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20 November 2021, Volume 48 Issue 11
Biotechnology
Co-expression Regulation of Cdc42 Gene in the Bone of Rumpless Chickens at Different Developmental Stages
QI Wangmei, HU Sile, XU Siriguleng, HA Dengchuriya, BAI Xiaoying, ZHANG Wenguang
2021, 48(11):  3905-3917.  doi:10.16431/j.cnki.1671-7236.2021.11.001
Abstract ( 283 )   PDF (4722KB) ( 107 )  
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To elucidate the potential regulatory mechanism of cell division cycle 42 (Cdc42) gene in the skeletal development of rumpless chicken, in this study, the hereditation of Cdc42 gene on tail development of rumpless chicken was revealed by genome pool resequencing and RNA sequencing, combined with genomics and bone development transcriptomics. Under the strong positive selection of the rumpless varieties, highly differentiated genes were significantly enriched in biological process such as neural development, basal metabolism and cytoskeleton rearrangement between rumpless and tailed chickens. The number of differentially expressed genes during bone development increased gradually next to decreased from the 8th, 11th and 14th day of embryo, on the 16th day it reached the lowest level and then rose again and reached the peak on the 21st day of embryo. The common candidate Cdc42 gene was identified at the genomics and transcriptomics levels. The common biological processes enriched in regulation of GTPase activity and the regulation of extent of cell growth. Common signaling pathways include tight junctions and adherens junctions. The weighted gene co-expression network analysis by differentially expressed genes showed that the density of gene network was more than 6 times higher and the average connectivity was more than 8 times higher in rumpless chicken than that of tailed chicken. The correlation between Cdc42 gene and other genes was low in the co-expression network of rumpless chicken. In contrast, Cdc42 gene was at the core of the network and acted as a bridge for the co-regulation of multiple genes in tailed chicken. In addition, Cdc42 gene was proved to be conserved among multiple species. In this study, it was finally determined that the formation of the rumpless trait in Piao chicken might co-regulate the tight junction signaling pathway through Cdc42 and its co-expressed genes, and interact with the Wnt signaling pathway, thus interfering with the development of growth plate and coccyx, and ultimately affecting the important link of premature termination of coccyx. These results would be provided theoretical guidance for the early initiation and genetic basis of rumpless phenotype.
Cloning and Tissue Expression Characterization Analysis of RPL27A Gene in Goats
ZHENG Jianying, WANG Jinling, WANG Yong, ZHU Jiangjiang, ZHANG Hao
2021, 48(11):  3918-3928.  doi:10.16431/j.cnki.1671-7236.2021.11.002
Abstract ( 257 )   PDF (3536KB) ( 82 )  
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In order to study the gene structure and biological function of ribosome protein L27A (RPL27A) in goats, 14 kinds of tissue samples (i. e, heart, liver, spleen, longissimus dorsi muscle, subcutaneous fat, etc.) of Jianzhou Big-eared goats were collected. The sequence of RPL27A gene in goats was obtained by RT-PCR amplification and cloning, and its biological characteristics were analyzed using online tools. Real-time quantitative PCR was used to detect the expression of RPL27A gene in the collected different tissues as well as the subcutaneous preadipocytes at different differentiation stages. The results showed that the CDS region of RPL27A gene in goats was 447 bp, which encoded 148 amino acids. The bioinformatics analysis results showed that RPL27A gene was highly conserved among different species. RPL27A protein was an unstable alkalinity protein, and had obvious interactions with the ribosomal proteins such as RPS18, RPL26, RPL34, RPL32 and RPL37A, which related to lipid metabolism and fat deposition. RPL27A protein had no transmembrane domain and signal peptide, and subcellular localization indicated that it mainly existed in cytoplasm. Real-time quantitative PCR results reveled that RPL27A gene was widely expressed in 14 tissues of goats, such as heart, liver, spleen, kidney, lung, longissimus dorsi muscle and subcutaneous fat, while the expression of RPL27A gene was the highest in rumen, and the expression level in triceps brachii and biceps femoris was significantly higher than that in other tissues (P<0.05). The expression level of RPL27A gene in subcutaneous adipocytes of goats induced for 60 h was significantly higher than that before differentiation (P<0.05). In this study, the CDS sequence of RPL27A gene in goats was successfully cloned, and the expression characteristics of RPL27A gene in 14 tissues of goats were revealed, which could provide materials for elucidating the regulatory effect and mechanism of RPL27A gene on the differentiation of adipocyte in goats.
Bioinformatics Analysis of SUN2 Gene in Sheep and Its Expression in A549 Cells
DU Xiaoyue, ZHANG Liang, LI Huiping, LIU Shuying
2021, 48(11):  3929-3939.  doi:10.16431/j.cnki.1671-7236.2021.11.003
Abstract ( 216 )   PDF (3523KB) ( 70 )  
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This study was aimed to clone Sad1 and UNC84 domain containing 2(SUN2) gene in sheep and conduct bioinformatics analysis, construct eukaryotic expression vector and detect its expression in A549 cells. The specific primers were designed according to sequence of SUN2 gene in sheep (GenBank accession No. :XM_015095026.2), and the CDS sequence of SUN2 gene in sheep was amplified by RT-PCR method and cloned. After sequencing and identification, the sequence were analyzed by bioinformatics, and pEGFP-C1-SUN2 recombinant plasmid was constructed. The pEGFP-C1-SUN2 recombinant plasmid was transfected into A549 cells by liposome transfection method and its expression was detected. The results showed that the total length of SUN2 gene CDS was 2 187 bp, encoding 728 amino acids, the theoretical molecular mass was 81.06 ku, the molecular formula was C3593H5639N1031O1110S14, the isoelectric point was 6.20, the total number of atoms was 11 387, and the instability coefficient was 56.88, which was unstable protein. The similarity of the nucleotide sequence of SUN2 gene between sheep and Capra hircus, Bostaurus, Susscrofa, Equuscaballus, Canis lupus familiaris, Rattus norvegicus, Mus musculus and Homo sapiens were 98.9%, 97.2%, 91.6%, 90.6%, 87.1%, 82.4%, 82.1% and 86.1%, respectively. The gene similarity among different species was relatively high and conservative in the evolution process. The phylogenetic tree results showed that sheep were closely related to Capra hircus, Bos taurus, Sus scrofa, Equus caballus and Canis lupus familiaris, but far related to Gallus gallus. The prediction of structural domain and transmembrane domain showed that SUN2 protein in sheep contained a highly conserved SUN domain, and there was a transmembrane domain. The signal peptide prediction showed that there was no signal peptide site, and the hydrophobicity analysis was a hydrophilic protein. The secondary structure of the amino acid sequence was mainly alpha helix (51.65%), and the rest were random coil (31.46%), extended chain (12.36%) and beta turn (4.53%). The eukaryotic expression vector pEGFP-C1-SUN2 of SUN2 gene in sheep was successfully constructed, which was transfected into A549 cells and the protein expression was detected. The results would provide a reference for the functional study of SUN2 gene in sheep, and laid a foundation for the subsequent exploration of the role of SUN2 gene in ovine pulmonary denocarcinoma.
Study on Cloning and Expression Characteristic of NLRC3 Gene in Rabbit
GUO Mengjiao, LI Mingtao, TAN Huihui, ZHANG Chengcheng, ZHANG Xiaorong, WU Yantao
2021, 48(11):  3940-3949.  doi:10.16431/j.cnki.1671-7236.2021.11.004
Abstract ( 243 )   PDF (5710KB) ( 40 )  
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This study was aimed to investigate the sequence, molecular structure, and expression characteristics of Oryctolagus cuniculus NOD-like receptor family CARD domain containing 3 (NLRC3) in vivo and in vitro. The primers were designed according to the predicted sequence published by GenBank (accession No. :XM_017338739.1), NLRC3 gene in rabbit was amplified by PCR and cloned, and its molecular structure was predicted and analyzed by bioinformatics method. The pcDNA3.1-rNLRC3-HA vector was constructed, and the subcellular localization of NLRC3 gene in rabbit was carried out by indirect immunofluorescent assay. The tissue distribution of NLRC3 gene in rabbit was detected by Real-time quantitative PCR. After challenged with enterohemorrhagic Escherichia coli (EHEC), the expression level of NLRC3 gene in rabbit was further analyzed. The results showed that the CDS sequence of NLRC3 gene in rabbit was 3 192 bp. The similarity and phylogenetic tree analysis showed that NLRC3 gene in rabbit had highly similarity with other mammalian species in the same branch. NLRC3 protein in rabbit consisted of N-terminal domain, NACHT domain and LRR domain. Tertiary structural model of NLRC3 protein in rabbit formed a typical horseshoe-like structure in a single curvature, with alpha helix forming the convex surface and beta sheet in the concave faces. Indirect immunofluorescence assay showed that NLRC3 protein in rabbit was located in the intracytoplasm, and did not colocate with mitochondria. NLRC3 protein was detected in all tested tissues of rabbit, with the highest level in spleen, followed by mesenteric lymphnodes and lymphoid follicles. The mRNA expression of NLRC3 gene in rabbit was upregulated in spleen, liver and kidney after EHEC infection. The results showed that NLRC3 gene in rabbit was involved in the immune response after EHEC infection. In summary, NLRC3 gene in rabbit was successfully cloned and analyzed by bioinformatics, it was an intracellular receptor and widely distributed in various tissues, it participated in the immune response after bacterial infection. This study provided basic materials for further exploring the regulatory mechanism of NLRC3 in rabbit in inflammatory response.
Animal Nutrition and Feed Science
Effects of Short-term Addition of High-level Flaxseeds on Dairy Performance, Fatty Acid Profiles of Milk and Rumen Fermentation
LI Ning, ZHANG Yangdong, HUANG Guoxin, LIU Kaizhen, ZHENG Nan, WANG Jiaqi, ZANG Changjiang
2021, 48(11):  3950-3961.  doi:10.16431/j.cnki.1671-7236.2021.11.005
Abstract ( 225 )   PDF (1309KB) ( 142 )  
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The purpose of this experiment was to study the effects of short-term addition of high-level flaxseed on production performance, fatty acid profiles of milk and rumen fermentation. Thirty healthy mid-lactation Holstein dairy cows with similar body weight were selected and divided into 3 groups randomly, and fed basal pasture diet (CK group), 3 000 g/d (13.01% of DM) whole flaxseed diet (WF group), and 3 000 g/d (13.01% of DM) ground flaxseed diet (GF group) respectively. The experiment lasted for 7 weeks, first week was aim to adapt the feeding environmental, 2 to 6 weeks was the feed adaptation period, and the 7th week was the test period. Daily feed intake and milk production were recorded during the test period. Milk samples and rumen fluid samples were collected on the last day of the test, and the fatty acid composition of the milk and rumen fluid was determined by GC-MS. The results showed that:①Compared with CK group, the addition of 3 000 g/d flaxseed had no significant effect on dry matter intake and milk production for dairy cows (P>0.05). The milk fat rate and milk protein rate of GF group had a lower tendency than that of CK group (0.05<P<0.1). ②The 3 000 g/d flaxseed addition increased the production of alpha-linolenic acid (ALA) and ω-3 PUFA of milk compared with CK group (P<0.05), and the production of ALA and ω-3 PUFA in GF group were significantly higher than that in WF group (P<0.05). ③The rumen pH of WF group was significantly lower than that of CK and GF groups (P<0.05), and the NH3-N concentration of WF group was significantly higher than that of CK and GF groups (P<0.05). The acetic to propionic acid ratio in GF group was significantly lower than that in CK group (P<0.05). In summary, short-term addition of high level whole flaxseed and ground flaxseed in the diet did not affect the dry matter intake and milk production of dairy cows, but the ALA content in milk was increased, and the improvement effect of GF group was more obvious than that of WF group. However, the addition of high-level ground flaxseed had a tendency to reduce the milk protein rate and milk fat rate, and the high-level whole flaxseed affects the internal environment of the rumen of dairy cows. Therefore, the amount of flaxseed added and its feeding form should be controlled to increase the ALA content in milk while reducing the impact of flaxseed on rumen fermentation.
