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20 April 2020, Volume 47 Issue 4
Biotechnology
Bioinformatics Analysis of Transcriptome Sequencing of Bovine Skeletal Muscle Satellite Cells After Interference with lnc23
SONG Yingshen, GUO Yiwen, MIAO Manning, ZHANG Linlin, LI Xin, DING Xiangbin, GUO Hong
2020, 47(4):  973-983.  doi:10.16431/j.cnki.1671-7236.2020.04.001
Abstract ( 278 )   PDF (2382KB) ( 127 )  
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This study was aimed to analyze the changes of transcriptional group level of a muscle highly expressed and functional lncRNA (lnc23),and screen out the regulatory pathways and related mRNAs,which might be involved in regulating the growth of skeletal muscle satellite cells.The second generation sequencing technique (next-generation sequencing,NGS) was used to sequence the successfully constructed lnc23 inhibition model and the corresponding control group of bovine skeletal muscle satellite cells based on Illumia hiSeq sequencing platform.After sorting,filtering and evaluating the data,the transcriptional differentially expressed genes between the samples were analyzed by bioinformatics,and the differential genes were analyzed by GO and KEGG enrichment methods.The results of the transcriptome data were verified by Real-time quantitative PCR.The results showed that 19 358 genes were sequenced and 1 297 differential expression genes were screened,of which 856 genes were up-regulated,and 441 genes were down-regulated.The GO function of the differential genes,including the cellular components,the molecular function and the biological process,were divided into 3 categories (222 branches),among which a large number of differential genes were related to the catalytic activity,the molecular function regulation,the signal transduction,the biological adhesion and the metabolism.KEGG analysis results showed that the differential genes were involved in 190 pathways,which were significantly enriched in PI3K-Akt,P53,TNF,RIG-Ⅰ-like receptors,neuroactive ligand receptor interactions,cytokine interactions and other signaling pathways,and further screened out differential genes,such as CCNA2,RRM2,IL-6 and MYL2,which might be involved in cell growth and muscle development.Real-time quantitative PCR results showed that 9 of 11 differential genes were consistent with the sequencing results of the transcriptional group,indicating the reliability of the sequencing results.This study successfully finished the interference with lnc23 and its control,completed the transcriptional group sequencing analysis of bovine skeletal muscle satellite cells,obtained the functional annotation information of differential genes and preliminarily revealed the potential genes and pathways of lnc23 regulating the differentiation of bovine skeletal muscle satellite cells,which laid a good foundation for further exploring the differentiation mechanism of bovine skeletal muscle satellite cells regulated by lnc23.
Establishment of a Method for Directed Evolution and Enzymatic Activity Enhancement of Transposase Tn3 in Vitro
SONG Shangqiao, MA Weiwei, LI Xiaojian, ZENG Suxian, LI Xin, YAN Jin, SUN Cuicui, LI Zongqiang
2020, 47(4):  984-991.  doi:10.16431/j.cnki.1671-7236.2020.04.002
Abstract ( 233 )   PDF (1625KB) ( 163 )  
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The aim was to explore the improvement and evolution of the enzyme activity of the transposase Tn3.The in vitro directional evolution of transposase Tn3 was studied by primers design and synthesis,PCR amplification,restriction enzyme digestion,error-prone PCR optimization and construction,DNA rearrangement construction and optimization, D-value determination and enzyme digestion identification.The results showed that the recombinant plasmids were digested with SacⅠ and XbaⅠand 450 bp enzyme digestion products were obtained.After 24 h culture in LB medium containing kanamycin, D-value was measured.After three rounds of repeatability studies,D-value and proliferation ability increased from 0 to 0.18,0.42 and 0.60,indicating that Tn3 activity was significantly improved.The selected evolution-type recombinant plasmid was extracted by plasmid extraction,and the endonuclease XhoⅠ and XbaⅠ were used for double digestion identification.It was found that the bands of the cleavage products of the evolutionary recombinant plasmids were relatively small.After that,gene sequencing analysis of the recombinant plasmid of evolutionary type showed that mutations occurred in multiple sites of Tn3 gene sequence and CCR5-delta32 gene in the middle of Tn3-Gal4 targeted sequence was removed.It showed that the cloning of the transposase Tn3 gene was successfully completed in this study.The optimal reaction system for error-prone PCR was established,it was feasible to use the LB medium containing kanamycin and D-value of the bacterial solution for screening.Mutations in the gene sequence of Tn3 and excision of the gene indicate that Tn3 had changed both in function and in gene sequence,which was exactly in the direction of need.
