were described by the established ssRT-qPCR. Results were as follows:The standard curve of ss(+)RT-qPCR and ss(-)RT-qPCR had good linear relationships in the range of 10²-10⁷ copies of template with the linear correlation coefficients up to 0.998 1 and 0.995 3, respectively. Sensitivity test showed that the lowest detection limit of ss(+)RT-qPCR was 10 copies of template, while that of ss(-)RT-qPCR was 100 copies of template. The ssRT-qPCR was reproducibility with less than 1% of the coefficient of valuation. There were no cross-reactions with bovine parainfluenza virus type 3, infectious bovine rhinotracheitis virus, and bovine respiratory syncytial virus. The dynamic changes of the positive and negative strand RNAs of BVDV during replication in MDBK cells infected with different multiplicity of infection were analyzed using ssRT-qPCR. The results showed that the positive and negative strand RNAs of BVDV increased firstly, then decreased, and then gradually increased in MDBK cells inoculated with BVDV at 10 and 0.1 MOI. Meanwhile, the dynamic change of positive and negative strand RNAs of BVDV was different in MDBK cells infected with BVDV at 1 MOI which the overall upward trend. The positive and negative strand RNAs of BVDV eventually reached a plateau in MDBK cells inoculated with BVDV at 10 and 1 MOI after 36 hours. However, the positive and negative strand RNAs of BVDV eventually reached a plateau in MDBK cells inoculated with BVDV at 0.1 MOI after 24 hours. The applicability of ssRT-qPCR was further validated by BVDV chain specificity test. ssRT-qPCR of BVDV was established in this study and the dynamic changes of positive and negative strand RNAs of BVDV during intracellular replication were described. This study provided a research tool for the researches of the antiviral mechanisms of host proteins and replication and regulation mechanisms of BVDV.

At present, the mechanism of how the host cell responds to Eimeria tenella infection is highly limited. In our previous study, isobaric tags for relative and absolute quantitation were used to study the proteomics changes in host cells after infection with E. tenella sporozoites. In this study, one of the up-regulated proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was selected to study its effect on the invasion of Eimeria tenella sporozoites. Gallus gallus GAPDH gene was cloned, and the prokaryotic recombinant expression plasmid pGEX-4T-1-GAPDH was constructed. The recombinant GAPDH protein (rGAPDH) was expressed and identified by Western blot. Expression of GAPDH in DF-1 cells during E. tenella sporozoites infection was detected by q-PCR, Western blot and immunohistochemistry. The effect of GAPDH polyclonal antibody on sporozoites invasion of cells was detected by antibody inhibition assay. Results were as follows:A 1 123-bp GAPDH was cloned and the rGAPDH was recognized as a~61.7 kDa protein by Western blot. The expression of GAPDH in E. tenella sporozoites-infected DF-1 cells were up-regulated by q-PCR and immunohistochemistry detection. The antibody inhibition assay showed that antibodies against GAPDH at 50, 100, 200, 300 and 400 μg·mL^-1 could significantly inhibit sporozoite invasion compared with the same does of normal rabbit IgG. These results show that E. tenella sporozoites infection could induce the up-regulation of host GAPDH, and host GAPDH plays an active role in Eimeria invasion.

The human amniotic membrane, type Ⅰ collagen were used as the scaffold, and the hair follicle stem cells and dermal cells from goat were used as the seed cells to construct the tissue engineered skin which would be have skin appendages such as hair follicles and sweat glands. Tissue engineered skin was constructed in vitro, and then was transplanted into goats to observe and test its repairing effect on damaged skin(as experimental group,and the transplantation of amniotic membrane instead of the tissue engineered skin was set as control). The results showed that hair follicle was formed when the tissue engineered skin was cultured for 25 days in vitro and the epidermal cells were keratinized by the scanning electron microscopy. The scar area was measured at 30, 60, and 90 days after transplantation. Results showed that the scar area in the experimental group was significantly lower than the control (P<0.01). When transplanted for 270 days, histological section of the transplanted skin showed that the dermal, epidermal and the hair follicles formed in the experiment group, and the control group were just connective tissues. In conclusion, the tissue engineering skin with hair follicles was successfully constructed using goat hair follicle stem cells and dermal cells as seed cells.