Effect of Different Energy Levels of Pregnant Sow on the Testicular Development and Immunity of Male Offspring
WU Junzhe, XU Xueyu, WU De, CHE Lianqiang, FANG Zhengfeng, FENG Bin, XU Shengyu, LI Jian, ZHUO Yong, LIN Yan, TAO Zhiyong
2021, 48(11):  3962-3974.  doi:10.16431/j.cnki.1671-7236.2021.11.006
Abstract ( 230 )   PDF (1824KB) ( 217 )  
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The experiment was aimed to study the effect of energy level during pregnancy on testicular development and immunity of offspring and its mechanism. 30 Landrace×Yorkshire(LY) sows (7-9 parity) with similar weight and backfat thickness were selected, and randomly divided into 2 groups, each group had 15 repetitions and 1 sow per repetition. From the day of pregnancy, normal energy (CON) and low energy diet (LE, 12.55 MJ/kg) were fed respectively until delivery. All sows were fed the same diet during lactation. After parturition, 15 offspring boars with an average weight ±0.05 kg were selected respectively from CON and LE groups, and fed with the same diet during the experiment. The experiment was ended at 120 day of age. The body weight of boars in each month was recorded, the daily gain and average daily feed intake were calculated. Blood samples were collected at 28 and 120 d for serum biochemical indexes and immune-related cytokines, and testis were collected at 28 and 120 d for testicular cell count and immune-related genes detection. The results showed that compared with control group, the average daily gain from 0 to 59 d in LE group was significantly decreased (P<0.01), the average daily feed intake from 28 to 89 d and the testicular weight on 0 day were significantly decreased (P<0.05), and the testicular index on 28 d was significantly increased (P<0.05). The number of Leydig cells at 0 and 120 d, germ cells at 120 d and Sertoli cells at 28 and 120 d in LE group were significantly decreased (P<0.05). TG content 28 d and T content at 120 d of serum were significantly increased (P<0.01), E2 content was significantly decreased (P<0.01) in LE group. The contents of TG, T and E2 were influenced by time and energy (P<0.01). The contents of TC and HDL-C in blood of LE group were significantly decreased at 120 d (P<0.01), and there was no interaction between time and energy among LDL-C, TC and HDL-C (P>0.05). The results of immune-related cytokine assay showed that the concentrations of IL-1α, TNF-α, IL-1β and IgG in the blood of boars increased significantly with the increasing of boar age (P<0.01), and TNF-α and IL-1β had the interaction of energy and time. Compared with control group, the content of TNF-α in LE group at 28 d was significantly decreased (P<0.05), and the level of IL-1β in LE group at 120 d was significantly increased (P<0.05). The relative expression level of ZO1 gene at 28 d and Occludin gene at 0 and 28 d in LE group were significantly decreased (P<0.05), and the relative expression level of JAM1 gene at 28 and 120 d was significantly increased (P<0.05). The relative expression level of CCL4 gene at 0 d and IL-1α gene at 28 d was significantly decreased (P<0.05), and the relative expression level of CCL2 gene at 0 and 120 d and ICAM1 and JAK2 genes at 28 d was significantly increased (P<0.05). Compared with control group, the relative expression of TLR1 gene at 28 d and TNFRSF1A gene at 0 d were significantly decreased (P<0.05), while the relative expression levels of NFKBIA, IL-1β and IFNG genes at 0 d were significantly increased (P<0.05). In summary, 12.55 MJ/kg energy intake during pregnancy could reduce the daily gain, average daily feed intake and testicular weight of offspring boars, reduce the number of germ cells in testis of offspring boars and the expression of immune-related cytokines and testicular genes, which had a profound impact on the immune ability and reproductive performance of adult offspring.
Study on Protective Effect of Celery Fermentation Broth on Ulcerative Colitis in Mice
ZHAO Dong, CAO Jinhu, JIN Huiqin, SHAN Yanke, LIU Xiaoming, LIU Fei
2021, 48(11):  3975-3984.  doi:10.16431/j.cnki.1671-7236.2021.11.007
Abstract ( 200 )   PDF (15431KB) ( 50 )  
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The purpose of this experiment was to study the changes in the main functional components of celery juice after fermentation and the effect of celery fermentation broth on the prevention and immunoregulation of dextran sodium sulfate (DSS) induced ulcerative colitis (UC) in mice. Fifty male 4-week-old C57BL/6 mice were randomly divided into blank group, model group, and low, medium, and high concentration of cetery fermentation broth groups, with 10 mice in each group. The mice in blank and model groups were given sterile normal saline, the others were given different concentrations of celery fermentation broth by gavage for 7 days, according to the dose standard of 10 μL/g BW. Beginning on the 8th day, except for the blank group to drink sterile water freely, the mice in other groups drunk 3% DSS solution for 7 days. During the induction process, the mice were weighed daily and scored by the disease activity index (DAI). On the 14th day, blood was obtained through the eyeball method, and then the mice were killed by dislocation of spine, and the colon tissue was taken to measure the length and made into pathological sections by the hematoxylin eosin (HE) staining method for histopathological analysis. CD4+ and CD8+ T lymphocyte ratios in peripheral blood of mice was detected by flow cytometry sorting technology. The serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interferon-γ (IFN-γ) and interleukin-10 (IL-10) were detected by enzyme-linked immunosorbent assay (ELISA). The results showed that:①After fermentation, the contents of various active ingredients such as total acid, total sugar, total polyphenols, flavonoids, vitamin C and superoxide dismutase (SOD) in celery juice increased significantly (P<0.05). ②Compared with the model group, high concentration group could reduce the weight loss (P<0.01), colon shortening (P<0.01) and DAI reduction (P<0.05) of mice induced by DSS. ③Histopathological analysis showed that the symptoms of UC in the fermentation broth groups were improved to varying degrees, the intestinal gland structure was relatively complete, the goblet cells were slightly reduced, and only a few lymphocytes and neutrophils were infiltrated. ④The results of flow cytometry analysis showed that CD4+/CD8+ T lymphocytes in the whole blood of the fermentation broth groups were increased (P<0.01) compared with the model group. ⑤The ELISA test results showed that the content of IL-6, TNF-α and IFN-γ in the fermentation broth groups were lower, and the content of IL-10 was higher than that of the model group (P<0.01). In summary, the main active components of celery juice after fermentation were generally higher than those of unfermented ones. Celery fermentation broth had preventive and immunoregulatory effects on DSS-induced UC in mice. The mechanism might be related to maintaining the balance of CD4+ and CD8+ T lymphocytes in the peripheral blood, inhibiting the expression of pro-inflammatory factors (IL-6, TNF-α and IFN-γ), and increasing the expression of anti-inflammatory factors (IL-10).
Effects of Fermented Shiitake Residues on Growth Performance, Colonic Volatile Fatty Acids Contents and Microbial Structure in Weaned Piglet
PENG Qiaoli, QI Qien, ZHOU Guoyong, TANG Min, CHEN Dongling, LIU Shen, ZHANG Huihua
2021, 48(11):  3985-3995.  doi:10.16431/j.cnki.1671-7236.2021.11.008
Abstract ( 204 )   PDF (1230KB) ( 57 )  
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This study was carried out to investigate the effects of fermented shiitake residues (FSR) on growth performance, intestinal digestive enzyme activities, colonic volatile fatty acid contents and microbial structure in weaned piglets. A total of 100 weaned piglets (Small ear flower pigs×Duroc pigs, 50 males and 50 females) were allocated to two treatment groups with five replicates of ten pigs each. The piglets in control group were fed with the basal diet without FSR, while in experimental group were fed with the basal diet supplemented with 5% FSR. The experiment lasted for 33 days. At the end of the experiment, 6 piglets (half male and half female) were selected from each treatment and slaughtered for sampling. The growth performance, duodenal mucosal digestive enzyme activity, relative expression of jejunal tight junction protein (TJP) mRNA, colonic volatile fatty acid content and intestinal microbial structure in weaned piglets were detected. The results showed that compared with control group, in experiment group, the final weight and average daily gain (ADG) were significantly increased, and the F/G was significantly decreased (P<0.05), the activities of trypsin and β-amylase were significantly increased (P<0.05), the levels of propionic acid, butyric acid, isobutyric acid and isovaleric acid in colonic were significantly increased (P<0.05), the relative abundance of Fibrobacteres and Streptococcus in colonic were significantly increased (P<0.01;P<0.05). The expression of TJP1 and TJP2 genes between the two groups was not significant difference (P>0.05). In conclusion, dietary supplementation with 5% FSR enhanced growth performance, improved intestinal digestive enzyme activities and production of volatile fatty acids and favorably modulated intestinal microbiota composition in weaned piglets.
Effect of N-acetylcysteine on Immune Response and Intestinal Mucosal Energy Status in Piglets Challenged with Lipopolysaccharide
WANG Qian, WANG Manli, NING Nan, WANG Shuaijie, TAN Zihan, WANG Lei, ZHAO Di, ZHANG Qian, HOU Yongqing
2021, 48(11):  3996-4003.  doi:10.16431/j.cnki.1671-7236.2021.11.009
Abstract ( 183 )   PDF (835KB) ( 28 )  
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This experiment was conducted to determine the effects of N-acetylcysteine (NAC) on the immune response and intestinal mucosal energy status in piglets challenged with lipopolysaccharide (LPS). Twenty-four healthy weaned piglets (11.58 kg±0.26 kg) were randomly allocated into 3 groups (control, LPS and NAC+LPS groups), with 8 replicates in each group. After a 3-day adaptation period, the piglets in each group were fed with basic diet (the NAC+LPS group was supplemented with 500 mg/kg NAC). On the 21st day of the trial, all the piglets were weighed and injected intraperitoneally with LPS (for LPS and NAC+LPS groups, injected with 100 μg/kg BW LPS) or the same amount of normal saline (for control group). 3 hours later, blood samples were collected from the anterior vena cava. Then all piglets were sacrificed, and samples from the small intestinal mucosal, liver and lymph node were collected. Blood cell counts, the level of adenylate in small intestinal mucosal and the relative expression levels of genes were detected. The results showed that:Compared with the control group, the number of white blood cells and neutrophils in the blood was significantly increased (P<0.05), the number of lymphocytes was significantly reduced (P<0.05). AMP level and AMP/ATP ratio in the small intestinal mucosal were increased (P<0.05), and the ATP level was decreased (P<0.05). The relative expression of dsRNA dependent protein kinase R (PKR), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) and myxovirus resistance protein 1 (MX1) genes in liver and lymph nodes were up-regulated in the LPS group (P<0.05). Compared with the LPS group, the number of white blood cell and neutrophils, the percentage of eosinophils and lymphocytes were decreased (P<0.05), the ileal mucosal AMP level and the AMP/ATP ratio decreased (P<0.05), the relative expression of IFIT1, PKR and MX1 genes in liver were up-regulated (P<0.05), and the relative expression of IFIT1 gene in lymph nodes was down-regulated (P<0.05) in the NAC+LPS group. These results suggested that NAC could alleviate the immune response of piglets and improve the energy state of the intestinal mucosa in LPS-challenged piglets.
Safety Evaluation of Zhenling Zengmian Powder on Tilapia
HE Wenna, XIAO Ting, HE Ying, ZHOU Yingning, JIANG Dongfu, LU Jingzhuan, CHEN Zhongwei, HU Tingjun
2021, 48(11):  4004-4013.  doi:10.16431/j.cnki.1671-7236.2021.11.010
Abstract ( 152 )   PDF (1011KB) ( 77 )  
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The purpose of the trial was to determine the safety of Zhenling Zengmian powder on tilapia at the recommended clinical dose, study the safe range of tilapia dose and comprehensively evaluate the efficacy of the drug, so as to provide important basis and guidance for clinical rational drug use. Five groups were set up in this experiment, including blank group (basic feed), 1 time dose group (including 0.4% of Zhenling Zengmian powder), 3 times dose group (including 1.2% of Zhenling Zengmian powder), 5 times dose group (including 2.0% of Zhenling Zengmian powder), and 10 times dose group (including 4.0% of Zhenling Zengmian powder). The tilapia were fed continuously for 30 days, and the clinical signs, blood physiological and biochemical indexes, organ index and histopathology of tilapia were detected on the 15th and 30th day, respectively. The results showed that the activity, feeding, body color and excretion of tilapia in each dose group were good, which was not significantly different from that in the blank group. The weight gain rate and specific growth rate were the highest in the 1 time dose group, which were significantly or extremely significantly higher than those in the blank group (P<0.05;P<0.01), but decreased with the increase of the amount of Zhenling Zengmian powder from 0 to 15 days. From 15 to 30 days, the weight gain rate of each dose group of Zhenling Zengmian powder was significantly higher than that of the blank group (P<0.01). Some of the blood physiological and biochemical indexes of tilapia in each dose group were significantly different from those in the blank group, but they were all within the normal range. The tilapia spleen index of each dose group at 0-15 days and the 1 time dose group at 15-30 days were significantly higher than that of the blank group (P<0.05). The liver and spleen sections of tilapia in each group were observed under microscope, and no cytotoxic changes caused by drugs were found. The above results showed that Zhenling Zengmian powder was safe to feed tilapia for continuously 30 days at or below the 10 times of recommended dose. As a feed additive, it could improve the growth performance and enhance immunity of tilapia, which was of great significance to promote the rapid and efficient development of tilapia culture.