Effects of Different Fatty Acids on Adipogenic and Transdifferentiation of Skeletal Muscle Satellite Cells in Yanbian Yellow Cattle
ZHANG Junfang, YAN Yan, CUI Yan, SUN Bin, WANG Ying, SUN Jianfu, JIN Xin, YAN Changguo, LI Xiangzi
2020, 47(4):  992-999.  doi:10.16431/j.cnki.1671-7236.2020.04.003
Abstract ( 215 )   PDF (3374KB) ( 72 )  
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The purpose of this study was to investigate the effects of different kinds of fatty acid monomers (oleic acid,stearic acid,palmitoleic acid and palmitic acid) on the skeletal muscle satellite cells (BSC) of Yanbian Yellow cattle,and analyze the expression of genes involved in lipogenesis and the effects on the formation of lipid droplets.Skeletal muscle satellite cells were isolated from 18-month-old Yanbian Yellow cattle semi-membrane muscle for in vitro culture,and 100 mmol/L oleic acid,stearic acid,palmitoleic acid and palmitic acid were added to the differentiation medium for 96 h,Real-time quantitative PCR was used to detect the expression of peroxisome proliferator-activated receptor gamma (PPARγ),sterol-regulated binding protein 1 (SREBP1),stearyl coenzyme A desaturase (SCD) and CCAAT/enhancer binding protein alpha (C/EBPα).Oil red O staining results showed that lipid droplets were formed in all fatty acid treatments compared with control cells.Oleic acid and palmitoleic acid treatments had more lipid droplets and larger lipid droplets than palmitic acid and stearic acid treatments.Real-time quantitative PCR results showed that the addition of unsaturated fatty acids such as oleic acid and palmitoleic acid to the skeletal muscle satellite cells of Yanbian Yellow cattle increased the expression of PPARγ,SREBP1 and C/EBPα genes,and inhibited the expression of SCD gene.The addition of saturated fatty acids (stearic acid and palmitic acid) also significantly increased the expression of SCD gene while promoting the expression of PPARγ,SREBP1 and C/EBPα genes(P < 0.05).The results indicated that the addition of fatty acids could induce the transdifferentiation of the skeletal muscle satellite cells to adipocytes in Yanbian Yellow cattle.
Polymorphism and Bioinformatics Analysis of BMPR-ⅠB Gene Promoter Region in Guizhou Local Goats
AI Jinxin, LONG Anju, LUO Weixing, CAI Huifen
2020, 47(4):  1000-1008.  doi:10.16431/j.cnki.1671-7236.2020.04.004
Abstract ( 232 )   PDF (1273KB) ( 111 )  
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In order to explore the polymorphism of bone morphogenetic protein receptor ⅠB (BMPR-ⅠB) gene promoter region and its correlation with reproductive traits,three Guizhou local goats,including Qianbei Ma goats,Guizhou Black goats and Guizhou White goats were selected as experimental materials.SNPs were screened by constructing a mixed DNA pool combined with PCR product direct sequencing technology,and various bioinformatics software were used to predict the effects of SNPs on the core promoter region,CpG island and transcription factor binding sites.The results showed that there were 4 SNPs in the promoter region of BMPR-ⅠB gene,which were g.29893005 T > C,g.29893016 C > T,g.29893729 C > G and g.29894370 C > T.The bioinformatics software predicted the core promoter region and CpG island of BMPR-ⅠB gene,and the SNPs site caused the transcription factor binding site to change.The g.29893005 T > C and g.29893016 C > T mutations caused the original transcription factors site binding Sp1 to disappear,g.29893729 C > G mutation caused the original transcription factor binding site AP-1 to disappear,resulting in a new transcription factor binding site USF.g.29894370 C > T mutation produced a new transcription factor binding site C/EBPalp changed the original transcription factor binding site Sp1 to C/EBPalp and Sp1.It was speculated that SNPs might have important effects on regulatory promoter functional elements.