In order to provide a basis for systematically studying the mechanism of Staphylococcus aureus infection of bovine mammary epithelial cells, the damages of producing panton-valenine leukocidin (PVL) S. aureus ATCC49775, pvl-knockout mutant △pvl 49775, complemented mutant C-△pvl 49775 and recombinant PVL (rPVL) in vitro on bovine mammary epithelial cells were studied. Recombinant plasmids of S. aureus LukS-PV and LukF-PV were transformed into E. coli BL21, respectively. After expression and purification, they were identified by Western blot. The effects of ATCC49775, △pvl 49775, C-△pvl 49775 and rPVL on the apoptosis of bovine mammary epithelial cells were detected by Live/Dead cell fluorescence staining and Annexin V-FITC/PI double staining. The recombinant expression vectors pET28a-LukS-PV and pET28a-LukF-PV were successfully constructed. The concentration of LukS-PV obtained after induction and purification was 0.6 mg·mL^-1, and the concentration of LukF-PV was 1.15 mg·mL^-1. rPVL mainly induced apoptosis (P<0.01). Both S. aureus ATCC49775, △pvl 49775 and C-△pvl 49775 induced significant apoptosis and necrosis on bovine mammary epithelial cells, but ATCC49775 and C-△pvl 49775 induced worse (P<0.05). The results showed that rPVL of prokaryotic expression had a strong toxicity on bovine mammary epithelial cells. The pvl-knockout mutant △pvl 49775 induced the apoptosis and necrosis of the bovine mammary epithelial cells low than the pvl positive strain ATCC49775 and C-△pvl 49775. Our study indicates that the PVL plays an important role in S. aureus infected bovine mammary epithelial cells.

In this study, a recombinant canine serum albumin-canine interferon alpha 2(albumin-CaIFN-α2) with antiviral activity were expressed by a Bac-to-Bac baculovirus expression system. The gene HBM-Alb-CaIFN-α2, encoding canine serum albumin and the mature protein gene of canine interferon alpha 2 and linked with a honeybee signal peptide (HBM), was inserted into the pFastBac1 vector, and then transformed into DH10Bac competent cells. The shuttle plasmid rBac-HBM-Alb-CaIFN-α2 was obtained after three times of blue and white spots screening and purification, and transfected into Sf9 cell. Cytopathic changes could be observed 72 h later. Under infection with the third generation recombinant baculovirus rBac-HBM-Alb-CaIFN-α2, cells and supernatant were harvested at 3 days after infection. Indirect immunofluorescence (IFA) results showed that infected cells could show green fluorescence. Western blot analysis showed that there were about 90 kDa expression products in culture supernatant, indicating that secretory expression was consistent with the expected results. The MDCK/VSV system was used to detect the antiviral activity of recombinant Alb-CaIFN-α2,the antiviral activity was 1.70×10⁶ U·mL^-1. This study laid a foundation for further study on novel interferon products for dogs.