Effects of Plant Essential Oil-Citric Acid Complex on Intestinal Barrier Function and Inflammatory Response of Broilers
WEI Qing, WEI Zhaoyang, WANG Kewei, WU Zhiwei, QIAO Jiayun, LI Haihua
2021, 48(11):  4014-4024.  doi:10.16431/j.cnki.1671-7236.2021.11.011
Abstract ( 231 )   PDF (1554KB) ( 53 )  
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The effects of adding different levels of plant essential oil-citric acid complex in the diet on the intestinal barrier function, serum cytokine content and key immune protein gene expression in broilers were studied in this experiment, in order to explore the appropriate additive dosage of the complex for broilers. A total of 1 344 one-day-old healthy broilers (Cobb) were divided into 4 groups with 12 replicates per group and 28 birds per replicate. Broilers in the control group were fed with basal diet, and those in the experimental groups (T1, T2, T3) were fed the basal diets supplemented with 0.8, 1.0 and 1.5 kg/t plant essential oil-citric acid complex, respectively. The experiment lasted for 42 days. On the ending of the test period, 6 broilers close to the average weight were selected for each repetition to collect blood and slaughtered. The jejunum, spleen, mesenteric lymph nodes (MLNs) were taken and the contents of D-lactic acid (D-LA), cytokine and activity of diamine oxidase (DAO) in serum were measured. Meanwhile, the mRNA expression of tight junction protein in the jejunum mucosa and Toll-like receptors (TLRs) in spleen and MLNs were measured. The results showed that:Compared with the control group, the contents of serum D-lactic and diamine oxidase(DAO) activity of broilers were extremely significantly decreased in T2 group (P<0.01) and significantly decreased in T3 group (P<0.05). The relative expression of Claudin-1 mRNA in broilers in both T2 and T3 groups were significantly increased (P<0.05), and the relative expression of ZO-1 mRNA in T2 group was increased significantly (P<0.05). The content of serum IL-1β was decreased significantly (P<0.05), and the relative expression level of TLR4 mRNA in MLNs was decreased significantly (P<0.05) of broilers in T1 group. The content of serum IL-1β was decreased extremely significantly (P<0.01), the content of serum IL-2 was increased significantly (P<0.05), and the relative expression level of TLR4 mRNA in spleen (P<0.01) and MLNs (P<0.05) were decreased of broilers in T2 group. The contents of serum IL-1β (P<0.01) and TNF-α (P<0.05) were decreased, and the relative expressions of TLR4 mRNA in spleen and MLNs was decreased extremely significantly (P<0.01) of broilers in T3 group. Compared with T1 group, the contents of serum D-lactic, DAO and TNF-α in T2 group were decreased significantly (P<0.05), and the content of serum IL-1β of broilers in T3 group was decreased significantly (P<0.05). In conclusion, adding plant essential oils-citric acid complex to broiler diets could increase the expression of intestinal tight junction proteins, reduce intestinal permeability, and down-regulate the expression of pro-inflammatory cytokines, thereby enhancing the intestinal barrier function and alleviating inflammation. Under the experimental conditions, the appropriate addition level of plant essential oil-citric acid complex was 1.0 kg/t.
Proteome Identification and Potential Function Analysis of Milk Fat Globule Membrane in Horses
CHANG Huaxiang, LI Xiaobin, XIONG Suyu, ZHANG Junyu, CHEN Kaixu, LIU Jiancheng, LI Ning, ZANG Changjiang, LI Fengming
2021, 48(11):  4025-4034.  doi:10.16431/j.cnki.1671-7236.2021.11.012
Abstract ( 220 )   PDF (6039KB) ( 18 )  
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The purpose of this study was to investigate the protein composition and potential biological function of milk fat globule membrane (MFGM) in horses. Twelve healthy thoroughbred mares with 5 years old and body weight of 450-500 kg were selected as experimental animals, they were mainly fed with hay. The feeding-and-management conditions of all experimental horses were the similarity. 12 samples were mixed with 4 samples in each group, and MFGM proteins were isolated and extracted from horse milk on the 60th day after delivery. Of these, 4 individual sample was mixed and 3 biological samples were prepared. The tryptic peptides of samples were identified by high-resolution mass spectrometry and bioinformatics analysis. The results showed that 310 proteins were identified in MFGM fraction of horses, the identified MFGM proteins were mainly involved in biological regulation, stimulus response, localization, multicellular biological processes, transportation, signaling, cell communication, developmental processes, cell differentiation and immune system process on the basis of biological processes. They were mainly located in the extracellular area, membrane, vesicle, nucleolus and mitochondria according to cellular components. They were mainly involved in catalytic activity, protein binding, carbohydrate derivative binding and small molecule binding regarding of molecular functions. KEGG pathway analysis of MFGM proteins indicated that several proteins were involved in platelet activation pathway, endocytosis, fatty acid biosynthesis and Ras signaling pathways. The protein-protein interaction network analysis of MFGM protein in horses contained 215 proteins. The results of this study revealed the complexity of MFGM proteome in horses, and provided scientific data for analyzing its nutritional and biological functions.
Genetics and Breeding
Transcriptomic Analysis of Mink Ovaries at Embryonic Diapause and Activation Stages Based on High-throughput Sequencing Technology
HAN Yuping, ZHAO Xiangyuan, FAN Bingfeng, LIU Lixiang, SHAO Jing, XU Baozeng
2021, 48(11):  4035-4046.  doi:10.16431/j.cnki.1671-7236.2021.11.013
Abstract ( 181 )   PDF (6506KB) ( 24 )  
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This experiment was aimed to obtain the mink ovarian transcriptome at the embryonic diapause and activation stages, dig the differentially expressed genes (DEGs) between them, and explore the biological functions of DEGs, which could provide a reference for investigating the molecular mechanisms underlying ovarian signals regulate the embryonic diapause and activation. Ovaries were collected randomly from eight healthy female mink (four mink for embryonic diapause and activation stages) were processed for transcriptomic sequencing using Illumina HiSeq platform RNA-Seq technology. Ovarian DEGs were screened and analyzed through bioinformatics. The results showed that a total of 661 195 568 raw reads were obtained by sequencing, 650 834 900 clean reads were obtained by filtering, and 389 895 unigenes were obtained by assembly. unigenes was annotated by comparing with Nr, GO, KOG and KEGG databases. 156 419, 122 657, 58 320 and 72 653 unigenes were annotated in Nr, GO, KOG and KEGG databases, respectively. There were 1 797 DEGs in mink ovaries at the embryonic diapause and activation stages. Compared with the embryonic diapause mink ovary, 1 298 DEGs in the activation stage ovaries were significantly up-regulated and 499 DEGs were significantly down-regulated. GO function analysis showed that the biological processes of DEGs significantly enriched mainly included transmembrane signaling receptor activity, signaling receptor activity, G-protein coupled receptor activity, enzyme linked receptor protein signaling pathway, cell surface receptor signaling pathway, cell cycle arrest, arylesterase activity. KEGG pathway analysis showed that the DEGs in the mink ovaries during embryonic diapause and activation stages were significantly enriched in the neuroactive ligand-receptor interaction. In this study, high-throughput sequencing technology was used to obtain transcriptome information of mink ovaries during embryonic diapause stage and during embryonic activation stage, which laid a foundation for further investigating the roles of mink ovary in regulating embryonic diapause and activation.
Cloning and Bioinformatics Analysis of XCL1 Gene CDS in Gushi Chicken
TIAN Huihui, DING Mengxia, GUO Yujie, SU Aru, SUN Guirong, TIAN Yadong, JIANG Ruirui, HAN Ruili, KANG Xiangtao, YAN Fengbin
2021, 48(11):  4047-4054.  doi:10.16431/j.cnki.1671-7236.2021.11.014
Abstract ( 184 )   PDF (1848KB) ( 61 )  
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The aim of this study was to clone the CDS region of lymphotactin (XCL1) gene in chicken and explore its biological characteristics. Using the total RNA of thymus tissue in Gushi chicken as the template, the CDS of the gene was amplified by RT-PCR and cloned, and the related bioinformatics analysis was carried out by bioinformatics software. The results showed that XCL1 gene CDS in Gushi chicken was 294 bp in length and encoded 97 amino acids. The homology analysis showed that the homology of XCL1 gene CDS sequence in chicken with Bos taurus, Capra hircus, Ovis aries, Homo sapiens, Mus musculus, Sus scrofa and Rattus norvegicus were 53.7%, 53.0%, 52.9%, 51.8%, 51.1%, 50.9% and 50.4%, respectively. The results of phylogenetic tree showed that chicken was a non mammal, and XCL1 gene was far from mammalian. The molecular formula of the XCL1 encoded protein was C491H822N150O132S6, the theoretical isoelectric point was 10.95, which was a basic protein. The molecular mass was 11.13 ku, the fat coefficient was 102.37, and the instability coefficient was 51.44, which was an unstable protein, and the half-life was 30 h. It had a transmembrane structure (amino acids 5-27) and a signal peptide region (amino acids 1-18), and was a hydrophilic secreted protein. There were 8 potential phosphorylation modification sites and 3 potential glycosylation modification sites in XCL1 protein. The secondary structure of XCL1 protein was composed of alpha helix (35.05%), beta turn (5.15%), extended chain (26.80%) and random coil (32.99%). The tertiary structure prediction was consistent with the secondary structure. Subcellular localization analysis showed that XCL1 protein mainly functions outside the cell;the protein had a conserved SCY domain at amino acid residues 31-88. The protein could form an interaction network with OXT, PTAFR, GPR132 and XCR1. The results of this experiment provided a theoretical reference for further study of XCL1 gene function in chicken.
Identification and Function Prediction Analysis of circ_CSPP1 in Ovarian Tissues of Xiang Pigs
HUANG Yali, NIU Xi, LU Huan, HUANG Shihui, WANG Jiafu, RAN Xueqin, LI Sheng
2021, 48(11):  4055-4066.  doi:10.16431/j.cnki.1671-7236.2021.11.015
Abstract ( 132 )   PDF (12286KB) ( 55 )  
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The purpose of this study was to explore the potential functions of circ_CSPP1 in the ovarian tissues of Xiang pigs. In this study, the sequence of circ_CSPP1 was spliced by CIRI-full, the conservative analysis of circ_CSPP1 was calculated by online blat of circBase database, and the characteristics of circ_CSPP1 in the ovarian tissues of Xiang pigs were identified and analyzed by RT-PCR technology and Sanger sequencing technology. Moreover, miRanda, RNAhybrid and PITA softwares were used to predict and analyze the circ_CSPP1 bound miRNA molecular and miRNA target genes. Then GO and KEGG enrichment analyses were performed for the target genes. The results showed that the isolated circ_CSPP1 was conservative and originated from exons 8-12 of CSPP1 gene, which could be served as molecular sponges of 14 miRNAs, such as ssc-miR-324, ssc-miR-10a-5p, ssc-miR-9847-3p, ssc-miR-365-5p, ssc-miR-92b-5p, ssc-miR-885-3p, ssc-miR-7139-3p, ssc-miR-9851-3p, ssc-miR-33b-3p etc, and targeted 755 protein genes. GO enrichment analysis showed that the target genes of circ_CSPP1 were significantly enriched in 153 GO terms, in which cell maturation, cell division sites, negative regulation of cell migration, Wnt signaling pathway, cyclin binding, uterine development, activation of MAPK activity, focal adhesion and cleavage groove were related to ovarian function. KEGG enrichment analysis showed that the target genes of circ_CSPP1 were enriched into 260 signal pathways, of which 29 signal pathways were significantly enriched, and the target genes mainly concentrated in cancer pathway, cell cycle, estrogen signaling pathway, PI3K-AKT signaling pathway, ovarian steroidogenesis, oxytocin pathway and apoptosis etc. In conclusion, circ_CSPP1 might act as molecular sponges of many miRNAs to regulate the biological processes including proliferation and apoptosis of ovarian granulosa cells, follicle maturation, ovulation process, ovarian steroidogenesis and premature ovarian failure in the ovarian tissues of Xiang pigs.