Analysis of DNA Methylation and mRNA Expression Levels of ITGB2 Gene in Different Fineness of Subo Merino Sheep
DU Jianwen, YANG Xuemei, WANG Dan, ZHU Hua, HE Junmin, ABLAT Sulayman, TIAN Yuezhen, ZHAO Bingru, XU Xinming, FU Xuefeng, HANIKEZI Tulafu, WU Weiwei, TIAN Kechuan, HUANG Xixia
2020, 47(4):  1009-1017.  doi:10.16431/j.cnki.1671-7236.2020.04.005
Abstract ( 209 )   PDF (2006KB) ( 118 )  
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This study was aimed to analyze the DNA methylation and mRNA expression levels of ITGB2 gene in different fine skin tissues of Subo Merino sheep.Using Subo Merino sheep aged ewe as experimental animal and skin tissue samples of different fineness as experimental samples,the promoter CpG island of ITGB2 gene (GenBank accession No.:NC_040252.1) were predicted,the BSP primer,the primers of the mRNA sequence of ITGB2 gene (GenBank accession No.:NM_001009485.1) and GAPDH gene (GenBank accession No.:NM_001190390.1) were designed,respectively.The bisulfite sequencing method (BSP method) was used to amplify and purify,and then inserted into pMD19-T vector and transferred to JM109 cells for overnight culture to form a single colony.The positive clones were screened for sequencing and the obtained sequences were analyzed.The methylation pattern of CpG island in the promoter region of ITGB2 gene in skin tissue of aged ewe,and the mRNA expression level of ITGB2 gene in different fineness skin tissues of Subo Merino sheep was detected by Real-time quantitative PCR.The results showed that the methylation rate of CpG island of extremely fine group in Subo Merino sheep (94.29%) was higher than that of extremely thick group (87.62%).Among them,the methylation rate of CpG2,CpG3,CpG4 and CpG7 island in extremely fine group of Subo Merino sheep (100%,100%,100% and 80.00%) were higher than that in extremely thick group (86.67%,93.33%,80.00% and 73.33%).The expression of ITGB2 gene in extremely thick skin tissues of Subo Merino sheep was extremely significantly higher than that in extremely fine skin tissues(P < 0.01),and there was a significant negative correlation between the level of DNA methylation and the mRNA expression of ITGB2 gene.This results indicated that DNA methylation had a role in skin growth and development,and could be used as a candidate epigenetic marker for Subo Merino sheep.
Physiology and Biochemistry
Recombinant Expression and Activity Analysis of Phage Lyase Lysep3
YAN Wei, YANG Na, MAO Ruoyu, WANG Xiumin, HAO Ya, MA Xuanxuan, TENG Da, WANG Jianhua
2020, 47(4):  1018-1027.  doi:10.16431/j.cnki.1671-7236.2020.04.006
Abstract ( 245 )   PDF (2268KB) ( 142 )  
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In order to break through the application bottleneck of phage lyase Lysep3,Lysep3 was recombinantly expressed using yeast expression system for its low-cost production,and in vitro activity and safety of Lysep3 were explored preliminarily to provide data reference for clinical experiments.Lysep3 gene was optimized according to the codon bias of Pichia pastoris and synthesized,and ligated into the expression vector pPICZαA.After verification of colony PCR and sequencing,the linearized recombinant vector pPICZαA-Lysep3 was electro transformed into P.pastoris X-33.After colony PCR and recombinant protein screening at shake flask level by methanol induction,recombinant yeast were fermented with high-density in 5 L fermenter,and fermentation supernatant was purified by His-trap HPGE purification column.The antibacterial activity of rLysep3 was analyzed by inhibition zone test and turbidity test.The safety of rLysep3 was analyzed by cytotoxicity of rLysep3 against macrophage RAW264.7 and hemolytic activity of rLysep3 against mouse red blood cells were assayed for its safety evaluation.In 5 L fermenter,Lysep3 was heterologously expressed in P.pastoris and its expression level reached 749.2 mg/L.The activity analysis of rLysep3 showed that it had antibacterial activity against Salmonella enteritidis,Staphylococcus aureus,Staphylococcus hyicus and Streptococcus agalactiae,and the number of S.enteritidis colonies was reduced by 1.6 to 1.7 orders of magnitude through the combination of rLysep3 and EDTA.In addition,cytotoxicity assays showed that rLysep3 still had a cell viability of 78.75% at a concentration of 1 024 μg/mL;hemolytic analysis showed rLysep3 only had a hemolysis rate of 1.58% at a concentration of 1 024 μg/mL.The above results demonstrated that the recombinant expression of Lysep3 could be achieved by the P.pastoris expression system,and rLysep3 had in vitro antibacterial activity,low cytotoxicity and low hemolysis.From the aspects of antibacterial activity,industrial preparation and safety,it showed that rLysep3 had the potential to develop a new antibacterial drug for the clinical treatment of S.enteritidis infection.
