According to the sources, polysaccharides can be divided into plant polysaccharides, animal polysaccharides and microbial polysaccharides. They not only have the characteristics of high efficiency, low toxicity and wide sources, but also have various physiological functions, such as regulating immune function, anti-tumor, anti-virus, anti-oxidation, anti-diabetes, anti-bacterial and so on. At present, people have been successfully extracted a large number of polysaccharides from higher plants, animal cell membranes and microbial cell walls and they are widely used in food, feed, medicine and other industries. With the continuous development of bacterial drug resistance, polysaccharides have become the focus of research at home and abroad as a class of antibiotic alternatives. This review intends to elaborate on the antibacterial activity and main mechanisms of polysaccharides, in order to provide a theoretical basis for further research on the biological activities of polysaccharides and their mechanism, and to develop safer, greener and more efficient antibiotic alternatives.

The aim of this study was to investigate the effect of lncRNA TCONS_00791383 on the proliferation and differentiation of porcine skeletal muscle satellite cells. qRT-PCR was used to detect the expression levels of TCONS_00791383 in 6 tissues (heart, spleen, lung, kidney, dorsal muscle and leg muscles) of Yorkshire piglets within 7 days of birth; At the same time, the expression levels of TCONS_00791383 before and after proliferation and differentiation of porcine skeletal muscle satellite cells were examined. The expressions of the proliferation and differentiation marker genes were examined after knocking down TCONS_00791383 in porcine skeletal muscle satellite cells using antisense oligonucleotides(ASO) fragment. Target genes of TCONS_00791383 were predicted by using trans (co-expression) method, GO enrichment and KEGG pathway analysis of these target genes were performed by DAVID. The results showed that TCONS_00791383 had the highest expression level in heart, but no expression in spleen and kidney tissues. During the process of proliferation and differentiation of skeletal muscle satellite cells, the expression level of TCONS_00791383 gradually increased and reached the peak at 30 h of differentiation. Compared with the control group, after knocking down TCONS_00791383 using the ASO fragment, the expression levels of the proliferation marker genes Pax3 and Pax7 significantly or extremely significantly decreased (P<0.05,P<0.01), and the expression level of the differentiation marker gene MyoG extremely significantly decreased (P<0.01) at 24 h of differentiation, the expression level of the proliferation marker gene Pax3 extremely significantly decreased (P<0.01) and Pax7 gene significantly decreased (P<0.05), the expression level of the differentiation marker gene MyHC significantly decreased (P<0.05) at 48 h of differentiation. The predicted target genes were enriched in AMPK, ATP and other important signaling pathways involved in the proliferation and differentiation of skeletal muscle satellite cells. This study shows that lncRNA TCONS_00791383 probably promotes the proliferation and differentiation of porcine skeletal muscle satellite cells.

This experiment aimed to explore the effective SNPs loci and functional genes related to blood glucose traits in chickens, and provide effective theoretical support for molecular breeding of high quality broilers. In this experiment, 407 Jing Xing Yellow hens were slaughtered at 98 days of age, blood DNA was extracted by phenol-chloroform method, and whole genome resequencing was performed at a depth of 10×; Glucose oxidase method was used to determine blood glucose levels in serum. The genome-wide association study (GWAS) were performed based on whole genome resequencing and glycemic phenotype data. GWAS screened a total of 6 blood glucose-related SNPs loci (associated threshold P<1.43×10^-6). The gene annotation revealed that rs734134177 was on the 8th intron of UBE3D gene, and its encoded protein was ubiquitin protein ligase. The blood glucose level of wild-type (AA) individuals at this locus was extremely significantly higher than that of mutant (GG) individuals (P<0.01); rs794554022 was extremely located at D 93.5 kb of ACAD9 gene downstream. ACAD9 protein was a member of the acyl-CoA dehydrogenase family and was the rate-limiting enzyme in the process of β-oxidation of fatty acyl-CoA in mitochondria. The blood glucose level of the wild-type(AA) individuals at the rs794554022 locus was extremely significantly lower than that of the mutant(CC) individuals (P<0.01). The above loci may be related candidate SNPs for regulating blood glucose level. The genes containing the two loci may be involved in the regulation of blood glucose metabolism in broilers. These results will provide candidate molecular markers for regulating the blood glucose metabolism of broilers and improving the meat quality, which will provide a new idea for the regulation of blood glucose metabolism in broilers.

The aim of this study was to clone quinoid dihydropteridine reductase (QDPR) gene and detect its expression in different tissues, different growth stages of Wumeng Crested chicken of different genders, so as to study the regulatory role of QDPR gene in the growth and development of Wumeng Crested chicken. In this experiment, the CDS region of QDPR gene of Wumeng Crested chicken was amplified and cloned by PCR, bioinformatics analysis of the sequences was carried out, and real-time fluorescent quantitative PCR analysis was performed to detect the expression of QDPR gene in heart, liver, spleen, lung, kidney, chest muscle and leg muscle of Wumeng Crested chicken in 4, 12, 20, and 28 weeks old. The results showed that the length of the open reading frame of QDPR gene was 417 bp, and 139 amino acids were encoded in total. The encoding protein was acidic and unstable. The QDPR gene sequence of Wumeng Crested chicken was highly homologous with the Gallus gallus, Coturnix japonica and Numida meleagris; QDPR gene was expressed in all tissues of Wumeng Crested chickens of different genders at different stages, the relative expression level of QDPR gene was the highest in the lung tissue of cock in 4,12 and 20 weeks old, which was extremely significantly higher than that in other tissues at the same age (P<0.01). The relative expressions of QDPR gene in liver, spleen and lung of 4-week-old hens were the highest in all stages of growth, which was extremely significantly higher than those in the stages of 12- and 20-week-old (P<0.01). The relative expressions of QDPR gene in the most of tissues of cock generally showed a trend of increasing first and then decreasing with time, while decreasing first and then increasing in hens. The results indicated that the expressions of QDPR gene in visceral tissues of Wumeng Crested chickens were higher than that in muscle tissues, and the expression in cock and hen were different in different growth stages. The experimental results can provide a reference for further research on the regulation mechanism of QDPR gene in chicken growth and development.