Expression and Regulation of NEAT1 in Mouse Uterine Tissue During Early Pregnancy
ZHANG Xinyan, JIA Yanni, CAI Rui, ZHANG Ruixue, JIN Yaping, LIN Pengfei
2021, 48(11):  4067-4073.  doi:10.16431/j.cnki.1671-7236.2021.11.016
Abstract ( 228 )   PDF (7996KB) ( 466 )  
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The test was aimed to study the expression and regulation of nuclear paraspeckle assembly transcript 1 (NEAT1) in uterine tissue during early pregnancy of mice, and provided scientific evidence for revealing the mechanism of NEAT1 in mouse embryo implantation. The models of early pregnancy, pseudopregnancy, artificial decidualization and steroid hormone treatments were constructed respectively, and the expression of NEAT1 in the uterine tissue was detected by RNAscope and Real-time quantitative PCR techniques. The results showed that the expression of NEAT1 was dynamically changing in uterine tissue in early pregnancy. NEAT1 was detected in endometrium and muscular layer on days 1-5 of pregnancy and mainly observed in endometrial luminal and glandular epithelial cells. NEAT1 was highly expressed in decidual cells on days 6-8 of pregnancy. There was no detectable NEAT1 staining at conceptus. Similar with those of early pregnancy, the expression of NEAT1 in pseudopregnant uteri was observed in endometrium and muscular layer. In the artificial decidulization model, NEAT1 was mainly localized in the decidualised cells and the expression level was extremely significant higher than that on the control group (P<0.01). In the steroid hormone treatment model, progesterone (P4) and estradiol (E2) could significantly up regulate the expression of NEAT1 (P<0.05), and had a more significant effect on the up regulation of NEAT1 expression in endometrial stromal cells. The above results indicated that NEAT1 was involved in regulating early pregnancy and decidulization processes in mice, and the expression level was mainly regulated by steroid hormones.
Effect of Sphingomyelin on Myogenic Differentiation of Mouse Muscle Satellite Cells C2C12
HOU Naipeng, WANG Yu, TAO Cong, WANG Yanfang
2021, 48(11):  4074-4083.  doi:10.16431/j.cnki.1671-7236.2021.11.017
Abstract ( 210 )   PDF (3690KB) ( 37 )  
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The study was aimed to provide a theoretical basis for investigating the effect of sphingomyelin on development of skeletal muscle of UCP1 knock-in (UCP1-KI) pigs, through detection the function of sphingomyelin in myogenic differentiation of C2C12 mouse satellite cells. ELISA method was used to measure the content of total sphingomyelin in the back muscle and serum from both wild type and UCP1-KI pigs. CCK8 was used to detect the effects of different concentrations of sphingomyelin (0, 5, 20, 50 and 100 μg/mL) on the proliferation of C2C12 cells. The successful differentiation was established and confirmed by morphological observation, the immunofluorescence of Myogenin and the mRNA levels of the myogenic differentiation markers, including Myf5, MyoD, Myogenin and MRF4. Then, the C2C12 mouse satellite cells were treated with different concentrations of sphingomyelin during myogenic differentiation. Cells were harvested after 6 days of differentiation and the optimal sphingomyelin concentration was determined based on the formation of myotubes, and immunofluorescence staining signal of Myogenin and the upregulation of differentiation markers (Myf5, MyoD, Myogenin and MRF4). Furthermore, the gene expression levels of cyclins (CyclinD1, CyclinE, CDK2 and CDK4) in C2C12 cells at 2, 4 and 6 d of myogenic differentiation were detected by Real-time quantitative PCR, and cell viability was detected by CCK8 at 2 d of myogenic differentiation. The results showed that the content of total sphingomyelin in the back muscle of UCP1-KI pigs was significantly increased (P<0.05) compared with those from wild-type pigs after cold stimulation. No significant difference in serum sphingomyelin content (P>0.05) was observed. Sphingomyelin had no effect on the proliferation of C2C12 cells before myogenic differentiation (P>0.05). After 6 days of myogenic differentiation, myotubes was formed obviously and the mRNA levels of myogenic differentiation markers (Myf5, MyoD, Myogenin and MRF4) were significantly up-regulated (P<0.01). Compared with control group without sphingomyelin, the Myogenin positive cells and myotube fusion index were significantly increased in the 20 μg/mL sphingomyelin group (P<0.05). Furthermore, the expression levels of Myogenin and MRF4 genes were significantly increased (P<0.05). With 20 μg/mL sphingomyelin, the expression levels of CyclinD1 and CDK4 in the treatment group were significantly higher than those from control group at 2 d of differentiation (P<0.05), and the cell viability of the treatment group was significantly higher than that of the control group (P<0.05). The mRNA expression levels of CyclinD1, CyclinE, CDK2 and CDK4 in the treatment group were significantly lower than those of control group at 6 d of differentiation (P<0.05). In this study, the results revealed that the addition of 20 μg/mL sphingomyelin could improve the cell viability at the early stage of myogenic differentiation and promote the myogenic differentiation efficiency of C2C12 cells, providing some references for the effect of sphingomyelin on muscle formation.
Cloning and Tissue Expression of Myf6 Gene in Nubian Goat
SONG Ying, ZHANG Sanbao, SHEN Yujian, HU Yan, ZHANG Yu, ZOU Juhong, XU Jianjian, LIAN Zitong, ZOU Jianwei, WEI Yingming, ZHENG Zihua, JIANG Qinyang
2021, 48(11):  4084-4093.  doi:10.16431/j.cnki.1671-7236.2021.11.018
Abstract ( 233 )   PDF (2467KB) ( 43 )  
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The research was aimed to clone myogenic factor 6 (Myf6) gene in Nubian goat and conducted bioinformatics analysis on it. The expression difference of Myf6 gene in different tissues of Nubian goat and the expression difference of longissimus muscle tissue of Nubian goat and Longlin goat were detected. Using the cDNA from the longissimus dorsi muscle tissue of Nubian goat as a template, PCR amplified the complete coding region (CDS) of the Myf6 gene and then verified the bacterial solution. Sequence similarity alignment and phylogenetic tree construction were carried out. The physicochemical properties of Myf6 protein were analyzed and its hydrophilicity/hydrophobicity was predicted. The protein secondary structure and tertiary structure were predicted. The expression of Myf6 gene in heart, liver, spleen, kidney, longissimus dorsi muscle, subcutaneous fat, rumen and Longlin goat longissimus dorsi muscle were detected by Real-time quantitative PCR. The results showed that the CDS sequence of Myf6 gene in Nubian goat was 729 bp and encoded 242 amino acids. The similarity of Myf6 amino acid sequence between Nubian goat and Ovis aries, Bos taurus, Sus scrofa, Equus asinus, Homo sapiens and Mus musculus were 99.3%, 98.8%, 94.1%, 93.8%, 93.4% and 89.0%, respectively. The analysis of the physical and chemical properties of the protein showed that the molecular weight of the goat Myf6 protein was 26.98 ku, which was an unstable acidic protein. The hydrophilic and hydrophobic analysis found that the protein was a hydrophilic protein. The transmembrane structure and signal peptide prediction results showed that the protein did not contain transmembrane structure and signal peptide, and was not a transmembrane protein and secreted protein. The prediction results of secondary structure showed that the protein was mainly composed of random coil (49.59%), followed by alpha helix (33.88%), extended chain (9.50%) and beta turn (7.02%). The prediction results of the tertiary structure were consistent with those of the secondary structure. The results of Real-time quantitative PCR showed that the expression of Myf6 gene in the longissimus dorsi muscle of Nubian goat was the highest, significantly higher than that of other tissues (P<0.05), and the expression in the kidney was the lowest, but there was no significant difference with heart, liver, spleen, subcutaneous fat and rumen (P>0.05). Compared the expression of Myf6 gene in the longissimus dorsi muscle of Nubian goat and Longlin goat, the expression level of Nubian goat was significantly higher than that of Longlin goat (P<0.05). The experiment provided a reference for further revealing the role of Myf6 gene in muscle differentiation.
Preparation and Characterization of Altrenogest-β-cyclodextrin Water-soluble Derivative Inclusion Complex
YANG Yushuai, LI Ze, JIN Tianming, YANG Sheng
2021, 48(11):  4094-4104.  doi:10.16431/j.cnki.1671-7236.2021.11.019
Abstract ( 188 )   PDF (2613KB) ( 33 )  
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In order to improve the water solubility of the poorly soluble drug altrenogest(ALT), this experiment used freeze-drying to prepare altrenogest-hydroxypropyl-β-cyclodextrin (ALT-HP-β-CD) and its derivative 2, 6-dimethyl-β-cyclodextrin (DIME-β-CD) inclusion complex drug-carrying system, the linear relationship and specificity of ALT, the precision of the instrument, and the rate and stability of the inclusion complex were measured. Based on the yield of the inclusion complex and the inclusion rate of ALT in the inclusion complex, the orthogonal test design was used to optimize the optimal preparation conditions of the two types of inclusion complexes, and Fourier transform infrared spectroscopy, thermogravimetric analysis and microscopy imaging methods were used to characterize the prepared ALT inclusion complex, and the solubility method was used to determine the solubility of ALT in the inclusion complex. The results showed that:ALT had a good linear relationship in the range of 1~12 μg/mL, and the specificity was good, and the precision of the instrument was good. The average recoveries of ALT-HP-β-CD inclusion complex with 0.1, 0.2 and 0.3 mL ALT standard solution were 97.78%, 99.38% and 99.90%, respectively. The average recoveries of ALT-DIME-β-CD inclusion complex with 0.1, 0.2 and 0.3 mL of ALT standard solution were 93.86%, 97.72% and 94.93%, respectively. The best preparation process for the inclusion complex of ALT-HP-β-CD was at 55 ℃, the molar ratio of ALT to HP-β-CD was 1:7, the inclusion time was 4 h, the pH of the solution was 8, and the amount of solvent water added was 110 mL/g drug. The best preparation process of the inclusion complex of ALT-DIME-β-CD was at 55 ℃, the molar ratio of ALT to DIME-β-CD was 1:4, the inclusion time was 4 h, the pH of the solution was 9, and the amount of solvent water added was 100 mL/g drug. The inclusion complex was characterized by Fourier infrared spectroscopy, thermogravimetric analysis and microscopy imaging methods. The solubility test showed that the solubility of the inclusion complex was about 1 012 times and 1 354 times of the ALT drug, respectively, indicating that the two inclusion complexes could effectively increase the solubility of ALT. The preparation method of the inclusion complex was simple, the conditions were mild, and it was easy to industrialized production.
Estimation of Genetic Parameters of Body Size and Body Weight in Xinjiang Brown Cattle at Different Growth Stages
XU Lei, ZHANG Menghua, WANG Dan, ZHANG Xiaoxue, FAN Shoumin, LIU Jianming, LIU Jiangwei, YAN Chengxu, HUANG Xixia, WANG Yachun
2021, 48(11):  4105-4114.  doi:10.16431/j.cnki.1671-7236.2021.11.020
Abstract ( 171 )   PDF (1050KB) ( 84 )  
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This study was aimed to construct the genetic parameter estimation model of body size and body weight traits in Xinjiang Brown cattle at different growth stages and estimate the genetic parameters of growth and development traits in Xinjiang Brown cattle, in order to provide a theoretical basis for the determination of breeding target traits and the formulation of comprehensive selection index in Xinjiang Brown cattle. DMU software was used to build multi-trait animal models by taking the body size and body weight data of 2 504 Xinjiang Brown cattle of the progenies of 81 bulls from four Xinjiang Brown Cattle core breeding farms collected from 1983 to 2017 as research materials, taking the body weight, body height, body oblique length and chest circumference at birth, and 6, 12 and 18 months of age as the research objects, and estimate the heritability and genetic correlation of traits by taking field, birth time, birth season and sex as fixed effects, and additive effect and maternal effect as random effects. The results showed that the value of estimated body weight heritability of Xinjiang Brown cattle from birth to 18 months of age was from 0.22 to 0.61, the value of estimated body height heritability was from 0.43 to 0.46, the value of estimated body oblique length heritability was from 0.29 to 0.52, and the value of estimated chest circumference heritability was from 0.35 to 0.61. There were positive genetic correlation and phenotypic correlation among body size and weight traits at the same and different growth stages, the genetic correlation coefficient between different body size and body weight traits at the same growth stage was from 0.11 to 0.92, and the phenotypic correlation coefficient was from 0.05 to 0.92. The genetic correlation coefficient between different body size and body weight traits at different growth stages was from 0.08 to 0.92, and the phenotypic correlation coefficient was from 0.01 to 0.72. The genetic correlation coefficient of body size and body weight traits between 18 months of age and other growth stages were high, both of which belong to middle and high heritability traits. Therefore, in order to further improve the genetic progress of the growth and development traits of Xinjiang Brown cattle, we should pay more attention to body size and body weight at 18 months of age when formulating the comprehensive selection index of Xinjiang Brown cattle.