Research Progress on the Regulation Mechanism of Exosomal lncRNA on Lipid Metabolism
ZHANG Jiasu, XIA Guangjun, YIN Baozhen, ZHANG Luomeng, SHAO Jing, GENG Chunyin
2020, 47(4):  1028-1034.  doi:10.16431/j.cnki.1671-7236.2020.04.007
Abstract ( 286 )   PDF (804KB) ( 99 )  
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Exosomes are a kind of nanometer extracellular vesicle secreted by cells and they are able to transmit biological information in a long distance.Exosomes are secreted and released by cells,then transmitted in body fluids,finally swallowed in another cells to transmit biological information.Lipid metabolism has always been a hotspot of the field of life science research.Although there are many lncRNAs affecting lipid metabolism,there are relatively few lncRNAs studies affecting lipid metabolism in exosomes.For this reason,the study of exosomal-lncRNAs shows it’s necessity.As one of the important genetic material carried by the exosomes,the long non-coding RNAs are a kind of RNA molecule of which length for more than 200 nt,and perform many important biological functions such as transcriptional activation,chromatin modification and so on.In this paper,the biological characteristics of the exosomal-lncRNAs have been introduced,and the examples of the effect of exosomal-lncRNAs on lipid metabolism by different molecular mechanisms have been listed.The lipid metabolism disease caused by the abnormal expression of exosomal-lncRNAs has analyzed,and the principle of the exosomal-lncRNA acted as lipid metabolism disease’s biomarker has also illustrated.Finally,the prospect of exosomal-lncRNA as a biomarker for animal fat deposition has prospected.If more exosome-derived lncRNAs are detected in the future research,and it’s sufficiently to label abnormal conditions of lipid metabolism,there will undoubtedly be positive implications for the prediction and regulation of fat deposition in livestock.
Animal Nutrition and Feed Science
Study of L-citrulline on Growth Performance of Swimming Exhausted and Serum Biochemical Effect of Mice
ZHAO Guodong, LI Xiaobin, LI Tingting, CHEN hui, MA Chen, ZENG Wushuang, ZANG Changjiang
2020, 47(4):  1035-1040.  doi:10.16431/j.cnki.1671-7236.2020.04.008
Abstract ( 199 )   PDF (628KB) ( 81 )  
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Effects of adding different doses of L-citrulline (L-Cit) in drinking water on the growth performance and serum biochemical indexes of swimming exhausted Kunming mice was studied in this paper.36 weaned females with average weight of (14.58±0.77) g were selected and randomly divided into 3 groups with 6 replicates in each group.The mice in control group drank from tap water,and 0.5% and 1% of L-Cit were added in the drinking water for test groupⅠand Ⅱ,respectively.The trial lasted 21 d.Every 3 d,the mice were weighed in an empty stomach and then swam with weight loading in (31±2)℃ bucket until exhausted.Blood was collected from the eyeballs at day 21.The results showed that:Compared with control group,at the day 6,9 and 12,the body weights of the female mice in test groups Ⅰ and Ⅱ increased by 12.88% (P < 0.05),20.30% (P < 0.01),7.99% (P < 0.05) and 21.27% (P < 0.01),24.47% (P < 0.01),19.46% (P < 0.01) respectively.And it increased by 13.71% (P < 0.01) at day 15 in test group Ⅱ.The serum activity of anti superoxide anion free radical (ASAFR) increased by 31.49% (P < 0.01) in test group Ⅰ.The serum activity of GSH-Px and inhibit hydroxyl free radical (IHFR) ability were increased by 54.35%(P < 0.01)and 13.10%(P < 0.01)in test group Ⅱ,respectively.Compared with the control group and test groupⅠ,the serum levels of serum albumin (ALB)content were respectively increased by 13.16% (P < 0.01) and 6.28% (P < 0.01).In conclusion,Adding L-Cit in drinking water could improve the growth performance and antioxidant capacity,increase serum total protein (TP) and albumin contents in mice after exercise,and adding 0.1% L-Cit (69.09 mg/d) was better.