This study aimed to reveal the regulating role of miR-128-1-5p in the proliferation and differentiation of ovine preadipocytes. The target genes of miR-128-1-5p were theoretically predicted and experimentally validated. After overexpressing miR-128-1-5p, the expression of proliferation marker genes were detected by qPCR, and the cell proliferation was examined by CCK-8 and EdU. The expression trends of miR-128-1-5p and its target genes during the differentiation of ovine preadipocytes were detected by qPCR and Western blotting. The regulatory mechanism of miR-128-1-5p on the ovine preadipocyte differentiation was revealed by overexpressing or inhibiting miR-128-1-5p. The cell adipogenic effect was tested by Oil Red O staining. The results showed that KLF2 was a target gene of miR-128-1-5p. During the proliferation of ovine preadipocytes, after miR-128-1-5p was overexpressed, the expressions of proliferation marker genes were significantly or extremely significantly decreased (P<0.05 or P<0.01), the cell vitality was also significantly or extremely significantly decreased (P<0.05 or P<0.01), and the newborn cell number was significantly reduced (P<0.05). During the differentiation of ovine preadipocytes, a negative correlation was found between the expressions of miR-128-1-5p and KLF2; After miR-128-1-5p was overexpressed, the mRNA and protein expression of KLF2 were extremely significantly (P<0.01) and significantly (P<0.05) decreased, respectively; The mRNA expressions of differentiation marker genes were significantly or extremely significantly increased (P<0.05 or P<0.01) in the miR-128-1-5p overexpressing cells; More lipid droplets were deposited; The results were opposite after miR-128-1-5p was inhibited. In conclusion, there is a binding site between miR-128-1-5p and KLF2. miR-128-1-5p inhibits the proliferation of ovine preadipocytes. miR-128-1-5p promotes the differentiation of ovine preadipocytes and lipid droplets deposition by targeting and negatively regulating KLF2 in the process of differentiation.

This study aimed to explore the effect of miR-106b-5p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-106b-5p played its roles via targeting KLF4. The quantitative real-time PCR (qRT-PCR) was used to detect the expression pattern of miR-106b-5p during the differentiation of goat intramuscular preadipocytes. The miR-106b-5p mimic and miR-106b-5p inhibitor were transfected into goat intramuscular preadipocytes cultured in vitro by liposome transfection. The effect of miR-106b-5p on lipid droplet accumulation was verified using the Oil Red O Staining. The expression levels of predicted target gene KLF4 targeting miR-106b-5p and adipogenic marker genes were detected using qRT-PCR. The target relationship between miR-106b-5p and KLF4 was detected by dual luciferase reporter system. The result of qRT-PCR showed that the highest expression level of miR-106b-5p was at day 3 of induced differentiation of goat intramuscular preadipocytes. The Oil Red O staining result showed that lipid droplet accumulation was decreased after treated by miR-106b-5p inhibitor, however, it had an opposite result after treated by miR-106b-5p mimic in goat intramuscular adipocytes. The expression level of PPARγ was significantly decreased (P<0.05) and the expression level of KLF4 was extremely significantly increased (P<0.01) in the miR-106b-5p inhibitor treatment. The expression levels of LPL and PPARγ were extremely significantly increased (P<0.01) in the miR-106b-5p mimic treatment. The luciferase activity assay result showed that luciferase activity of KLF4 was significantly inhibited in the miR-106b-5p mimic treatment. miR-106b-5p promotes the differentiation of goat intramuscular preadipocytes via targeting and negatively regulating the expression of KLF4.

The study aimed to predict and verify the key miRNA regulating the expression of microphthalmia-associated transcription factor (MITF). The online softwares were used to predict miRNAs with a targeted relationship to MITF in this study. In the melanocytes of alpaca, the expression of MITF gene after overexpression and inhibition of miR-96-5p was detected by fluorescence quantitative PCR. MITF-specific antibody staining was performed on cells in which the expression of miR-96-5p was overexpressed and inhibited by immunocytochemical techniques. The results showed that miR-96-5p was a key miRNA regulating the expression of MITF. The results of fluorescence quantitative PCR and immunocytochemical detection showed that when miR-96-5p was overexpressed, MITF mRNA level had no significant change, but the expressions of its downstream genes were extremely significantly reduced (P<0.01). MITF protein and its downstream proteins levels were extremely significantly lower than those in the blank groups (P<0.01). When miR-96-5p was inhibited, MITF mRNA level had no significant change, but the expressions of its downstream genes were extremely significantly increased (P<0.01). MITF protein and its downstream proteins levels were extremely significantly higher than those in the blank groups (P<0.01). In conclusion, miR-96-5p can target MITF gene and play a regulatory role at the post-transcriptional level of MITF, inhibiting the transcription of MITF and the expressions of its downstream genes of TYR, TYRP1 and TYRP2.

The aim of this study was to investigate the effects of amphiregulin (AREG) on in vitro maturation (IVM) of oocytes from sheep small antral follicles. In this study, oocytes from small and medium antral follicles of sheep ovary from the slaughterhouse were collected. Autofluorescence was used to detect NAD(P)H and FAD⁺⁺ levels of oocytes, mitochondrial membrane potential of oocytes were detected by JC-1. After IVM, the cumulus expansion index (CEI) and first polar body expulsion rate (MII%) of oocytes from small and medium antral follicles were compared. The effects of AREG, AREG + GDF9, AREG + BMP15, AREG + GDF9 + BMP15 and GDF9 + BMP15 on CEI, MII% and mitochondrial membrane potential of small antral follicular oocytes were compared after IVM. After IVM, the effects of AREG + GDF9 + BMP15 on the levels of NAD(P)H and FAD⁺⁺ of small antral follicular oocytes and their development ability after in vitro fertilization (IVF) were detected. The results showed that, before IVM, the mitochondrial membrane potential and FAD⁺⁺ level of small antral follicular oocytes were significantly lower than those of medium antral follicular oocytes(P<0.05). After IVM, the CEI and MII% of the small antral follicular oocytes were significantly lower compared to medium antral follicular oocytes (P<0.05). Compared with the control group, AREG + GDF9 + BMP15 treatment significantly increased the CEI, MII% and mitochondrial membrane potential of small antral follicular oocytes after IVM (P<0.05), and there was no significant difference compared with the medium antral follicular oocytes(P>0.05). Moreover, AREG + GDF9 + BMP15 treatment significantly increased the levels of NAD(P)H and FAD⁺⁺ of small antral follicular oocytes after IVM (P<0.05), and there was no significant difference compared with the medium antral follicular oocytes(P>0.05). Compared with the control group, supplemented AREG + GDF9 + BMP15 into IVM medium could significantly increase the cleavage rate and blastocyst rate of small antral follicular oocytes after IVF((43.79 ±3.69)%, (28.54 ±4.31)% and (78.99 ±1.12)%, (47.46 ±2.50)%, respectively, P<0.05), while there was no significant difference compared with the medium antral follicular oocytes(P>0.05). These results indicated that the metabolic level and IVM quality of oocytes from sheep small antral follicles were relatively low. With the synergistic effect of GDF9 and BMP15, AREG can significantly improve the metabolic level and IVM quality of oocytes from small antral follicles, and further improve their development ability after IVF.