Advances in Mechanism of Aneuploidy in Mammalian Oocytes
LIU Lixiang, ZHAO Xiangyuan, SHAO Jing, FAN Bingfeng, HAN Yuping, YANG Yifeng, XU Baozeng
2021, 48(11):  4115-4124.  doi:10.16431/j.cnki.1671-7236.2021.11.021
Abstract ( 191 )   PDF (2666KB) ( 31 )  
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During meiosis, the female germ cells are prone to chromosomal separation errors, resulting in aneuploid oocytes. After fertilization, aneuploid embryos will be produced, resulting in birth defects or embryo death, which is an important factor affecting mammalian reproduction. Homologous chromosome synapsis occurs in oocytes at the early stage of the first meiosis, when DNA double strand breaks trigger recombination. The lack of crossover during recombination, the reduction of the number of recombination events and the proximity of crossover to telomeres or centromeres lead to co-oriented segregation or no segregation of chromosomes, then bringing about aneuploid oocytes. During meiosis, when there is an error in the connection between the chromosome kinetochore and the spindle microtuble, the spindle assembly checkpoint (spindle assembly checkpoint, SAC) is activated, the E3 ubiquitin ligase APC/Cyclome (APC/C) is silenced, and the APC targeting proteins securin and cyclin B are protected from degradation, thus inhibiting the separase and the continued separation of chromosomes. Until all chromosomes and spindles achieve stable bipolar orientation and are correctly arranged on the equatorial plate, SAC is closed and chromosomes are separated correctly. The absence of SAC protein in oocytes leads to SAC unable to effectively monitor the correct attachment of telomeres to the spindle, resulting in chromosome segregation errors and aneuploid oocytes. Therefore, analyzing the mechanism involved in aneuploid oocytes by modern molecular technology is an important goal to protect mammalian fertility. This article mainly introduced the characteristics of oocyte meiosis, and expounded in detail the molecular mechanism of chromosome segregation error in oocyte aneuploidy, in order to provide reference for the development of treatment methods for aneuploid oocytes.
Effects of Partially Replacing Glycerol with Glutamine on Capacitation and in vitro Fertilization Ability of Frozen-thawed Pig Sperm
ZHANG Yuyang, WANG Qiuyue, LYU Yanqiu, CHEN Xuan, XU Haifeng, ZHANG Zhengwen, JIN Yi
2021, 48(11):  4125-4132.  doi:10.16431/j.cnki.1671-7236.2021.11.022
Abstract ( 183 )   PDF (1666KB) ( 34 )  
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The purpose of this experiment was to explore the effect of glutamine (Gln) instead of partial glycerol on capacitation and in vitro fertilization ability of frozen-thawed pig sperm. The experiment was divided into 6 groups:3% glycerol control group and 5 treatment groups (groups Ⅰ-Ⅴ:2% glycerol+glutamine (0, 20, 40, 80 and 100 mmol/L, respectively)). The sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, protamine deficiency, capacitation and in vitro fertilization of frozen-thawed Songliao Black pig sperm were detected. The results showed that replacing part of glycerol with glutamine could improve the quality of frozen thawed sperm, and the degree of improvement was affected by the concentration of glutamine. Compared with control group Ⅰ, the sperm quality parameters in group Ⅰ decreased significantly (P<0.05). Compared with group Ⅰ, the sperm motility, acrosome integrity and viability in group Ⅱ increased significantly (P<0.05), the sperm mitochondrial membrane potential in group Ⅲ increased significantly (P<0.05), and the sperm motility, plasma membrane integrity, acrosome integrity, viability and mitochondrial membrane potential in group Ⅴ increased significantly (P<0.05). It showed that replacing part of glycerol with glutamine had a great impact on sperm quality, and higher quality sperm could be obtained when glutamine was 100 mmol/L. Therefore, 100 mmol/L glutamine was used in subsequent experiments. Compared with control group, the sperm protamine deletion rate of 2% glycerol+100 mmol/L glutamine treatment group decreased significantly (P<0.05), there was no significant difference in sperm capacitation(P>0.05), but the cleavage rate of embryo was increased significantly (P<0.05). In conclusion, glutamine could be used as a new cryoprotectant replacing part of glycerol to improve the quality of pig semen, reduce the toxic effect of glycerol on pig semen, and provide technical support for cryopreservation and commercial production.
Research Advances on Regulation of Animal Follicular Development by miRNA
FENG Guanghang, JIANG Shengwei, LI Yaokun, LIU Guangbin, ZOU Xian, LIU Dewu
2021, 48(11):  4133-4142.  doi:10.16431/j.cnki.1671-7236.2021.11.023
Abstract ( 185 )   PDF (2803KB) ( 38 )  
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Fertility of female animals is based on follicular development, which is regulated by complex genetic networks. microRNA (miRNA) is a kind of non-coding RNA that regulates gene expression primarily at the post-transcriptional levels. In the past decade, studies on miRNA in follicles have revealed the key roles of miRNA in follicular development and function. This paper focuses on the follicular development process of female animals, and analyzes the function of miRNA carried by follicular fluid exosomes. The mechanism of miRNA regulating follicular development was summarized, including activation of primordial follicles and selection of growing follicles. The authors also introduced the growth regulation of miRNA on follicular granulosa cells and theca cells. However, most of the current follicular miRNA studies are focused on cellular knockdown model studies, and knockout models are less studied. Therefore, current research should increase the application of cellular and in vivo knockout models, which is helpful to better understand the function of miRNA in follicular development. These results can provide a reference for in-depth analysis of the molecular mechanism of reproductive traits regulation in female animals.
Preventive Veterinary Medicine
Pathogenicity and Whole Genome Sequence Analysis of Porcine Deltacoronavirus CH7328 Strain
WANG Zhengfan, ZHU Lisai, LI Fei, XIANG Rui, LI Yiyun, YING Biyun, WANG Guiping, BAI Aiquan, JIA Aiqing
2021, 48(11):  4143-4153.  doi:10.16431/j.cnki.1671-7236.2021.11.024
Abstract ( 212 )   PDF (5539KB) ( 51 )  
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In order to understand the pathogenicity of Porcine deltacoronavirus (PDCoV) CH7328 strain, and analyze its genetic variation and genetic evolution, this test used virus culture, indirect immunofluorescence assay (IFA), virus growth curve and piglet pathogenicity test to identify the growth characteristics and pathogenicity of the virus, and analyze the entire genome sequence of the virus strain. The results showed that ST cells infected with PDCoV CH7328 strain showed swelling and rounding, the cells were broken glass-like and brushed, and the cells clustered and fell off. The results of the IFA test showed that the PDCoV CH7328 strain was mainly distributed in the cytoplasm. The growth curve results showed that the PDCoV CH7328 strain began to proliferate at 6 h, and reached a peak at 36 h, the corresponding TCID50 was 10-8.0/mL, and then gradually decreased. The pathogenicity results of piglets showed that the piglets in challenge group showed clinical symptoms such as diarrhea, dehydration and anorexia, the small intestine was obviously thinner, transparent and full, and the small intestinal villi were severely shed, atrophy or reduced, and intestinal villi cells were necrotic. The results of fecal pathogen detection showed that the virus could be detected on the second day of the challenge, after which the detoxification continued to increase, while the normal control piglets did not have any clinical signs. Whole genome sequencing and sequence analysis showed that the full length of the genome of the PDCoV CH7328 strain was 25 420 bp, which was the highest similar to the domestic GD strain, reaching 99.9%. The genetic evolution results showed that the PDCoV CH7328 strain was in the same evolutionary branch with domestic HKU15-155, CHN-HN-2014 and GD strains, and the genetic relationship was the closest. The results of this study provided a reference data and theoretical basis for the screening of PDCoV vaccine candidate strains and the prevention and control of pig farm diseases in my country.
Prokaryotic Expression of African Swine Fever Virus pS273R Protein and Preparation of Its Polyclonal Antibody
ZHAO Gaihong, LI Tingting, ZHANG Taoqing, CHEN Xin, WANG Xiao, LI Changyao, ZHANG Zhaoxia, ZHENG Jun, WENG Changjiang
2021, 48(11):  4154-4161.  doi:10.16431/j.cnki.1671-7236.2021.11.025
Abstract ( 212 )   PDF (3481KB) ( 68 )  
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To provide the material for the functional study of ASFV pS273R, ASFV pS273R protein was expressed and polyclonal antibody against pS273R protein was prepared in present study. The recombinant pS273R protein expressed in E. coli expression system was purified by Ni-affinity column and molecular sieve, respectively. Specific polyclonal antibodies against the pS273R protein were obtained by the immunized BALB/c mice, and the anti-pS273R polyclonal antibody (pAb) was purified from the mouse serum by using a Protein G affinity chromatography column. The reactivity and specificity of polyclonal antibodies were evaluated by Western blotting, IFA and IP. The results showed recombinant pS273R protein was efficiently expressed in E. coli as when induced with 16 ℃ and the concentration of IPTG at 0.1 mmol/L. The Flag-tagged pS273R protein in HEK293T cells and the ASFV-encoded pS273R in porcine alveolar macrophage (PAMs) were detected by Western blotting, IFA and IP using the mouse anti-pS273R pAb. In summary, ASFV pS273R was successfully expressed in E. coli, preparation and purification of mouse anti-S273R pAb had good specificity and reactivity. The availability of anti-pS273R pAb laid an important foundation for further research studying the biological function of pS273R protein.
Preparation of Polyclonal Antibody of SipA in Salmonella Enteritidis and Analysis of Its Expression Regulation by Non-coding Small RNA RyhB-1/2
MENG Xia, HE Mengping, WEI Huaxu, ZHANG Ke, WANG Heng, ZHU Guoqiang
2021, 48(11):  4162-4169.  doi:10.16431/j.cnki.1671-7236.2021.11.026
Abstract ( 206 )   PDF (2200KB) ( 18 )  
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The purpose of this study was to express Salmonella virulence protein SipA through pET prokaryotic expression system and prepare polyclonal antibodies against SipA protein, so as to analyze the regulatory effect of noncoding small RNAs RyhB-1 and RyhB-2 of Salmonella Enteritidis SE50336 on SipA. pET28a-sipA recombinant plasmid was constructed by pET prokaryotic expression system and transformed into E. coli BL21(DE3) competent cells. After optimizing and obtaining the best protein induction conditions, the protein was induced and expressed. The expression of recombinant protein was analyzed by 10% SDS-PAGE. The recombinant protein was purified by nickel affinity chromatography. BALB/c mice were immunized with purified SipA protein and Freund adjuvant to obtain specific polyclonal antibody against SipA protein, and the specificity of the prepared serum was analyzed. Western blotting technique was used to detect wild strain SE50336 and RyhB single deletion strains SE50336ΔryhB-1, SE50336ΔryhB-2 and RyhB double deletion strain SE50336ΔryhB-1ΔryhB-2 so as to analyze the regulatory effects of RyhB-1 and RyhB-2 on SipA protein. The results showed that the recombinant plasmid pET28a-SipA was successfully constructed and transformed into E. coli BL21(DE3) competent cells. SDS-PAGE showed that the recombinant protein was mainly expressed in the form of inclusion body, with a size of 75 ku. After purification by affinity chromatography, the purity of SipA protein was high. The results of Western blotting showed that the polyclonal antibody could specifically detect the SipA recombinant protein of Salmonella Enteritidis. Compared with wild strain, the expression of SipA protein in RyhB single deletion strain SE50336ΔryhB-1 and SE50336ΔryhB-2 decreased, especially in double deletion strains, indicating that RyhB-1 and RyhB-2 could upregulate the expression of SipA protein. The purification of SipA recombinant protein and the preparation of SipA polyclonal antibody laid a foundation for further study on the regulatory mechanism of RyhB-1 and RyhB-2 on SipA protein.
Differential Analysis of miRNA Expression Profiles in Liver of Duck Infected with Duck Enteritis Virus
ZENG Maoqin, LIU Yanhan, ZHANG Qiandong, BI Wenwen, YE Ni, WANG Yizhou, YANG Ying, CHENG Zhentao, WEN Ming
2021, 48(11):  4170-4181.  doi:10.16431/j.cnki.1671-7236.2021.11.027
Abstract ( 185 )   PDF (3506KB) ( 16 )  
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In order to explore the differential expression of miRNA in liver of duck infected by Duck enteritis virus (DEV), in this experiment, 30-day-old hemp ducks were inoculated with the DEV-GZ strain through the leg muscles. Duck liver tissue samples were collected at 66, 90 and 114 h after infection, and total RNA was extracted from the tissue. After passing the quality inspection, high-throughput sequencing technology performed miRNA sequencing on the samples of control and test groups, and screened out the differentially expressed miRNA in liver of DEV-infected duck, performed bioinformatics GO functional classification and KEGG signal pathway analysis, and randomly selected some differentially expressed miRNA for Real-time quantitative PCR verification. The results showed that when DEV infected ducks at 66, 90 and 114 h, the number of differentially expressed miRNAs in liver were 227, 225 and 231, respectively. GO function annotations showed that the differentially expressed miRNA in liver of infected ducks were mainly classified into cellular processes, single-organism processes and metabolic processes in the classification of biological processes. In the classification of cell components, they were mainly classified into cells, cell parts and organelles. In molecular functions, the classification mainly included binding molecular function and catalytic activity function category. KEGG pathway enrichment showed that differentially expressed miRNA mainly involved PI3K-Akt, JAK-STAT, phosphatidylinositol signaling pathway system, ECM-receptor interaction, MAPK, Wnt, Toll-like receptors, IL-17, lipid metabolism, calcium signal pathway, cAMP and other signaling pathways. Among them, differentially expressed miRNA mainly played a role in the biological system and nervous system after 66 h of infection, mainly played a role in endocrine system and digestive system after 90 h of infection, and mainly played a role in the systemic biology, immune and digestive system after 114 h of infection. 10 differentially expressed miRNAs were selected for Real-time quantitative PCR verification, and the results were consistent with the results of high-throughput sequencing. It indicated that DEV infection had a significant impact on the expression of miRNA in liver of duck, and provided a reference for revealing the pathogenic mechanism of DEV from the perspective of host miRNA.