Effects of Ginkgo Leaves on Growth,Slaughter Performance and Nutrient Ultilization in Sheep
HU Gaojie, YANG Gaiqing, WANG Linfeng, FU Tong, LIAN Hongxia, GAO Tengyun, LI Ming, WANG Fuzhou
2020, 47(4):  1041-1049.  doi:10.16431/j.cnki.1671-7236.2020.04.009
Abstract ( 311 )   PDF (961KB) ( 97 )  
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Ginkgo leaves contain not only conventional nutrients,but also special medicinal active ingredients such as flavonoids and azlactones,which can be used as a special feed resource.This experiment was conducted to study the effects of Ginkgo leaves feed on growth performance,slaughtering performance,nutrient digestion and utilization,and serum biochemical indexes in sheep.Twenty-one Dupo hybrid rams weighted about 35 kg were randomly divided into three groups,which named control group (fed diet without Ginkgo leaves),low-level Ginkgo leaves group group (L group,7.5% Ginkgo leaves) and high-level Ginkgo leaves group (H group,15% Ginkgo leaves).The body weights of Dupo rams were weighed every two weeks,and digestion and metabolism test and blood collection were carried out.Sheep were slaughtered at the end of the experiment to detect the carcass traits.The results showed that Ginkgo leaves had no significant effect on average daily feed intake (ADFI),final weight and average daily gain (ADG) (P > 0.05).The energy digestibility and deposition rates of low-level Ginkgo leaves group were significantly lower than those of control group (P < 0.05).The nitrogen deposition rate of high-level Ginkgo leaves group was higher than the other two groups,and the difference with low-level group was significantly (P < 0.05).Ginkgo leaves had no significant effect on digestibility and retention of calcium and phosphorus (P>0.05).Biochemical test results showed that AST activity in low-level group was significantly lower than that in control group (P < 0.05),and high-level group had no significant change compared with control group (P > 0.05).The serum total protein and globulin contents in low-level group were significantly higher than those in control group (P < 0.05),but there was no significant difference between high-level group and control group (P > 0.05).There was no significant difference in total cholesterol (TC) and triglyceride (TG) levels among three groups (P > 0.05),but they had a certain reduction trend in two Ginkgo groups.The slaughter test showed that the carcass weight and net meat weight of sheep fed with Ginkgo leaves were significantly reduced (P < 0.05),and there was no significant difference in slaughter rate and net meat rate among three groups (P > 0.05).Compared with control group,the perirenal fat weights of Ginkgo leaves groups were significantly lower than control group (P < 0.05),were 31% and 49% lower than control group,respectively.In conclusion,Ginkgo leaves had certain effects on nutrients digestion and metabolism,and significantly reduce the perirenal fat in carcass,and the specific effect was related to the amount of feeding.