The study was conducted to investigate the effects of silencing goat beta-defensin 124 (gBD124) on the expression of key genes in p38MAPK/AP1 pathway and their downstream target genes of the cytokines and chemokines. Three designed gBD124-shRNAs were loaded into LV10-U6/RFP&Puro shuttle plasmid, then cotransfected into 293T cell with packaging plasmid for lentivirus packaging. Virus titer was detected using the method of suspension gradient dilution. Three recombinant LV10-gBD124 vectors and LV10-gBD124-NC were transfected into epididymal caput cells of Taihang goat, respectively for screening the effective silent vectors. The effective recombinant LV10-gBD124 vectors were co-transfected into epididymal caput cells, and the blank cell group, LV10-NC group and effective silent vector group were setup, respectively. The epididymal caput cells and culture mediums were collected separately after screening by 2 μg·mL^-1 puromycin. The mRNA and protein expression levels of gBD124, some key genes in MAPK signaling pathway, cytokines and chemokines were detected by qRT-PCR, Western blot and the high-specific ELISA kits, respectively. The results showed that LV10-gBD124 recombinant vectors were constructed and two valid recombinant vectors of LV10-gBD124-51 and LV10-gBD124-161 were screened for silencing gBD124. The epididymal caput cells with gBD124 silenced were successfully constructed. The results of qRT-PCR indicated that silencing of gBD124 in epididymal caput cells significantly decreased the expressions of MAPK1, c-JUN and c-FOS(P<0.05),and significantly increased the expression of RASA1 in the MAPK signaling pathway(P<0.05). The result of Western blot showed that protein levels of total p38MAPK, total c-JUN, total c-FOS, phosphor-p38MAPK and phosphor-c-JUN were significantly reduced in epididymal caput cells with gBD124 silenced (P<0.05). The expressions of IL-1β, IL-1R2, IL-8, CCL6, CCL21 were markedly promoted (P<0.05), the expressions of CCL5 and IL-1α were significantly reduced in epididymal caput cells with gBD124 silenced (P<0.05). The concentration of CCL5 in the culture medium was significantly reduced in the group of silencing gBD124 (P<0.05). Compared with the blank control group, the concentrations of IL-1β and IL-8 in the culture medium were enhanced, and the concentration of IL-1α was significantly decreased in the group of silencing gBD124(P<0.05). The silencing of gBD124 in epididymal caput cells could regulate the expression of cytokines and chemokines through inhibiting the p38MAPK/AP1 signaling pathway.

This study was conducted to elucidate the physiological basis of active substances and their mechanism of chicken embryos based on exosomes and their miRNAs. Eighteen Huiyang Beard chicken embryos with 13 embryo age were selected (3 groups, 6 eggs per group), and embryos-derived exosomes were isolated by enzymatic digestion, sucrose cushion gradient and differential ultracentrifugation, and their characteristics were identified by transmission electron microscopy, nanoflow and labeled protein flow detection. High-throughput sequencing was used to study the expression profile of miRNAs in exosome samples, and target genes prediction and functional analysis were performed for top 10 and co-expressed miRNAs. The results showed that extracellular vesicles with typical characteristics of exosomes were obtained from 3 buoyant density ranges (S1:1.13 g·mL^-1, S2:1.21 g·mL^-1 and S3:1.32 g·mL^-1), respectively. A total of 675, 661 and 845 known chicken miRNAs of S1, S2 and S3 samples were detected via alignment to miRBase V21, in which 488 (52.6%) miRNAs were co-expressed in the 3 samples. These co-expressed miRNAs might participate in the cell proliferation, differentiation, and immune response, etc. Based on both TargetScan 7.2 and miRDB systems, a total of 3 650 genes could be targeted the 56 miRNAs which were top 10 and co-expressed in the 3 samples. In addition, the 3 650 target genes were analyzed by DAVID, it was found that 24 pathways related to growth, development and immunity were significantly enriched by these genes (P<0.05), which including MAPK signaling pathway, mTOR signaling pathway, Notch signaling pathway, Wnt signaling pathway and GnRH signaling pathway, etc. In this study, an effective method to isolate exosomes from chicken embryo tissue was established, and chicken embryos-derived exosome miRNAs could affect biological pathways related to growth, development and immune regulation.

The purpose of this study was to explore the whole transcription pattern of bovine in vivo blastocysts. Three lactating cows over 15-month-old and in good health were selected, superovulation technology was used to obtain in vivo blastocysts. Single-cell whole transcriptome sequencing technology was used to detect the transcription pattern of mRNA, lncRNA, and circRNA of bovine blastocyst, and bioinformatics tools were used to analyze the related informations. As a result of the experiment, bovine blastocysts had about 19 975 mRNAs, 12 715 novel lncRNAs, and 887 circRNAs. There were 12 alternative splicing methods for mRNAs, mainly the transcription start site (TSS). GO and KEGG were used to analyze mRNAs. It was found that biological functions were mainly enriched in metabolism, cell membrane and protein binding, cell communication, cellular component organization and developmental process in GO enrichment analysis; The pathways were enriched in cell cycle, membrane transport, and chromosome tissue regulation. There were 6 types of circRNAs, of which the CLASSIC type had the largest number. This type of splicing would form a circular exon circRNA (EcircRNA). The whole transcriptome pattern of bovine in vivo blastocyst was clarified in this study, which provided new ideas for a comprehensive understanding of bovine in vivo blastocysts.

This experiment investigated the effects of resveratrol (RES) on methane (CH₄) emissions, nutrient degradation and microbial community in two types of substrate, aiming to explore the mechanisms of RES regulating rumen fermentation and reducing CH₄ emissions. This experiment was a two-factor design. Three rams with similar body weight((50±2.3) kg) and health status installed with permanent rumen fistula were selected as rumen fluid donors. In experiment one, 0%, 7.7%, 14.3% and 25.0% RES were added to the substrates with a concentrate:forage ratio of 68:32 (high concentrate, HC) and 28:72 (high forage, HF), respectively, for testing gas production. High-throughput sequencing of 16S amplicons was performed in the control group and 14.3% group. In experiment two, 0% and 14.3% RES were added to the above two substrates for degradation rate test. Five replicates were set for the above experimental treatments. The results showed that:1) The gas production (GP) and CH₄ production of the two substrates decreased linearly with the increase in RES level (P<0.05); At the same RES level, compared with HC, GP and CH₄ production in HF were significantly reduced (P<0.05), and the two factors had an interactive effect on the changes of the two indicators (P<0.05). 2) The degradation rates of dry matter(DM), organic matter(OM), crude protein(CP), neutral detergent fiber(NDF), and acid detergent fiber(ADF) of the two substrates were significantly decreased after supplemented with RES (P<0.05). Compared with HC, the degradation rate of each nutrient in HF was significantly reduced at the same RES level, and there was an interaction between the two factors on degradation rate of neutral detergent fiber (NDFD) and degradation rate of acid detergent fiber (ADFD) at 24 h of fermentation (P<0.05). 3) At phylum level, the addition of RES in both substrates resulted in a significant increase in the Proteobacteria (P<0.05); compared with HC, the abundance of the Proteobacteria in HF increased significantly (P<0.05). In addition, at 24 h of fermentation, the addition of RES in HF caused a significant decrease in the Synergistetes (P<0.05); at 12 h of fermentation, Compared with HC, the abundance of Actinomycetes in HF significantly decreased at the same RES level. At genus level, the addition of RES and the change of substrate types all resulted in a significant decrease in the abundance of Prevotella_1, and it was statistically significant at HC (12 h fermentation) and HF (24 h fermentation) (P<0.05); Ruminobacter and Succinivibrio, the dominant strains of Proteobacteria, increased significantly with RES supplemetation (P<0.05). In addition, at 12 h of fermentation, RES was added to HF, the abundance of Rikenellaceae_RC9_gut_group, Prevotellaceae_NK3B31_group and Christensenellaceae_R-7_group increased significantly (P<0.05). Compared with HC, Prevotellaceae_UCG-001, Prevotellaceae_NK3B31_group and Christensen ellaceae_R-7-_group decreased significantly in HF (P<0.05). At 24 h of fermentation, the addition of RES in HF resulted in a significant decrease in the abundance of Veillonellaceae_UCG-001 and Lachnospiraceae_XPB1014_group (P<0.05), Prevotellaceae_UCG-001, Phocaeicola, Prevotellaceae_NK3B31_group, Succiniclasticum, Christensenellaceae_R-7_group, Ruminococcaceae_NK4A214_group and Ruminobacter were significantly increased (P<0.05); and the two factors had an interaction effect on the abundance changes of Prevotella_1, Succiniclasticum, Veillonellaceae_UCG-001 and Lachnospiraceae_XPB1014_group (P<0.05). The results showed that,high level of RES suppplementation effectively reduced the CH₄ emission, but inhibited the degradation of nutrient in the rumen, which might be related to the significant decline of Prevotella_1 in the rumen flora.