Construction and Identification of Recombinant Pseudorabies Virus Expressing the p54 Protein of African Swine Fever Virus
HE Xinglin, ZOU Zhong, GONG Wenxiao, ZHANG Yufei, LI Chengfei, XU Ting, CHEN Huanchun, JIN Meilin
2021, 48(11):  4182-4191.  doi:10.16431/j.cnki.1671-7236.2021.11.028
Abstract ( 187 )   PDF (9284KB) ( 68 )  
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The objective of this study was to construct a recombinant Pseudorabies virus (PRV) with high expression of the p54 protein of African swine fever virus (ASFV). A universal transfer vector pCAGIG-TK(l+r) containing homologous arm of PRV TK gene and green fluorescent protein (GFP) reporter gene was constructed by seamless cloning. The E183L gene was optimized and synthesized according to the gene sequence of China/2018/AnhuiXCGQ strain, and inserted into the universal transfer vector to construct the recombinant intermediate transfer plasmid pCAGIG-TK(l+r)-p54. The recombinant plasmid was linearized by ScaⅠenzyme and then transfected into BHK-21 cells by lipofectamine. 6 h later, 0.1 MOI of PRV was infected. After the lesion appeared, the recombinant PRV was purified by plaque selection and PCR. Furthermore, the expression of foreign protein was detected by Western blotting and indirect immunofluorescence assay (IFA), and the genetic stability and proliferation characteristics of the recombinant virus were studied. The results showed that green fluorescence could be observed in BHK-21 cells transfected with pCAGIG-TK(l+r)-p54 after 24 h, indicating that the recombinant plasmid was successfully transferred into BHK-21 cells. The purified recombinant virus expressed green fluorescent protein and contained ASFV E183L gene. Western blotting and IFA results showed that the recombinant PRV could express the p54 protein in BHK-21 cells, which showed a specific band at 26 ku, and had a specific reaction with ASFV positive serum, showing good immunogenicity. The ASFV E183L gene could be detected in BHK-21 cells after 20 successive passages, showing good genetic stability. One-step growth curve showed that there was no significant difference in the proliferation characteristics between the recombinant virus and the parental virus, and their highest viral titers were 107.34/0.1 mL and 107.61/0.1 mL, respectively. The insertion of foreign fragments did not affect the proliferation ability of the recombinant virus in cells. In this study, recombinant virus rPRV-p54 was successfully obtained, which could express ASFV p54 protein efficiently, and laid a foundation for the further study of the immunogenicity of p54 protein and the development of multigene recombinant PRV vector vaccine for ASFV.
Analysis of Key Action Sites of Salmonella Typhimurium STM LT2 Hfq
DUAN Shiyu, PAN Yong, YANG Yang, ZHANG Jiali, LINGHU Yuanfeng, YANG Qi, ZHOU Bijun
2021, 48(11):  4192-4203.  doi:10.16431/j.cnki.1671-7236.2021.11.029
Abstract ( 195 )   PDF (2891KB) ( 25 )  
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To explore the possible key action sites of Hfq, GcvB and their target gene oppA, in this study, λ-Red homologous recombination technology was used to construct mutant strains with core amino acids on the distal and proximal faces of Hfq and Hfq C-terminal truncated strains. On this basis, the corresponding Hfq-His6 protein tag fusion strains were constructed. P22 phage transduction technology was used to construct corresponding GcvB gene deletion strain and oppA::lacZ gene fusion strains. The protein level of Hfq was detected by Western blotting test, the transcription level of GcvB and oppA gene was detected by Real-time quantitative PCR, and the level of oppA protein was detected by β-galactosidase test. Western blotting test results showed that the mutation of core amino acids on the distal and proximal faces of Hfq and the truncation of the C-terminus of Hfq all increased the protein level of Hfq to varying degrees. The results of Real-time quantitative PCR showed that compared with control group, the mutation of the core amino acid of the proximal face of Hfq down-regulated the transcription level of the GcvB gene. The results of β-galactosidase test showed that compared with control group, the mutations of core amino acids on the distal and proximal faces of Hfq and the truncation of the C-terminus of Hfq did not cause significant changes in the transcription level of oppA gene, but they all increased the level of oppA protein to varying degrees. Among them, when the core amino acid mutation of Hfq proximal face and the C-terminus of Hfq were truncated to amino acid at positions 65 and 72, the protein level of oppA was up-regulated most obviously, up-regulating 2.9, 3.3 and 2.0 times, respectively. The results indicated that the proximal face of Hfq was very important for Hfq to maintain the stability of GcvB, the amino acid at position 56 of the proximal face of Hfq and the amino acid at positions 65 to 87 of the C-terminus of Hfq were negatively correlated with the level of oppA protein.
Prokaryotic Expression and Immunogenicity Analysis of the Structural Protein σB of a Novel Duck Reovirus DH13 Strain
YANG Huihu, HUANG Huilan, YUAN Sheng, LI Wenjun, YAO Xinyan, ZHANG Yuqian, LIANG Zhaoping, HUANG Shujian, ZHANG Xuelian
2021, 48(11):  4204-4212.  doi:10.16431/j.cnki.1671-7236.2021.11.030
Abstract ( 207 )   PDF (3871KB) ( 24 )  
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The purpose of this study was to express recombinant σB protein of DH13 strain of Novel duck reovirus (NDRV) using prokaryotic expression system, and detect its immunogenicity, so as to provide material basis for the development of NDRV gene engineering vaccine. σB gene of NDRV DH13 strain was amplified by RT-PCR and prokaryotic expression recombinant plasmids pET-30a-σB and pET-32a-σB were constructed and transformed into E. coli BL21 (DE3) and with IPTG induction corresponding recombinant proteins were obtained and analyzed by SDS-PAGE. The recombinant protein without His tag was purified by digestion and as immunogen to immunize New Zealand White rabbits to prepare polyclonal antibody. The recombinant protein with His tag was purified by Ni2+ affinity chromatography and as detection antigen to measure rabbit polyclonal antibody titer by indirect ELISA. It was determined whether the polyclonal antibody specifically recognize the recombinant protein by Western blotting. The results showed that σB gene with a size of about 1 100 bp was amplified. The results of SDS-PAGE showed that two recombinant σB proteins were efficiently expressed, the sizes of which were about 41 and 46 ku respectively, and both existed in the form of inclusion bodies. The indirect ELISA results showed the titer of the prepared polyclonal antibody was up to 1:204 800, it could specifically recognize the recombinant protein, which further reflected that the recombinant His σB protein had good immunogenicity. Western blotting results showed that the recombinant σB protein could reacted with the rabbit polyclonal antibody, indicating that the recombinant σB protein expressed by prokaryotic expression had good reactivity. This experiment successfully expressed the recombinant σB protein using the prokaryotic expression system, and confirmed that the NDRV σB protein had good immunogenicity and reactogenicity. This result would help the subsequent identification of the biological function of the NDRV σB protein, preparation of diagnostic antigens and development of new NDRV vaccines.
Epidemiological Investigation of Viral Diarrhea of Dairy Calves in Tangshan City, Hebei Province
CHANG Liyun, LIU Zhiyong, QIN Jianhua, ZHAO Yuelan
2021, 48(11):  4213-4219.  doi:10.16431/j.cnki.1671-7236.2021.11.031
Abstract ( 241 )   PDF (1027KB) ( 143 )  
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The purpose of the experiment was to master the epidemic status of viral diarrhea pathogens of dairy calves in 13 counties (cities and districts) of Tangshan city, Hebei province, and to effectively prevent and control dairy calves diarrhea. 788 fecal samples of diarrhea calves from 38 dairy farms in different areas of Tangshan were collected to extract genomic RNA from diarrhea fecal samples. According to the E2 gene of Bovine viral diarrhea virus (BVDV), VP6 gene of Bovine rotavirus (BRV) and N gene of Bovine coronavirus (BCV) published in GenBank, specific primers were designed by Primer Premier 5.0 software, and the three genes in fecal samples were detected by PCR. The pathogen detection results of different areas, seasons and mixed infections were summarized and analyzed by Excel 2007. The positive rates of BVDV, BRV and BCV were 29.82% (235/788), 29.44% (232/788) and 18.02% (142/788), respectively. In all tested areas, Luannan county had the highest BVDV infection rate, with a positive detection rate of 45.38% (59/130). Hangu district had the highest BRV and BCV infection rates, with a positive detection rate of 55.00% (22/40) and 37.50% (15/40), respectively. From the sampling season, the BRV infection rate was the highest in spring, summer and autumn, with the positive detection rates of 27.71% (46/166), 39.58% (114/288) and 19.89% (39/196), respectively. In winter, BVDV infection was the main infection, with the positive detection rate of 52.17% (72/138). In terms of mixed infection, BRV+BCV double mixed infection was the main, and the infection rate was 8.37% (66/788). BVDV, BRV and BCV infection were found in diarrhea calves in Tangshan city, Hebei province, and the main infection was BVDV and BRV.
Research Progress on Vaccine Development for Lumpy Skin Disease
XIE Shijie, ZHU Junda, WANG Tiankun, HU Xin, WEI Wenjuan, WU Wenxue, PENG Chen
2021, 48(11):  4220-4230.  doi:10.16431/j.cnki.1671-7236.2021.11.032
Abstract ( 533 )   PDF (1096KB) ( 213 )  
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Lumpy skin disease causes permanent damage to the skin of infected animals and leads to serious symptoms such as reduction of milk production, growth retardation, infertility of male animals and pre-mature miscarriage of females. As a result, the disease poses an essential threat to economy. Currently, there are no specific antiviral treatments for the disease, and the use of safe and effective vaccine constitutes a major way to prevent and control the spread of the disease. Lumpy skin disease virus attenuated vaccines have been successfully used in the control in many countries. In addition, due to the high similarity of genome sequence in Sheeppox virus, attenuated vaccines of Goatpox virus, Goatpox virus and Sheeppox virus have also been used in the control of Lumpy skin disease virus. Lumpy skin disease virus codes a large number of genes, the current research results have not found that the gene deletion vaccine and subunit vaccine have good protective effect. Inactivated vaccines have shown encouraging results in laboratory studies in recent years, but further large-scale clinical trials are still needed to prove their effectiveness. Finally, as a kind of Poxvirus, Lumpy skin disease virus has been proved to be one of the effective vectors for expressing various pathogenic antigens. This article mainly discussed the current vaccines available for Lumpy skin disease virus, including the application, protection effect, research direction of attenuated vaccines, inactivated vaccines, gene-deleted vaccines, and subunit vaccines, so as to provide novel insights into the development of vaccines and therapeutics for Lumpy skin disease virus in the future.