In order to analyze the physiological function of submandibular gland of ruminant and provide information about histomorphology, we compared the histologic structure and the characteristic expression of EGF, EGFR and eNOS between Tibetan sheep and Big-tail sheep living in Tibet Plateau pastoral areas in this study. The tissue structure of submandibular gland and the localization and distribution of EGF, EGFR and eNOS in Tibetan sheep and Big-tail sheep were observed by special staining method, immunohistochemical SP method and immunofluorescence method. Results were as follows:The proportion of serous acinus in Tibetan sheep submandibular gland was larger than that in Big-tail sheep. The AB, PAS and AB-PAS staining were mainly positive in mucinous acinus and mixed gonadal vesicle but not in serous acinus. The expression of EGF in striated tube, intercalated duct epithelial cells and serous acinus of Tibetan sheep were significantly lower than that of Big-tail sheep, and the positive expression of EGFR in submandibular gland of Tibetan sheep was higher than that of Big-tail sheep. the eNOS was positive in serous acinus and striated duct in both of the two kinds of sheep. However, EGF, EGFR and eNOS were expressed negative in mucinous acinus. Statistical analysis showed that the expression levels of EGF and eNOS in Tibetan sheep were significantly lower than that in Big-tail sheep (P<0.01), while the EGFR in Tibetan sheep was significantly greater than that in Big-tail sheep in serous acinar striated tube basement membrane (P<0.05). Taken together, we suggest that the submandibular gland in Tibetan sheep mainly contain serous acinus and strongly positive expression of EGF in submandibular gland of both two kinds of sheep in striated duct basilar membrane, indicating that EGF is mainly secreted in striated duct and the binding sites of EGF and EGFR are mainly in striated duct basilar membrane. It plays an important role in proliferation and secretion of serous acinar cells. Furthermore, the eNOS showing strongly positive expression in both serous cells and striatum epithelial cells may be related to stress reaction under cold and low oxygen environment.

The effects of different transport time on pathological damage in the heart of goats (Capra hircus) were investigated with HE staining and transmission electron microscopy technique, and the expression of heat shock protein were analyzed with immunohistochemistry, Western blot and ELISA. The changes of microstructure, ultrastructure, localization and expression of HSP70, HSP27 and HSP90 in goat hearts under 2 and 6 hours of road transport stress were analyzed. The results showed that the myocardial fibers of goats in transport stress group exhibited diffuse granular degeneration of varying degrees, the intracellular mitochondria were swollen and broken, the edema, hemorrhage and inflammatory cell infiltration were observed in intercellular substance, capillaries were dilated and congested. And the lesion in the group transported for six hours was more serious. HSP70, HSP27 and HSP90 were mainly expressed in the cytoplasm of cardiac myocytes. HSP27 and HSP90 were also expressed in the nucleus of cardiac myocytes. This three proteins were unevenly distributed in myocardial fibers and myocardial cells of different regions. Compared with the control group, the expression levels of HSP27 protein in the heart of goats in group transported for two hours increased significantly, while the expression level of HSP27 after six-hour transportation stress decreased. Compared with the control group, the expression level of HSP70 and HSP90 protein in the groups transported for 2 and 6 hours increased significantly or increased extremely significantly. In conclusion, the pathological damage of cardiac tissue cells occurred in goats under transport stress, and the acute degeneration of cardiac cells was very obvious. The degeneration was more serious after six-hour transportation stress. The expression levels of HSP70, HSP27 and HSP90 proteins changed significantly after transport, which was related to the stress injury of heart.

In order to study the effects of dihydroartemisinin (DHA) on oxidative stress in weaned piglets, a model of oxidative stress was established by injection of lipopolysaccharide (LPS). Thirty healthy weaned piglets were randomly divided into control group (CON), model group (LPS) and treatment group (LD, LPS + DHA). Each group had 10 replicates. CON group and LPS group were fed with basal diet, LD group was fed with basal diet + 80 mg·kg^-1 DHA. Each group was fed with corresponding diet for 21 days. LPS group and LD group were intraperitoneally injected with 100 μg·kg^-1 LPS at 4 hours before slaughter. CON group was injected with the same dose of sterile saline. Compared with the CON group, the contents of malondialdehyde (MDA), protein carbonyl (PC), 8-hydroxy-2-deoxyguanosine (8-OHdG) and hydrogen peroxide (H₂O₂) in the liver of LPS group were significantly increased (P<0.05). Meanwhile, the activities of total antioxidant capacity (T-AOC), catalase (CAT), glutathione peroxidase (GPx) and the contents of reduced glutathione (GSH) in the liver of LPS group were significantly decreased (P<0.05). Compared with LPS group, the contents of MDA, PC, 8-OHdG and H₂O₂ in LD group decreased significantly (P<0.05), and the activities of CAT, T-AOC and GPx increased significantly (P<0.05). In addition, the expression of nuclear factor E2-related factor 2 (Nrf2) and CAT and the protein expression of Nrf2 and HO-1 in liver of LPS piglets were significantly lower than those of CON group (P<0.05). DHA treatment significantly reversed the above changes induced by LPS (P<0.05). The results showed that DHA could alleviate oxidative stress in weaned piglets induced by LPS by regulating Nrf2/ARE signaling pathway, which provided a theoretical basis for reducing oxidative stress in livestock production.