The effects of different dietary energy levels on production performance and slaughter indicators of 13-18 months old Holstein steers were investigated in this study. Sixty 13-month-old healthy Holstein steers with average body weight of (501.47±40.09) kg were selected and randomly divided into 3 groups with 20 replicates in each group and 1 steer in each replicate. According to the energy level of the diets, 3 groups were low energy group (LE), medium energy group (ME) and high energy group (HE), respectively. The whole experiment lasted for 154 days with a 10-day pretest period and a 144-day formal test period. The comprehensive net energy(NE_mf) of the diets in the early stage of the experiment (97 d) were 5.90, 6.10 and 6.30 MJ·kg^-1, respectively. The NE_mf of the diets in the later stage of the experiment (47 d) were 6.10, 6.30 and 6.50 MJ·kg^-1, respectively. The crude protein level of the diets in the whole experiment was 11.50%. At the end of the feeding experiment, the production performance and other indicators were measured. Five steers were randomly selected from each group to collect blood sample for determination of blood biochemical indicators and slaughtered for determination of slaughtering performance and meat quality. The results showed that the different dietary energy levels had no significant effect on the average daily gain (ADG) of the Holstein steers during the whole experimental period (P>0.05). Compared with LE group, the feed to gain ratio of HE group was significantly lower (P<0.05), but there was no significant difference between HE group and ME group (P>0.05); With the increase of dietary dietary energy level, the serum glucose content in the HE group was the lowest, which was 16.04% (P<0.05) and 8.99% (P<0.05) lower than those of LE group and ME group, respectively. The content of serum urea nitrogen of ME group was the lowest, which was 8.52% lower than that of LE group (P<0.05), and there was no significant difference between ME group and HE group (P>0.05). The content of β-hydroxybutyric acid and growth hormone decreased significantly with the increase of dietary energy (P<0.05), and the activity of serum lipoprotein lipase increased with the increase of dietary energy level (P<0.05); With the increase of dietary energy level, body height decreased significantly (P<0.05), but there was no significant effect on body length and heart girth (P>0.05); The loin eye area of Holstein steers increased significantly with the increase of dietary energy level (P<0.05), but the dietary energy level had no effect on dressing percentage, net meat rate and meat to bone ratio (P>0.05); The content of crude fat in longissimus dorsi muscle in HE group was significantly increased (P<0.05) and the content of water was significantly reduced (P<0.05) compared to LE group; The income of ME group was the highest, which was 0.97 yuan·(day·head)^-1 and 1.02 yuan·(day·head)^-1 higher than those of LE group and HE group, respectively. In summary, under the conditions of this experiment, when the dietary CP level of 13-18 months old Holstein steers was 11.50%, the suitable NE_mf level in the early period was 6.10 MJ·kg^-1, and the suitable NE_mf level in the later period is 6.30 MJ·kg^-1.

The purpose of this experiment was to study the effects of mono-dicalcium phosphate (MDCP) on growth performance, serum biochemical indices, tibia indices, metabolism of calcium and phosphorous in bone and cecal microecology of AA broilers and to evaluate the appropriate level of addition for the complete replacement of dicalcium phosphate (DCP) with MDCP in diet. A total of 450 one-day old AA broilers were randomly divided into 5 groups. DCP was used as the control group(CK). Four levels of MDCP were used to replace DCP to provide non-phytate phosphorus which was provided 60%, 80%, 100%, and 120% of the total non-phytate phosphorus labeled as treatment 1 (T1), treatment 2(T2), treatment 3(T3) and treatment 4(T4), respectively, with 6 replicates per group and 15 broilers per replicate for a 42-day trial period. Metabolic traits were conducted from day 19 to 21 and day 39 to 41, respectinely. Data of production performance, blood samples, and cecal microbes were collected and analyzed on 21 and 42 d, respectively. The results showed as follows:1) Compared with the CK group, the MDCP test groups improved the performance of broilers at 21 and 42 d significantly (P<0.05), and T2 group had the highest European index. 2) The apparent metabolic rate of calcium and phosphorus were decreased significantly in T2, T3 and T4 groups (P<0.05) at 21 d, and was increased significantly in T1 and T2 groups at 42 d (P<0.05). 3) Tibia calcium content of T1 group was significantly increased and phosphorus and maximum stress were significantly decreased at 21 d compared with CK group (P<0.05), tibia calcium and phosphorus content of T3 group were significantly increased compared with CK and T1 group at 42 d (P<0.05), the maximum stress of T2 and T3 group were significantly higher than the other groups at 42 d (P<0.05). 4) Compared with CK group, the blood phosphorus content was significantly decreased in T1 group (P<0.05), and was significantly increased in T4 group (P<0.05). 5) The results of high-throughput sequencing of cecal microbes in broiler chickens showed that there were significant differences in cecal microbes between the CK group and the T1-T4 groups, and the clustering distance was farther at 21 d. The diversity of cecal microbes at 42 d was significantly higher than at 21 d. The results of various indicators showed that MDCP could replace DCP as a source of non-phytate phosphorus in broiler chickens. According to European index, tibia indices, serum biochemical indices and cecal microbes of broilers, the MDCP at 80% replacement level had the best performance.