Basic Veterinary Medicine
Molecular Characterization of 3'-terminal Genome of Canine Coronavirus Strains JS1706 and JS1712
TANG Ye, CHEN Yi, TIAN Xiaoyan, LYU Haifeng, GAN Junji
2021, 48(11):  4231-4241.  doi:10.16431/j.cnki.1671-7236.2021.11.033
Abstract ( 236 )   PDF (3111KB) ( 32 )  
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In order to fully understand the molecular characteristics of the major structural protein genes and non-structural protein genes at the 3' end of the genomes of Canine coronavirus (CCoV) strains JS1706 and JS1712, eight sets of primers were designed for RT-PCR assays. PCR amplification products were sequenced and assembled, an 8.7 kb genome fragment was obtained. The genome organization and its encoded proteins were 5'-S-3abc-E-M-N-7ab-3' in order. The 8.7 kb genomic nucleotide sequences of CCoV strains JS1706 and JS1712 were compared with the same region nucleotide sequences of the reference strain of Alphacoronavirus. The results showed that the two isolates had the highest nucleotide identity with the reference strain of CCoV type Ⅱ (83.4%-93.1%). The nucleotide identities were followed by 87.1%-87.9% with the FCoV type Ⅱ reference strain, 86.1%-86.8% with the TGEV reference strain, 72.0%-72.1% with the CCoV type Ⅰ reference strain and 67.5%-69.9% with the FCoV type Ⅰ reference strain. The amino acid similarity of S, E, M and N protein genes of JS1706 and JS1712 strains with the same genus Coronavirus reference strains were 46.4%-95.2%, 75.6%-100%, 82.8%-99.2% and 78.5%-99.7%, respectively. These results indicated that the S gene within the same genus of Coronavirus was highly divergent, and the E, M, and N genes were relatively conservative. According to the genetic similarity comparison of 8.7 kb nucleotide sequence and S protein amino acid sequence, JS1706 and JS1712 strains had the highest similarity with the prototype pantropic strain CB/05, which was 93.0%-93.1% and 94.8%-95.2%, respectively. Amino acid sequences alignment of other structural proteins, including E, M and N, also showed highly similarity with CB/05 strain, which were 97.6%-100%, 92.4%-93.1% and 97.9%, respectively. Further analysis of S protein gene showed that JS1706 and JS1712 had some unique amino acids at the N terminal of S protein, and there was no obvious S1/S2 proteolytic cleavage site (RRARR), but there was a S2' cleavage motif (KRKYRS) at the 958-963 amino acid position. Phylogenetic analysis based on the amino acids of S protein showed that CCoV strains JS1706 and JS1712 clustered into a branch with CCoV subtype Ⅱa reference strains and FCoV type Ⅱ reference strains. The structure and size of ORF3abc and ORF7 genes encoding non-structural protein of JS1706 and JS1712 strains were similar to those of classical vaccine strains INSAVC-1, without obvious insertion, deletion and frameshift mutations. This study would contribute to understand the molecular characteristics of CCoV epidemic strains in China and laid a foundation for subsequent molecular epidemiological investigation, diagnostic reagents development and vaccine research.
Analysis of microRNA Expression Profile in Porcine Small Intestinal Epithelial Cells Infected with Enterotoxigenic Escherichia coli
LIU Yingying, ZHANG Di, LIU Guomei, LI Pei, LI Yan, ZUO Yuzhu
2021, 48(11):  4242-4253.  doi:10.16431/j.cnki.1671-7236.2021.11.034
Abstract ( 223 )   PDF (6584KB) ( 22 )  
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This study was aimed to explore the changes of microRNA (miRNA) expression profile in porcine small intestine epithelial cells (IPEC-J2) infected with enterotoxigenic Escherichia coli (ETEC), and provide a theoretical basis for the analysis of the regulatory role of host miRNA in the process of ETEC infection. IPEC-J2 pre- and post-infection of ETEC were sequenced by Illumina 6000 Novoseq SE50 sequencing platform, Bowtie was used to compare it with reference genome, and DESeq R Package was used to analysis the miRNA differential expression. The target genes of the differentially expressed miRNA were predicted jointly by miRanda and RNAhybid, and the GO and KEGG analysis of the target genes were performed. Five miRNAs were randomly selected to verify the sequencing results by Real-time quantitative PCR. The results showed that after filtration, 12 889 260 and 11 203 056 clean reads were obtained in the sRNA libraries of IPEC-J2 before and after ETEC infection, respectively. In the pre-infection and post-infection libraries, miRNA accounted for the highest proportion, which were 73.16% and 54.10%. A total of 97.98% and 69.83% of sRNA with length of 18-40 nt matched to the reference genome, indicating the sequencing quality was well controlled. Most of the first bases of the 22-24 nt sequences were inclined to U, and AGCUUAU was the most frequent base at 2-8 sites. A total of 311 known miRNAs and 128 novel miRNAs were discovered. The miRNA sequences of 23 nt length accounted for the highest proportion, taking up 41.42% and 23.56% in the two libraries, respectively. A total of 140 differentially expressed miRNAs were revealed in the comparison between the two libraries, among which 74 were up-regulated and 66 were down-regulated after ETEC infection. GO analysis showed that miRNA target genes were significantly enriched in metabolic processes, positive regulation of metabolic processes, cell composition or biosynthesis, immune system, intracellular parts and organelles. KEGG analysis showed that the differentially expressed miRNA target genes were significantly enriched in lysine degradation, IgA-producing intestinal immune network, NF-κB signaling pathway and T cell receptor signaling pathway. Verification by Real-time quantitative PCR showed that the expression trends of 5 randomly selected miRNAs were consistent with the sequencing results, indicating that the sequencing was accurate and reliable. In conclusion, the miRNAs in IPEC-J2 were involved in the process of ETEC infection, which provided a scientific basis for further revealing the key miRNA regulating ETEC infection and its mechanism of action.
Protective Effects of Resveratrol on Zearalenone-induced Liver Oxidative Damage and Inflammation in Mice
ZHU Gensheng, XIA Sugan, SHE Jinjin, BAI Yuni, ZOU Hui, GU Jianhong, YUAN Yan, LIU Xuezhong, LIU Zongping, BIAN Jianchun
2021, 48(11):  4254-4261.  doi:10.16431/j.cnki.1671-7236.2021.11.035
Abstract ( 174 )   PDF (13159KB) ( 166 )  
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In order to explore whether resveratrol (RSV) had a protective effect on the liver injury of mice induced by zearalenone (ZEA), 40 clean grade male BALB/c mice were randomly divided into 5 groups:Control group (normal saline), ZEA group (40 mg/kg), different concentrations of RSV (50, 100 or 200 mg/kg) and ZEA (40 mg kg) co-treatment groups, with 8 mice in each group. Each group was administered by gavage for 12 days. After the experiment, the liver samples of mice were collected, the liver coefficient was calculated, the liver pathological changes were observed by HE staining and immunohistochemical staining, and the liver NF-κB was detected. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and catalase (CAT) in liver tissue were detected by colorimetry. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in serum were detected by colorimetry. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-1β in serum were detected by ELISA method. The results showed that compared with control group, the liver tissue of ZEA group had obvious pathological changes. Compared with ZEA group, the liver histopathological changes of mice treated with different concentrations of RSV and ZEA were reduced. Immunohistochemical results showed that compared with control group, liver NF-κB protein expression was increased. Compared with ZEA group, liver NF-κB protein expression of different concentrations of RSV and ZEA co-treatment groups were decreased. Compared with control group, the liver coefficient and the content of MDA in liver tissue in ZEA group were extremely significantly increased (P<0.01), the activities of SOD and CAT in liver tissue were significantly or extremely significantly decreased (P<0.05; P<0.01), the activities of AST, ALT and LDH, and the contents of IL-6 and TNF-α and IL-1β in serum were extremely significantly increased (P<0.01). Compared with ZEA group, CAT activity in liver tissue of different concentrations of RSV and ZEA co-treatment groups were significantly or extremely significantly increased (P<0.05; P<0.01); Activities of AST, ALT and LDH and the contents of IL-6 and IL-1β in serum were significantly or extremely significantly decreased (P<0.05; P<0.01); SOD activity in liver tissue and the content of TNF-α in serum of 50 mg/kg RSV and ZEA co-treatment group were significantly increased (P<0.05) and extremely significantly decreased (P<0.01), respectively; The liver coefficient and MDA content in liver tissue were significantly or extremely significantly decreased in 100 and 200 mg/kg RSV and ZEA co-treatment groups (P<0.05; P<0.01). The results showed that ZEA had serious oxidative and inflammatory effects on the liver, and RSV had a certain protective effect on the liver injury caused by ZEA poisoning, 100 mg/kg RSV had the best protect effect.
Role of S100A12 in the Expression of Inflammatory Cytokines and Phagocytosis in Porcine Alveolar Macrophages Induced by Actinobacillus pleuropneumoniae
LIU Haiyao, LI Na, ZHANG Xiaoguang, LI Ziheng, BAO Chuntong, JIANG Hexiang, LIU Baijun, XIAO Jiameng, WANG Jun, LI Fengyang, LEI Liancheng
2021, 48(11):  4262-4273.  doi:10.16431/j.cnki.1671-7236.2021.11.036
Abstract ( 157 )   PDF (4565KB) ( 27 )  
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The purpose of this study was to explore the role of S100A12 in the expression of inflammatory cytokines and phagocytosis in porcine alveolar macrophages (PAM) induced by Actinobacillus pleuropneumoniae (APP). After PAM cells were inoculated with APP strain, Real-time quantitative PCR was used to detect the mRNA expression levels of inflammatory cytokines and S100A12 at different time points after infection. S100A12 recombinant protein expression vector was constructed and purified, and its cytotoxicity to PAM was detected by MTT method. The effects of different concentrations (0, 5, 50 and 500 ng/mL) of S100A12 recombinant protein on the expression level of inflammatory cytokine mRNA in PAM were detected by Real-time quantitative PCR. S100A12 interference plasmid was constructed, and its interference efficiency and effects on inflammatory cytokine mRNA expression and apoptosis of PAM after APP infection were detected by Western blotting, Real-time quantitative PCR and flow cytometry. Plate counting method was used to detect the influence of S100A12 expression on APP adhesion and invasion and survival in PAM cells. The results showed that APP infection promoted the expression of IL-6, IL-8, IL-18, IL-1β and TNF-α as well as the expression of S100A12 in PAM, and the expression increased significantly or extremely significantly with the increase of APP infection dose and time (P<0.05;P<0.01). 5, 50 and 500 ng/mL recombinant protein S100A12 had no toxicity to PAM. The low concentration of S100A12 recombinant protein significantly promoted the expression of inflammatory cytokines IL-6, IL-8, TNF-α, IL-1β, IL-21 and IL-5 in PAM (P<0.05), while the high concentration of S100A12 recombinant protein had no effect on the expression of the above-mentioned inflammatory cytokines (P>0.05). The interference plasmid could successfully block the expression of S100A12 protein. Compared with control group transfected with shNC, the transcriptional level of the cytokines IL-6, IL-8, IL-1β and IL-5 in PAM transfected with shS100A12 interference plasmid under APP induction were significantly or extremely significantly reduced, and apoptosis rate was also significantly or extremely significantly increased (P<0.05;P<0.01). Further research found that interfering with the expression of S100A12 protein in PAM could significantly enhance the adhesion and invasion ability of APP to PAM and its survival ability in PAM (P<0.05). On the contrary, adding S100A12 recombinant protein in vitro significantly inhibited the adhesion, invasion and survival of APP to PAM cells (P<0.05). The above results showed that S100A12 could enhance the expression of inflammatory cytokines after APP infection in PAM cells, inhibit APP-induced PAM apoptosis, and reduce APP's adhesion and invasion to PAM and its survival in PAM cells, which laid a foundation for further revealing the regulatory effect and mechanism of S100A12 on PAM in the process of APP infection.
Isolation, Identification, Virus Gene Detection and Antibiotic Resistance Analysis of Riemerella anatipestifer in Sansui County of Guizhou Province
WU Zhengzhuo, CHEN Guoquan, YAO Biqiong, SHI Dajun, YAN Chaohua, WANG Na, WANG Kaigong, ZHOU Bijun, CHENG Zhentao, WEN Ming
2021, 48(11):  4274-4283.  doi:10.16431/j.cnki.1671-7236.2021.11.037
Abstract ( 185 )   PDF (3818KB) ( 36 )  
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In order to understand the epidemic serotype, virulence and drug resistance of Riemerella anatipestifer (RA) in Sansui county, Guizhou province, 6 isolates were isolated from clinically suspected RA infected ducks in six large-scale duck farms in Sansui county, Guizhou province. The pathogenic bacteria were isolated and identified, and virulence gene detection and drug resistance analysis were carried out. The results of bacterial isolation and identification showed that the isolates were smooth, round and translucent drop-shaped colonies on chocolate agar medium. Gram staining was negative for short bacilli, Wright staining showed bipolar staining. The isolates did not have movement. The test of urea, catalase and oxidase were positive, which was in line with the biochemical characteristics of RA. The 6 strains of isolates were named SS-RA1 to SS-RA6. The 16S rRNA gene sequence of the 6 strains of isolates was compared with that of the RA reference strain on NCBI, the gene sequence similarity was ≥ 98%, indicating that all 6 strains were RA. SS-RA1 to SS-RA4 were serotype 2 and contain 8 virulence genes (OmpA、CAMP、wza、AS87_04050、Fur、SIP、TbdR1 and luxE genes), SS-RA5 and SS-RA6 were serotype 11, lacking the AS87_04050 gene, and only containing the remaining 7 virulence genes. The results of animal regression test showed that all the ducklings in the virus attack group died, and the ducklings in the control group did not show obvious clinical symptoms, indicating that the 6 strains of bacteria were pathogenic to the ducklings. The results of drug sensitivity test showed that the 6 strains of isolates were only sensitive to 7 antibiotics:Carboxybenzyl, piperacillin, ceftazidime, cefalexin, ceftriaxone, cefradine and cefoperazone, and showed different degrees of resistance to the other 13 antibiotics. The resistance rates to aminoglycosides and macrolides were 100%. In this study, the 6 RA strains were successfully isolated, providing a theoretical basis for vaccine selection and drug control of Riemerella anatipestifer infection in Sansui county, Guizhou province.