Diversity Analysis of Fecal Flora on Pre-weaning Foal and Mare
LI Xiaobin, LI Hai, ZANG Changjiang, LI Chao, HUANG Xinxin, MA Lixin, LI Jiahao, HUANG Jinlong, LI Fengming
2020, 47(4):  1050-1057.  doi:10.16431/j.cnki.1671-7236.2020.04.010
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This experiment was aimed to study the diversity of fecal flora on pre-weaning foal and mare,and provide a basis for enriching the diversity of the intestinal flora of foal.The test selected five thoroughbred mares with similar age of 5-year-old,2-foetus,the average body weight (453.34±54.23) kg healthy mare,and their five foals with similar birth dates(±5 d),the average body weight (185.65±9.54) kg,three males and two females.All horses were kept in the same environment.The same composition and nutrient levels of the mares feeding.The roughage and concentrate supplements purchased by the horses were the same except for breastfeeding.The foals were weaned at 6 months of age.The results showed as followed:Alpha diversity index of Chao1 and ACE in the feces of mares were significantly increased by 18.94% and 15.62% than that of the foals (P < 0.05),respectively.The total number of species shared by mares and foals was 1 399,and the unique species of mares and foals were 150 and 68,respectively.At the phylum level,the top 10 bacteria found in the feces of mares and foals were Firmicutes,Proteobacteria,Bacteroidetes,Actinobacteria,Euryarchaeota,Verrucomicrobia,Spirochaetes,Unidentified_Bacteria,Tenericutesin and Firmicutes abundance in feces of mares was significantly higher than that of foals (P < 0.05).The Bacteroides in feces of mares was 33.93% higher than that of foals,but there was no significant difference (P > 0.05).In conclusion,the diversity of fecal flora was significantly higher than that of pre-weaning foals.Firmicutes,Bacteroides and Proteobacteria were the main bacteria in the feces of mare and pre-weaning foals,and the abundance of Firmicutes and Bacteroides in feces of mare was higher than that of the pre-weaning foals.
Effects of Mixed Lipid on Breast Muscle Fatty Acid Composition, and Underlying Metabolomics Mechanism in Qingyuan Chickens
GOU Zhongyong, CUI Xiaoyan, FAN Qiuli, LI Long, LIN Xiajing, WANG Yibing, JIANG Shouqun, JIANG Zongyong
2020, 47(4):  1058-1069.  doi:10.16431/j.cnki.1671-7236.2020.04.011
Abstract ( 210 )   PDF (1403KB) ( 89 )  
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This study was conducted to examine the effects of dietary addition of soybean oil or a blend of oil and fat on the growth performance and fatty acid profile of breast muscle in chickens,and to explore the mechanism of metabolic changes occurring in the fatty acid profile of breast muscle based on metabolomics technology,which could provide new ideas for regulating the fatty acid composition of chickens.A total of 480 one-day-old female chicks (Qingyuan) were randomly assigned into two dietary treatments,each consisted of six replicates and 40 chicks per replicate.The diet of control group (CON) was added with soybean oil,and the experimental group (BOF) was added with the blend of oil and fat,soybean oil∶lard∶fish oil∶coconut oil=1.0∶1.0∶0.5∶0.5.The experimental period was 127 d.The results showed that the birds fed the BOF diet,compared to the controls,had similar daily body weight gain (P > 0.05),lower daily feed intake and feed∶gain ratio (P < 0.05);higher content of lauric acid (C12∶0),myristic acid (C14∶0),oleic acid (C18∶1n-9),eicosapentaenoic acid (EPA,C20∶5n-3) and docosahexaenoic acid (DHA,C22∶6n-3) (P < 0.05),and lower content of linoleic acid (C18∶2n-6) (P < 0.05) in breast muscle tissue.A total of 110 different metabolites (VIP > 1,P < 0.05) in the breast muscle were detected by liquid chromatography-mass spectrometry(LC-MS),due to the dietary treatment,63 of which were annotated and were mainly phospholipid metabolites,including phosphatidylcholine (PC),phosphatidylserine (PS) and phosphatidylethanolamine (PE).In addition,the contents of glutathione and carnosine in breast muscle were significantly increased in the BOF treatment (P < 0.05).Through the metabolic pathway enrichment analysis,it was found that fatty acid metabolism pathways,including alpha-linolenic acid metabolism,arachidonic acid metabolism,linoleic acid metabolism and glycerophospholipid metabolism were changed significantly (P < 0.05).In summary,the BOF diet improved the feed efficiency of chickens and promoted DHA and EPA deposition in the breast muscle compared to those birds fed traditional soybean oil diet.It had been proven that the dietary fat intervention modified the phospholipid metabolites,oxidative stability metabolites and lipid metabolism pathways in breast muscle.The identified differential metabolites and differential metabolic pathways could provide references for the future studies on regulating fatty acid composition in chicken’s breast muscle.