This experiment aimed to establish a method for simultaneous detection of multiple biogenic amines in pig intestinal chyme using liquid chromatography mass spectrometry (LC-MS). The experiment firstly optimized the conditions for derivatization of biogenic amines by dansyl chloride (DNS), and further determined the best method for purifying biogenic amines in intestinal chyme samples. The results showed that the derivatization conditions were optimal when the derivatization agent concentration was 8 mg·mL^-1 and the derivatization temperature was 40℃ for 90 min. The biogenic amine purification method was determined to be the best combination of extraction with ethyl acetate first and extraction with ethyl acetate after derivatization. The quantification results of 11 biogenic amines in the mass concentration range of 5-1 000 μg·L^-1 showed a good linear relationship (R²>0.99). The detection limit and limit of quantification were 0.01 - 0.23 μg·L^-1 and 0.03 - 0.76 μg·L^-1, respectively. The intra-day relative standard deviation of each bioamine was 0.73%-7.92%, and the inter-day relative standard deviation was 0.07%-8.67%. Finally, this method was used to detect biogenic amines in chyme samples from piglets' ileum, cecum, colon and rectum. The content distribution of 11 biogenic amines in different intestinal chyme was preliminarily determined. Detection of chyme samples in piglets' ileum, cecum, colon and rectum found that the biogenic amines in the chyme were mainly spermidine, putrescine, cadaverine, spermine and histamine. Tyramine, 1,7-diaminoheptane,serotonin, and agmatine were detected in very small amounts, and no tryptamine and phenethylamine were detected. In summary, the method established in this test has accurate results, high sensitivity, and good reproducibility, and can be used to detect multiple biogenic amines in chyme of different intestinal sections of piglets simultaneously.

This experiment was conducted to investigate the effects of tannic acid on digestive enzyme activity and digestibility of dry matter and crude protein of corn-soybean meal diet in the simulated gastric and small intestinal digestion for growing pigs, which will provide a reference for evaluating the biological effects of tannic acid. In the experiment 1, a single factor completely randomized arrangement was used to investigate the effects of 2 tannic acids without diet on digestive enzyme activities in simulated gastric and small intestinal fluid for pigs. Five treatments consisted of 0 mg tannic acid (the gastric fluid volume was 20 mL, the intestinal fluid volume was 22 mL);tannic acid 1,10 mg; tannic acid 1,20 mg; tannic acid 2,10 mg; tannic acid 2,20 mg. The activities of pepsin, amylase, trypsin and chymotrypsin were determined. The experiment 2 was to investigate the effects of tannic acid supplemention in corn-soybean meal diet on digestive enzyme activity in gastric and small intestinal simulated digestion phases and nutrient digestibility determined with the simulated digestion for growing pigs. Five treatments of dietary tannic acid concentration were 0 mg·(2 g)^-1; tannic acid 1, 10 mg·(2 g)^-1; tannic acid 1, 20 mg·(2 g)^-1; tannic acid 2, 10 mg·(2 g)^-1; tannic acid 2, 20 mg·(2 g)^-1, respectively. The activities of pepsin were measured at 0.5 and 4 h in the gastric digestion phase, and the activities of amylase, trypsin and chymotrypsin were measured at 0.5, 4, and 8 h in the small intestinal digestion phase. Simultaneously, the digestibility of dry matter and crude protein were determined for the diet. The results showed that:1) Without the diet, compared with the blank control group, the 2 tannic acids didn't significantly affect the activity of pepsin in the simulated gastric fluid(P>0.05); tannic acid 1 significantly reduced the activities of amylase, trypsin and chymotrypsin in the simulated intestinal fluid contrast to tannic acid 2 (P<0.05). 2) From 0.5 to 4 h of the simulated gastric digestion, except for the 10 mg·(2 g)^-1 supplementation level at 4 h, the activity of pepsin in simulated gastric fluid in the tannic acid 1 diet was significantly greater than that in the tannic acid 2 diet(P<0.05). Except for tannic acid 2 at 0.5 h, the pepsin activities in simulated gastric fluid in the 2 tannic acids added at 10 mg·(2 g)^-1 were significantly greater than that at 20 mg·(2 g)^-1(P<0.05). At 0.5 h of the simulated small intestinal digestion, except for the chymotrypsin activity at 20 mg·(2 g)^-1 supplementation level, the 2 levels of tannic acid 1 or 2 in the diet didn't significantly influence amylase activity in the digestive fluid(P>0.05), but both significantly reduced chymotrypsin activity(P<0.05). The trypsin activity of the digestive fluid in the tannic acid 1 was greater than that in the tannic acid 2(P<0.05). At 4 h of the simulated small intestinal digestion, except for the chymotrypsin activity at 20 mg·(2 g)^-1 supplementation level, the activities of amylase and chymotrypsin in digestive fluid was greater but the trypsin activity was lower in diet supplemented with tannic acid 1 than tannic acid 2(P<0.05). The supplement of tannic acids 1 and 2 reduced the activities of trypsin and chymotrypsin (P<0.05). At 8 h of the simulated small intestinal digestion, the concentration of tannic acid in diet affected the activity of amylase, but no significant difference was observed on the average activity of amylase between the treatments of tannic acid 1 and tannic acid 2(P>0.05). The supplement of tannic acids 1 and 2 reduced the activities of trypsin and chymotrypsin(P<0.05). 3) Campared with the control, the 2 tannic acids significantly reduced the digestibility of dietary crude protein at both levels (P<0.05). More reduction in digestibility of dietary crude protein was observed in tannic acid 2 than tannic acid 1 (P<0.05). In summary, inconsistent effect of tannic acid on digestive enzyme activities was presented in digestive fluid with or without diet, the tannic acid reduced the dietary crude protein digestibility maybe mainly related to the reduced activities of chymotrypsin in the digestive fluid and hydrolysis efficacy of digestive enzymes reduced by the formation of chelates between tannic acid and dietary components in the simulated digestion for growing pig.

This experiment was conducted to study heat stress on sheep in three regions (Yanshan Mountain of Hebei North, Taihang Mountain and Central-South plain) of Hebei province in summer, and the correlation was also investigated between ambient temperature-humidity parameters and physiological parameters of housed sheep in order to provide theoretical basis for improving the environment of sheep shed. Twelve large-scale sheep farms in 3 regions were selected to automatically detect and record the ambient temperature and relative humidity of sheep sheds in summer for 2 months. In the mean time, the rectal temperature and respiratory rate of sheep in each farm were detected. The results showed that the average daily temperature in sheep sheds of Yanshan Mountain of Hebei North, Taihang Mountain and Central-South plain reached 24.3, 28.5, and 28.7℃, and the temperature-humidity index (THI) was 72.40, 79.91, and 79.47, respectively. These results suggested that sheep in Yanshan Mountain of Hebei North region were suffering from mild heat stress, while sheep in the other two regions were suffering from moderate or serious heat stress. Thus, the heat stress prevention of sheep in summer in Hebei province should be paid more attention, and it was urgent to strengthen the heat prevention and cooling measures of sheep shed in summer. From physiological parameters of sheep in 3 regions, the range of rectal temperature was from 39.1 to 39.8℃ and respiratory rate was from 42 to 114 times·min^-1. There was no significant correlation between rectal temperature and ambient temperature or THI (P>0.05); however, there was a significant linear negtive correlation between rectal temperature and relative humidity (P<0.05, r=0.60). Also, an extremely significant positive correlation was observed between respiratory rate and ambient temperature (P<0.01, r=0.84) or THI (P<0.01, r=0.87); however, no significant correlation was observed between respiratory rate and relative humidity (P>0.05). In summary, the individual physiological condition of sheep can be inferred from ambient temperature, humidity parameters or THI, which can provide data for the prevention of heat stress and diagnosis of related diseases in housed sheep.