Inhibitory Effect of Astragalus Polysaccharide on Inflammatory Response of Chicken Macrophages by Inducing SOCS3 Expression
TAO Xinlei, LIU Danhua, TIAN Xu, ZHENG Shimin, GAO Xueli, LYU Xiaoping, LIU Chaonan
2021, 48(11):  4284-4291.  doi:10.16431/j.cnki.1671-7236.2021.11.038
Abstract ( 240 )   PDF (1375KB) ( 50 )  
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The aim of this study was to investigate the anti-inflammatory effect and action mechanism of Astragalus polysaccharide (APS) on LPS-induced chicken macrophage (HD11) inflammation model. HD11 cells were stimulated with different concentrations of LPS. Cell viability was detected by CCK8 assay and interleukin-1β(IL-1β) mRNA expression was detected by Real-time quantitative PCR in order to determine the optimal concentration of LPS for the construction of cellular inflammation model. HD11 cells were divided into control group (C), lipopolysaccharide (LPS) group (L), APS group (A) and APS inhibited LPS group (A+L). The mRNA expressions of IL-1β, tumor necrosis factor-α (TNF-α), NF-κBp65, p38MAPK and suppresser of cytokine signaling 3 (SOCS3) were detected by Real-time quantitative PCR, the protein contents of NF-κBp65, p38MAPK and SOCS3 were detected by Western blotting, and the protein contents of IL-1β and TNF-α were detected by ELISA at 2, 6, 12, 24 and 48 h after LPS stimulation. The results of cell viability and IL-1β detection showed that the optimal addition concentration of LPS for construction of cellular inflammation model was 0.5 μ g/mL LPS. Real-time quantitative PCR results showed that compared with control group, the mRNA expressions of IL-1β, TNF-α, NF-κBp65, p38MAPK and SOCS3 in L and A groups were significantly increased (P<0.05). Compared with L group, the mRNA expression levels of IL-1β, TNF-α, NF-κBp65 and p38MAPK in A+L group were significantly decreased (P<0.05), and the mRNA expression level of SOCS3 was significantly increased (P<0.05). Western blotting results showed that compared with control group, the ratios of P-NF-κBp65/NF-κBp65, P-p38MAPK/p38MAPK and SOCS3/α-tubulin in A group were significantly increased (P<0.05). Compared with L group, the ratios of P-NF-κBp65/NF-κBp65 and P-p38MAPK/p38MAPK in A+L group were significantly decreased (P<0.05), and SOCS3/α-tubulin ratio in A+L group was significantly increased (P<0.05). ELISA results showed that compared with control group, the protein contents of IL-1β and TNF-α in L group were significantly increased within 2-48 h (P<0.05), the protein contents of IL-1β and TNF-α in A+L group were significantly decreased within 12-48 h after LPS stimulation when compared with L group (P<0.05). The results suggested that in the LPS-induced HD11 cell inflammation model, APS could inhibit the overactivation of NF-κBp65 and p38MAPK signaling pathways by promoting SOCS3 expression, and thus played an anti-inflammatory role.
Establishment of the Epidemiological Cut-off Values of Haemophilus parasuis for Quinolones
CHEN Jiali, CHEN Chaoqun, WU Xue, HUANG Anxiong, HAO Haihong
2021, 48(11):  4292-4301.  doi:10.16431/j.cnki.1671-7236.2021.11.039
Abstract ( 201 )   PDF (1786KB) ( 50 )  
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In order to better understand the current situation of drug resistance of Haemophilus parasuis (Hps) to quinolones and establish the epidemiological cut-off values (ECOFFs) of Hps to quinolones, this test was done according to the method specified in the CLSI-VET. By searching from databases such as ScienceDirect, PubMed, CNKI and others, the MICs of 605 strains of ciprofloxacin, 74 strains of ofloxacin, 322 strains of levofloxacin, 211 strains of danofloxacin, 276 strains of nalidixic acid, 143 strains of lomefloxacin, 638 strains of enrofloxacin and 262 strains of mabofloxacin were collected and corrected. Then the statistical software ECOFFinder was used to fit the data, draw the nonlinear regression of MIC distribution and determine the ECOFFs and drug resistance rates of Hps to 8 quinolones. Among the 8 drugs, the drugs that could only be used for veterinary clinical use were danofloxacin, enrofloxacin, marbofloxacin and ciprofloxacin. As the first generation quinolone antibacterial, nalidixic acid had been withdrawn from the market. And the other three drugs should not be used in the veterinary clinic. The result of the experiment was that the ECOFFs of Hps to ofloxacin, nalidixic acid, lomefloxacin, levofloxacin, ciprofloxacin, danofloxacin, marbofloxacin and enrofloxacin were 8, 4, 4, 0.25, 0.25, 0.625, 0.625 and 0.03125 μg/mL, respectively. It showed that wild type strains of Hps had high sensitivity to ciprofloxacin, danofloxacin, marbofloxacin and enrofloxacin which were available in veterinary clinic and it's recommended to use them in veterinary clinical use. However, the drug resistance rate of these four drugs was also high, indicating that the existence of drug-resistant strains was serious. The data concluded in this paper was applicable in the era of frequent international exchanges and could be used as the criterion of sensitivity and resistance when there was no corresponding pharmacodynamics and clinical breaking point at home and abroad. And it also laid a foundation for further establishing clinical breaking point, guiding clinical medication and prolonging the clinical service life of quinolones.
Effects of Nicotine on LPS-induced Inflammatory Cytokines and Prostaglandin Secretion in Rabbit Endometrial Epithelial Cells
WEI Hongyu, BAO Zhaoxia, CHEN Hui, CAO Jinshan, HE Pengfei
2021, 48(11):  4302-4310.  doi:10.16431/j.cnki.1671-7236.2021.11.040
Abstract ( 181 )   PDF (3663KB) ( 65 )  
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The purpose of this study was to investigate the mechanism of nicotine which is the high affinity agonist of α7nAChR on the inflammation of rabbit endometrial epithelial cells induced by lipopolysaccharides (LPS). Epithelial cells were isolated from the endometrium of rabbits in late estrus and stimulated with 100 ng/mL LPS for 12 h. CCK8 method was used to detect the effects of nicotine in 5, 10 and 20 μg/mL on cell survival rates, and appropriate concentrations of nicotine were screened for subsequent tests. The cells were divided into control group (CON), LPS group, LPS+nicotine group, LPS+nicotine+methyllycaconitine citrate(MLA, specific antagonist of α7nAChR) group. The contents of interleukin-1β(IL-1β), IL-6, IL-8, tumor necrosis factor-α(TNF-α), prostaglandin E2(PGE2) and prostaglandin F2α(PGF2α) in the supernatant of cell culture were detected by ELISA. Rabbit endometrial epithelial cells were successfully isolated and maintained good growth until the fifth generation. CCK8 test results showed that the survival rate of 20 μg/mL nicotine group was significantly decreased (P<0.05), and 10 μg/mL nicotine had no significant effect on the survival rate of cells(P>0.05), so 10 μg/mL nicotine was selected for the following test. ELISA results showed that compared with control group, the content of IL-1β, IL-6, IL-8, TNF-α, PGE2 and PGF2α was significantly increased in LPS group (P<0.05). When compared with LPS group, the content of IL-1β, IL-6, IL-8, TNF-α, PGE2 and PGF2α was significantly decreased in LPS+nicotine group(P<0.05), and the content of inflammatory factors and prostaglandins in LPS+nicotine+MLA group had no significant difference (P>0.05). These results suggested that nicotine could down-regulate the secretion of IL-1β, IL-6, IL-8, TNF-α, PGE2 and PGF2α in LPS-induced rabbit endometrial epithelial cells. It was speculated that nicotine played an anti-inflammatory role by down-regulating inflammatory factors and prostaglandins mediated by α7nAChR. These results might provide a reference for the selection of α7nAChR as a therapeutic target for endometritis.
Effects of Selenium-oligochitosan Against Zearalenone on the Expression of Tight Junction Protein in IPEC-J2 Cells
FU Chunni, LI Yuanhui, LI Pengcheng, LI Liuan, QIN Shunyi
2021, 48(11):  4311-4318.  doi:10.16431/j.cnki.1671-7236.2021.11.041
Abstract ( 192 )   PDF (24399KB) ( 33 )  
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The aim of this study was to investigate the effects of selenium-oligochitosan against zearalenon (ZEN) on the expression of tight junction protein of IPEC-J2 cells. The experiment was divided into control group (C), ZEN (30 μg/mL), ZEN+0.5 Se (30 μg/mL ZEN+0.5 μmol/L selenium-oligochitosan, in terms of Se), ZEN+1.5 Se and ZEN+3 Se groups. When the confluence of cells reached 60%-70%, the culture medium of ZEN+0.5 Se, ZEN+1.5 Se and ZEN+3 Se groups was replaced with the culture medium containing the corresponding Se concentration, C and ZEN groups were replaced with normal culture medium. After 12 h, 30 μg/mL ZEN was added to the ZEN-containing groups, and the culture was continued for 24 h. The cell culture medium of each group was collected to detect the activity of alkaline phosphatase (AKP), the expressions of zonula occludens-1(ZO-1) and Occludin were detected by Western blotting, the expression and localization of ZO-1 and Occludin proteins were detected by immunofluorescence. The results of AKP activity showed that compared with control group, the AKP activity was significantly increased in ZEN, ZEN+0.5 Se and ZEN+1.5 Se groups (P<0.05), but there was no significant difference in ZEN+3 Se group (P>0.05). Compared with ZEN group, the AKP activity of ZEN+1.5 Se and ZEN+3 Se groups was significantly reduced (P<0.05). Western blotting results showed that compared with control group, the protein expression levels of ZO-1 and Occludin in ZEN group were significantly decreased (P<0.05). When compared with ZEN group, there were no significant differences in ZO-1 expression levels in ZEN+0.5 Se, ZEN+1.5 Se and ZEN+3 Se groups (P>0.05), but with the increase of Se concentration, the protein expression level of ZO-1 was gradually increased, the protein expression level of Occludin was significantly increased in ZEN+0.5 Se, ZEN+1.5 Se and ZEN+3 Se groups (P<0.05). The immunofluorescence results showed that selenium-oligochitosan could relieve the decreased fluorescence signal of ZO-1 and Occludin caused by ZEN, and restore the location distribution of the proteins. In conclusion, selenium-oligochitosan could reduce the increase of AKP activity in IPEC-J2 cells caused by ZEN, up-regulate the decreased expression levels of ZO-1 and Occludin in IPEC-J2 cells caused by ZEN, and adjust its location distribution.
Clinical Veterinary Medicine
Research Progress on Epigenetic Regulation Mechanism in Canine Tumor
REN Xiaoli, FAN Yuying, HUANGFU Heping, DONG Qing, SHI Dongmei, LIU Yun
2021, 48(11):  4319-4326.  doi:10.16431/j.cnki.1671-7236.2021.11.042
Abstract ( 236 )   PDF (787KB) ( 64 )  
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The canine neoplastic disease is common in the veterinary clinic, which has a high incidence and is one of the important causes of dog death worldwide. Because canine tumors are similar to human tumors in their pathological classification, spontaneity, genes, and signal pathways, dogs with tumors can be used as research models for human tumors. Epigenetics refers to heritable changes in gene function and expression levels that occur without a change in DNA sequence, mainly through the regulation of gene transcription and translation process influence the functions and features. Epigenetic changes mainly include changes in DNA methylation level, histone modification, chromatin remolding, and regulation of non-coding RNA. Abnormal DNA methylation has been studied in a variety of canine tumors, including canine leukemia, lymphoma, and melanoma. Among these tumors, the abnormal DNA methylation patterns of canine and human tumors are similar. The dysregulation of the expression of a variety of histone-modifying enzymes in tumors is the research hotspot of molecular targets for the development of anti-tumor drugs, but there are currently few studies in canine tumors. microRNA and lncRNA are currently research focuses on non-coding RNA. Many researches had been devoted to the development of targeted research drugs for non-coding RNA, but currently, there are few applications in the veterinary field. In this paper, the epidemiology, DNA methylation, histone modification, noncoding RNA, and other epigenetic changes in canine cancer were reviewed to reveal the relationship between epigenetic abnormalities and the occurrence and development of canine tumor, and to provide a reference for the development of specific markers for the diagnosis, targeted therapy and prognosis of canine neoplastic diseases.