This study was conducted to explore the effects of Yujin powder (YJP) on SIgA and inflammatory cytokines in ileal and colonic mucosa of Large Intestine Dampness-heat Syndrome (LIDHS) rat. Forty Wistar rats were randomly divided into 4 groups:normal control, LIDHS model, self-healing and YJP treatment group, with 10 rats in each group. The rat model of LIDHS was established through setting such complex factors as high-sugar and high-fat diet, improper diet, high temperature and high humidity environment, drinking and intraperitoneal injection of Escherichia coil to imitate the inducing conditions of LIDHS. After the model being successfully established, the rats of YJP treatment group were treated with YJP for 5 days. And then we collected ileum and colon, made histopathological slices and observed the pathological changes of ileum and colon. Mucous SIgA and tissue TNF-α, IL-1β, IL-6, IL-10, IL-17 and IL-23 in ileum and colon were detected by ELISA method. The results showed that in model rats, the ileal and colonic mucosal epithelium cells showed degeneration, necrosis and exfoliation, the integrality of epithelium was destroyed; and there were inflammatory cells infiltration. In YJP group, mucosal epithelium regenerated, the integrality was recovered, the continuity was good, epithelium cells have a regular arrangement, and inflammatory cells infiltration obviously decreased. The contents of mucous SIgA in ileum and colon in LIDHS model group were significantly higher than those in normal group (P<0.05); those in YJP treatment group were both significantly lower than those in self-healing group (P<0.05). The contents of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-17 and IL-23 in ileum and colon tissue were significantly or extremely significantly higher than those in normal group (P<0.05 or P<0.01). The contents of anti-inflammatory cytokine IL-10 were significantly or extremely significantly lower than those in normal group (P<0.05 or P<0.01). The contents of above inflammatory cytokines in ileum tissue in YJP treatment group were significantly different from those in self-healing group (P<0.05); the contents of TNF-α, IL-10 and IL-23 in colon were significantly different from those in self-healing group (P<0.05); the contents of IL-1β, IL-6 and IL-17 were extremely significantly different from those in self-healing group (P<0.01). It is demonstrated that ileal and colonic mucosal immune homeostasis of LIDHS model rat was destroyed, which manifested as hypersecretion of SIgA and secretion disorder of inflammatory cytokines. The treatment of YJP could back-regulate this phenomenon.