This study aimed to investigate the intracellular localization of 10 putative effector proteins via type Ⅲ secretion system (T₃SS) in Chlamydia psittaci, laying the basis for the next structure and function analysis. In this study, computer-aided bioinformatics methods such as Effective T3 and BPBAac were used to select the possible T₃SS effector proteins of C psittaci. The recombinant plasmid pGEX -6P-1-target genes were constructed and transformed into E. coli BL21, and the recombinant proteins were induced and purified. Then BALB/c mice were immunized with the recombinant proteins to produce polyclonal antibodies, and the titre of antibodies were detected by ELISA. Indirect immunofluorescence assay (IFA) was used to detect the subcellular localization of these proteins. In our study, fourteen possible Cps T₃SS effector protein-coding genes were predicted:e.g. CPSIT_0357, CPSIT_0429, CPSIT_0461, CPSIT_0463, CPSIT_0490, CPSIT_0594, CPSIT_0785, CPSIT_0844, CPSIT_0846, CPSIT_0019, CPSIT_0020, CPSIT_0968, CPSIT_0969, and CPSIT_0942. Ten target recombinant proteins were successfully prepared except CPSIT_0357, CPSIT_0429, CPSIT_0968 and CPSIT_0969. After immunization of BALB/c mice, the specific antibodies were prepared, and the titers were (1:32 000)-(1:64 000). Moreover, the IFA results showed that CPSIT_0844 and CPSIT_0846 were located in the inclusion membrane, while CPSIT_0461, CPSIT_0463, CPSIT_0490, CPSIT_0594, CPSIT_0785, CPSIT_0019, CPSIT_0020 and CPSIT_0942 were located in whole the inclusion bodies. In conclusion, two effector proteins localized in the Chlamydia inclusion membrane and eight effector proteins localized in the chlamydia inclusion were identified.

This research aimed to investigate the effect of Trichinella spiralis dipeptidyl-peptidase 1 (Dpp1) on rat peripheral blood mononuclear cell (PBMC) proliferation, migration, apoptosis, nitric oxide secretion and phagocytosis in vitro. A pair of primers were designed based on Dpp1 gene nucleotides in GenBank, Dpp1 gene was cloned by RT-PCR and the expression vector was constructed to obtain the recombinant protein (rDpp1). The reactogenicity of rDpp1 was analyzed by Western blot. PBMCs were separated from rats and co-incubated with rDpp1 in vitro,binding of rDpp1 with rat PBMCs was confirmed by immunofluorescence assay (IFA), and the effects of different concentration of rDpp1 (10, 20, 40 μg·mL^-1) on cell proliferation,migration,nitric oxide production,phagocytosis and apoptosis were determined. The results showed that rDpp1 at a concentration of 40 μg·mL^-1 significantly promoted the proliferation of PBMCs; rDpp1 at 10 μg·mL^-1 could promote cell migration, while 20 and 40 μg·mL^-1 concentrations showed a significant promotion; rDpp1 with 40 μg·mL^-1 concentration significantly promoted NO secretion; rDpp1 with 10 μg·mL^-1 promoted cell phagocytosis, and 20 and 40 μg·mL^-1 concentrations showed very significant promotion; each concentration of proteins could significantly promote the apoptosis of PBMCs. The functions of rat PBMCs were influenced by T. spiralis dipeptidyl-peptidase 1 via different pathways.

Previous research results showed that African swine fever virus (ASFV) MGF360-9L can significantly inhibit the host's innate immune response. Herein we compared and analyzed MGF360-9L gene sequences of ASFV and predicted the relationship between MGF360-9L structure and function. In this study, bioinformatics method was used to analyze and predict its pri-mary, secondary and tertiary structure of the expressed protein. Based on the sequence of Georgia 2007/1 published in GenBank (FR682468.1), the MGF360-9L gene was synthesized and its recombinant eukaryotic expression plasmid was constructed. The recombinant plasmid was transfected into PK-15 cells, then gene expression was identified by Western blotting, and subcellular localization of this protein was observed by indirect immunoinfluscent assay (IFA). Results showed that MGF360-9L gene is highly conserved among type II ASFV strains, Georgia 2007/1 is the representative virus strain. The homology analysis results of 54 different ASFV strains is consistent with its phylogenetic relationships of them, the conserved sequence is a 378 bp fragment at the 3'terminal of MGF360-9L. The advanced structure of this protein was mainly α-helix. Nuclear location sequence (NLS) prediction revealed that it might be located in the nucleus, and is consistent with the results of MGF360-9L IFA in PK-15 cells. This study accumulated significant data for further research on inhibition of innate immune response by ASFV MGF360-9L and pathogenic mechanisms of the MGF360 gene family in ASFV.

To understand the molecular epidemiology and genetic variation of porcine reproductive and respiratory syndrome virus (PRRSV) in Sichuan and Guizhou provinces, Southwest China in recent years, 8 PRRSV positive samples collected from the pig farms during 2016-2018 were used for viral isolation and full-length genomic sequences amplification, the genomic recombinations of these sequences were analyzed by using RDP4 and Simplot biological softwores. The results showed that the full length of the genome of 8 PRRSV strains were 15 010-15 321 nt, and the homologies among these strains were 80.9%-91.7%. The amino acid (aa) analysis of Nsp2 showed that four strains showed the same 30 aa deletion pattern as HP-PRRSV, and the other four strains showed the same 131 aa deletion pattern as NADC30. Among them, GZgy17 exhibited a novel "1+19+29 aa" deletion pattern. The ORF5 amino acid analysis showed that a new 1 aa deletion at site 33 was emerging in three isolates. Furthermore, recombination analysis showed that the eight strains exhibited six recombination patterns among different lineages:(1) SCnj16:L8+L1; (2) SCxyz17:L1+L5; (3) SCya18:L1+L3; (4) GZgy17:L8+L3; (5) SCcd16 and Cya17:L1+L8+L5; (6) SCcd16 and SCya17:L1+L8+L3. The results indicated that there are not only multiple lineages of PRRSV strains coexisted in Southwest China, but also complex genetic recombination patterns in the genomes of the isolates. The epidemic situation of PRRSV strains with the above complex genome in China deserves great attention.

Decreasing the circulation of the Japanese encephalitis virus (JEV) in pigs is critical for blocking JEV infection in human. In view of the urgency of JE prevention and control in China and the uncertainty of the commercial Genotype Ⅲ JEV attenuated vaccine to the immuno-protection of partial Genotype Ⅰ JEV (GⅠ JEV) endemic strains, it is urgent to develop a safe, effective and low-cost GⅠ JEV vaccine for pigs. The expression cassette of JEV prME gene was cloned into the baculovirus shuttle vector to generate a recombinant bacmid (Bacmid-prME), the recombinant baculovirus was produced by transfecting Sf9 cells with Bacmid-prME. The accurate expression of the recombinant prME protein was confirmed by Western blot and immunofluorescence analysis, and the formation of Virus-Like Particles (VLPs) was verified through observing under Electronic microscopy. The immunogenicity of GⅠ JEV VLPs was further evaluated in mice. The recombinant prME protein was efficiently expressed in Sf9 cells and assembled into VLPs. GⅠ JEV VLPs induced the higher neutralizing antibody titer in mice. In our study, GⅠ JEV VLPs obtained in the baculovirus expression system provides a material basis for the development of a safe and effective JEV subunit vaccine.

This study aimed to analyze the epitope characteristics of VP2 and NS1 proteins of raccoon dog and arctic fox amdoparvovirus (RFAV), and further select the conserved B- and T-cell epitopes on the VP2 and NS1 proteins of RFAV in Amdoparvovirus genus. In the present study, the RFAV genome except the 5' and 3' end of the genome was cloned and sequenced, then the bioinformatics programs were used to predict the physical and chemical properties, Secondary structure, post-translational modifications and potential B- and T-cell epitopes of VP2 and NS1 proteins, and further compared the potential epitopes and post-translational modifications with the typical members within Amdoparvovirus genus. The 4 327 bp RFAV genomic sequence in length, encoding 636 amino acids of VP2 protein and 641 amino acids of NS1 protein, was obtained. Both VP2 and NS1 proteins are hydrophilic proteins and the secondary structure were mainly composed of random coils. We predicted there were 3 conserved B-cell epitopes, 3 conserved T-cell epitopes and 8 conserved post-translational modification sites on VP2 protein within the Amdoparvovirus genus; while NS1 protein had 1, 2 and 7 in the respective conserved items. In this study, we cloned the main genome sequence of RFAV, and predicted the potential epitopes and post-translational modifications on VP2 and NS1 proteins of RFAV. This study provides fundamental basis for the development of a candidate vaccine and disease diagnosis of amdoparvoviruses.

In this study, we compared the virulence of the most prevalent Haemophilus parasuis (36 strains) serovars 4, 5, 12, and 13 in China in BALB/c mice and piglets models. The results of mice virulence tests showed that the LD₅₀values of the four serotype strains were 9.80×10⁷-4.60×10⁹CFU (serovar 4),2.10×10⁸-8.85×10⁹ CFU (serovar 5),4.81×10⁷-7.01×10⁹ CFU (serovar 12) and 1.75×10⁸-8.45×10⁸ CFU (serovar 13), respectively. Serovar 13 was the most highly virulent serovar in mice, followed by serovars 4, 12, 5. However, there was a significant difference in virulence only between serovars 5 and 13 (P<0.05).The results of piglet virulence tests showed that the virulence of different strains in the same serovar were remarkably different, including virulent strains and weak strains. Serovar 5 was the most highly virulent in piglet, followed by serovars 13, 4, 12,but there were significant differences between serovars 5 and 4 (P=0.039), serovars 5 and 12 (P=0.033), and there was no significant difference between serovars 5 and 13 (P=0.241). Comparing the results of HPS virulence tests on mice and piglets, three conclusions can be drawn:first, the overall virulence of the four domestic epidemic serotypes from strong to weak were serovars 5, 13, 4 and 12 in turn; Second, there were both strong and weak strains in the same serotype, so there was no correlation between the serovar and virulence of HPS. Third, although HPS can kill BALB/c mice, its virulence result was not consistent with the piglet virulence test, indicating that it has some defects as an alternative model.

The purpose of this study was to establish a rapid, specific and visual assay for the detection of Mycoplasma hyopneumoniae (Mhp) by the combination of the loop-mediated isothermal amplification (LAMP) method and lateral flow dipstick (LFD) system. Five sets of specific primers and one FITC labeled probe targeting to P36 gene of Mhp were designed for LAMP. Biotinylated LAMP amplicons were hybridized exclusively with the FITC-labeled probe and the amplification product was detected by LFD assay. After optimization, the optimal reaction condition of LAMP is 65℃, reaction time is 15 minutes. It only takes about 40 minutes from the extraction of genomic DNA to the visualization of the amplicons by LFD, which is nearly 2 hours shorter than that of conventional PCR. Mhp could be accurately detected by LAMP-LFD. LAMP-LFD can specifically detect Mhp, and the detection results of common pathogens of swine diseases such as Mycoplasma hyorhinis and porcine circovirus are negative. Sensitivity test showed that the sensitivity of LAMP-LFD to Mhp was approximately 1×10⁰ copies of DNA, which was 1 000 times higher than that of standard PCR. Using this method, 64 Mhp positive samples were detected from 88 clinical suspected samples, 56 positive samples were detected by parallel detection of national standard PCR method, and the coincidence rate was 90.9%. The results showed that the LAMP-LFD assay can be used in the detection of Mhp specifically and accurately, and it has advantages of high sensitivity, simple operation, low dependence of instruments and equipment, low detection cost and short time-consuming. It is suitable for the use of basic laboratory, emergency detection or on-site monitoring. It has high promotion value and is expected to develop into a rapid, easy-to-operate tool for detection of Mhp.

To shorten the detection time, a quadruple fluorescence RT-PCR method was established for detecting avian influenza virus with the related probes and primers designed and synthesized by analyzing the conservative sequences of M gene, HA gene of H5 subtype, H7 subtype and H9 subtype. This method can detect avian influenza virus, as well as H5 subtype, H7 subtype, and H9 subtype simultaneously. The results showed that the method was quick, specific, and the detection limitation was 10^-5 ng·μL^-1. Sixty avian influenza viruses were detected from 384 chicken swab samples from 13 living birds markets with the method. All the detected avian influenza viruses were H9 subtype. The results were consistent with the method reported in standard, NY/T 772-2013. The κ value for these two methods was 1 (P<0.001). This method can realize the detection of avian influenza virus which has the characteristics of safety, specificity, rapidity, sensitivity, simplicity and high throughput. It will play an important role in the rapid detection of avian influenza virus.