The objective of this study was to investigate the effects of different levels of yeast culture (YC) supplementation in high-concentration diet on nutrients apparent digestibility and rumen fermentation parameters of fattening Hu sheep. A total of 120 healthy male Hu sheep with an average body weight of 50 kg (4-5 months old) were randomly assigned into 4 groups (n=30) balanced on body weight. Sheep in control group (CON) were offered a basal diet (concentrate:forage=75:25) without YC supplementation, sheep in 3 experimental groups as group A, B and C were fed the basal diet supplemented with 10, 20 and 40 g·d^-1 YC, respectively. Each group had 5 replicates with 6 sheep. The whole experiment lasted 37 d with an adaptation period (7 d) and experiment period (30 d). At the end of the experiment, 5 sheep were selected from each group for digestive metabolism experiment for determination of apparent digestibility and rumen fermentation parameters. The results showed as follows:1)Group A and B had a tendency to increase dry matter intake(P=0.064); The apparent digestibility of DM and OM in group A, B and C were significantly higher than that in CON group(P<0.05), but there were no significant differences among group A, B and C (P>0.05); The apparent digestibility of CP in group A and B were significantly higher than that in CON group(P<0.05); In addition, the apparent digestibility of NDF and ADF in group A were significantly higher than that in CON group(P<0.05). 2) GE and ME intakes in group A, B and C were significantly higher than that in CON group(P<0.05);The metabolizability of GE in group A and B were significantly higher than that in CON group (P<0.05). 3) Apparent digestible nitrogen in group A, B and C were significantly higher than that in CON group(P<0.05), but there were no significant differences among group A, B and C (P>0.05);There were no significant differences in retained nitrogen, utilization rate and biological value of nitrogen among all groups (P>0.05). 4)There were no significant differences in rumen pH, TVFA, acetate, propionate, butyrate and acetate/propionate among all groups (P>0.05); NH₃-N concentration in group A and C were significantly higher than that in CON group (P<0.05). The results indicate that under the conditions of this experiment, the supplementation of 10 g·d^-1YC can improve the apparent digestibility of nutrients, energy and nitrogen utilization of fattening Hu sheep, but has no obvious effect in improving the rumen environment; The higher doses of YC did not show additional positive effects.

To quickly detect the content of lactic acid bacteria in fermented feed, a new method was established by combining ethidium bromide monoazide(EMA) with q-PCR technology, and the factors affecting the effects of EMA, such as the EMA concentration, the action time and the illumination time, were investigated in this experiment. The known number of lactic acid bacteria was treated with EMA under the experimental conditions and then the bacterial DNA was extracted. Finally, the standard curve between the Ct value and the number of lactic acid bacteria was established following q-PCR method. The unknown number of lactic acid bacteria can be calculated by replacing the Ct value into the standard curve equation. The results showed that EMA exposure at 3-4 μg·mL^-1 concentration for 10 minutes could effectively inhibit the DNA amplification of the dead bacteria and eliminate the influence of DNA amplification on Ct value. By measuring 8 parts of fermented feed, the results have a high degree of conformity with the plate count. Therefore, it is feasible to quickly and accurately detect the quantity of viable lactic acid bacteria in fermented feed with EMA-qPCR.

The aim of this study was to predict candidate genes for bovine microRNA-223 (bta-miR-223), and to perform protein interaction analysis and signal pathway enrichment analysis to construct a regulatory network in which it may participate. This study was conducted to investigate the relationship between mastitis resistance and bta-miR-223 by predicting the candidate target genes and analyzing the associated protein interaction, signal pathway enrichment and the regulatory networks. Targetscan and miRWalk were used to predict its candidate target genes. Protein association analysis and KEGG pathway analysis were performed using STRING, and DAVID, respectively. Cytoscape software was used to visualize the regulatory network in which the bta-miR-223 candidate target genes may be involved. The results indicated that there exist protein interactions among candidate target genes, for example FBXO30 and SMURF2, FBXW7 and UBA2, etc. In addition, the reactome pathways they participate in include antigen processing, the immune system, the innate immune system, and neutrophil degranulation. The results of KEGG pathway enrichment analysis showed that these candidate target genes are involved in epithelial cell bacterial invasion, endocytosis, PI3K-Akt, TGF-β, AMPK and other signaling pathways. The candidate target genes of bta-miR-223, FBXO30, SMURF2, FBXW7, UBA2, etc., may participate in antigen processing, innate immune system and neutrophil degranulation. Moreover, they may play an important regulatory role through bacterial invasion of epithelial cells, endocytosis, and PI3K-Akt signaling pathway.