This study aimed to investigate Bartonella and Anaplasma infection in ticks and plateau pika in Songpan county of Sichuan province. Plateau pikas were trapped and the ticks collected from yaks were classified by morphological identification. The total DNA of ticks and Plateau pika spleens was extracted, partial sequences of 16S rRNA of ticks and Anaplasma, and rpoB gene of Bartonella were amplified by PCR, respectively. The positive products were sequenced and compared through the NCBI database. Phylogenetic trees were constructed based on 16S rRNA and rpoB for determination species of ticks, Anaplasma and Bartonella, respectively. A total of 330 ticks were collected from 3 villages and only Haemphysalis qinghaiensis was found. As for tick samples, the infection rates of Bartonella of Jinan, Shanba, and Xiabazhai were 16.7%,8.2%, and 18.3%, respectively and only B. melophagi was detected. Compared with Jinan village, the infection rate of Bartonella in Xiabazhai was higher (P<0.05); The infection rates of Anaplasma in Jinan, Shanba, and Xiabazhai were 9.8%, 12.4%, and 26.7%, respectively and only A.bovis was detected. Compared with Jinan, the infection rate of Anaplasmain Xiabazhai was higher (P<0.01); As for plateau pika, B. grahamii was detected in Jinan, Shanba, and Xiabazhai with infection rates of 8.7%, 17.9%, and 13.3%, respectively, and no significant difference observed. B. queenslandens was detected only in Xiabazhai with an infection rate of 6.7%; Bartonella sp. (MN296294) and Bartonella sp. (MN296293) were detected in Jinan and Xiabazhai, respectively. No Anaplasma spp. were detected in plateau pika and no co-infection was observed in tick and plateau pika. In Songpan, H. qinghaiensis is biological vector of Anaplasma and plateau pika being a reservoir host of Bartonella spp. B. queenslandens was detected firstly in plateau pika and there exists a risk of human infection.

The study aimed to investigate the effects of avian reticuloendotheliosis virus (REV) on the number of T lymphocytes in blood and the expression of related cytokines in local lymphoid tissues of SPF chicks. Ninety-six 1-day-old SPF chicks were randomly divided into REV-infected group and control group. The above indexes were detected by Flow Cytometry, acid α-naphthyl acetate esterase (ANAE) and Quantitative Real-time PCR. The results showed that compared with the control group, the number of CD4⁺ T lymphocytes in the infected group was significantly decreased at day 7-35, and the number of CD8⁺ T lymphocytes was significantly increased at day 7-28, the CD4⁺/CD8⁺ ratio was significantly decreased at day 7-28 (all P<0.05 or P<0.01); the number of ANAE⁺ T lymphocytes in the local lymphoid tissues of Hader's gland (HG), Peyer's patch (PP) and caecal tonsil (CT) were significantly decreased (all P<0.05 or P<0.01); the transcription levels of cytokines IL-2, IFN-γ and TNF-α in HG, PP and CT were increased in various degrees. These data indicated that REV infection caused a decrease in the number of CD4⁺ T lymphocytes in the peripheral blood of chickens, an imbalance of the ratio of CD4⁺/CD8⁺ T lymphocytes, a relative decrease in the number of T lymphocytes in local lymphoid tissues, and a continuous increase in the expression of cytokines TNF-α are closely related to the significantly reduced cellular immune function of infected chickens caused by REV.

To study the relationship between the distribution of sex hormone receptors (AR and ER) in normal and cryptorchid testis and cryptorchidism of the Ziwuling black goat, the histochemical characteristics of normal testis and cryptorchidism were compared by immunohistochemistry, immunofluorescence and morphometric statistical software. The results of special staining showed that compared with the normal group, the interstitial tissue of cryptorchidism group was loose, the lumen area was significantly reduced, the content of glycogen was significantly decreased, and the contents of collagen and reticular fibers were increased. The results of immunohistochemistry and immunofluorescence showed that:1) AR was strongly expressed in Leydig cells of the normal testis and moderately positive in all levels of spermatogenic cells, while the expression of Leydig cells and Spermatogenic cells in cryptorchidism group was significantly decreased and occasionally expressed in Spermatogonia. 2) ER was strongly expressed in the normal testicular Leydig cells, moderately positive in Peritubular myoid cells, occasionally expressed in Sertoli cells and not expressed in Spermatogenic cells. 3) ER was moderately positive in cryptorchidism Leydig cells, primary Spermatocytes, Spermatogonia and Sertoli cells. 4) The average optical density of AR in cryptorchidism group was significantly lower than that in the normal group, while the average optical density of ER in cryptorchidism group was significantly higher than that in the normal group. The ratio of AR to ER expression in cryptorchidism group was close to 1:1. The distribution of collagen and reticular fibers in cryptorchidism of Ziwuling black goat is more than that of the normal testis, and the main component of seminiferous tubule basement membrane is the neutral glycoprotein. The decrease of the content of acid glycoprotein may affect the normal formation of spermatozoa. The expression of ER and AR is abnormal during cryptorchidism, especially the expression of spermatogonia and Sertoli cells is opposite to that of AR, which can provide some reference for the related research of cryptorchidism in mammals.

To evaluate the effect of Forsythia Fructus on the proliferation of H5N1 and H9N2 avian influenza viruses(AIVs) and their mediated inflammation in vitro. The aqueous extract of Forsythiae Fructus was prepared in this experiment, and CCK-8 method was used to examine the cytotoxicity of Forsythiae Fructus aqueous extract to DF-1 cells, firstly. Three methods of treatments including infection after co-incubation of the AIVs and Forsythiae Fructus aqueous extract, treatment of cells by Forsythiae Fructus aqueous extract post virus infection and administration method treatment of cells by Forsythiae Fructus aqueous extract prior to virus inoculation were used to screen the optimal administration method of the of Forsythiae Fructus aqueous extract. TCID₅₀ method was used to detect the proliferation of H5N1 and H9N2 avian influenza viruses and the expression changes of inflammatory chemokines and cytokines were detected by qRT-PCR under the optimal administration method. The results indicated that Forsythiae Fructus aqueous extract showed no cytotoxicity to DF-1 cells below the concentration of 4 mg·mL^-1 and treatment of Forsythiae Fructus aqueous extract post virus infection was the optimal administration method. Forsythiae Fructus aqueous extract significantly reduced the virus titers of H5N1 and H9N2 AIVs at various time points in a concentration-dependent manner. The expression of CX3CL1, IL8L1, CCL5, SCYA4, IFN-α, IL-1β, IL-6 and TNF-α decreased significantly in H5N1 AIV treated by the aqueous extract group compared with the control. While in H9N2 AIV group, the expression of CX3CL1, IL8L1, CCL5, IFN-β, IL-6 and TNF-α showed a similar trend. These results indicated that Forsythiae Fructus has shown promising antiviral and antiinflammation activity by inhibiting the proliferation of H5N1 and H9N2 AIVs in DF-1 cells and reducing the expression of inflammation related cytokine genes.