Evaluation of Forage Nutritional Value in Alpine Meadow Grassland Natural Pastures in Different Months
ZHANG Qunying, ZHOU Yixiu, HAO Lizhuang, LIU Shujie
2020, 47(4):  1070-1079.  doi:10.16431/j.cnki.1671-7236.2020.04.012
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The aim of this experiment was to explore dynamic changes in the nutritional value of forage in alpine meadow grassland natural pastures in different months (July to December) in Yushu county of Qinghai province.From July 2017 to the middle of December 2017,a typical local grazing grassland was selected to track and graze yaks to simulate the collection of edible forage.And conventional nutritional content determination and in vitro fermentation technology were used to measure the gas production (GP),digestibility of dry matter in vitro (IVDMD) and other indicators of 48 h.The nutritional value of forage in each month was comprehensively evaluated to provide basic data for pasture value.The results showed that the content of crude protein (CP),crude fat (EE),Ash,neutral washing soluble matter (NDS) and digestive energy (DE),metabolic energy (ME),IVDMD of forage in July and August were extremely significantly different than other months (P < 0.01),and they were low in November and December.While the content of neutral detergent fiber (NDF),acid detergent fiber (ADF) and hemicellulose (HC) were extremely significant higher in November and December than other months (P < 0.01),and at low level in July and August.The pH and NH3-N concentration of rumen fluid in different months had no significant difference (P > 0.05).The GP and the concentration of volatile fatty acids (VFA) were significantly different between different months(P < 0.05),the value of GP and VFA in July and August were extremely significantly higher than other months (P < 0.01),and were extremely significantly lower in November than in other months (P < 0.01).The concentration of total volatile fatty acid (TVFA) had no significant difference among different months (P > 0.05),but the value was higher in July and September.In summary,the pastures of the typical alpine meadow pastures had good nutritional quality in July and August,but the nutritional quality of pastures were low from October to December,which could’t meet the nutritional needs of grazing yaks.The accurate feeding herders and rational use of grassland resources was recommended to promote the development of local animal husbandry.
Effects of Bacillus subtilis,Oligomeric Chitosan and Sodium Butyrate on Growth Performance,Immune Function and Meat Quality in Yellow-feathered Chickens
FAN Qiuli, JIANG Shouqun, GOU Zhongyong, LI Long, LIN Xiajing, WANG Yibing, CHEN Fang
2020, 47(4):  1080-1091.  doi:10.16431/j.cnki.1671-7236.2020.04.013
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The aim of this experiment was to study the effects of Bacillus subtilis,oligomeric chitosan,sodium butyrate added alone or in pairs instead of antibiotic on growth performance,immune function,carcass performance and meat quality of Yellow-feathered chickens raised in cages.Seven hundred and twenty 1-day-old rapidly growing Lingnan Yellow-feathered male chickens were randomly assigned to eight groups according body weight (each group consisted of six replicates with 15 broilers per replicate),and they were control group (antibiotic-free),antibiotic group (200 mg/kg 4% eramycin),probiotics group (500 mg/kg Bacillus subtilis),oligomeric chitosan group (50 mg/kg oligomeric chitosan),sodium butyrate group (500 mg/kg sodium butyrate),antibiotic-free combination group 1 (500 mg/kg Bacillus subtilis+30 mg/kg oligomeric chitosan),antibiotic-free combination group 2 (30 mg/kg oligomeric chitosan+300 mg/kg sodium butyrate),antibiotic-free combination group 3 (500 mg/kg Bacillus subtilis+300 mg/kg sodium butyrate).The experiment was carried out in three stages,and the experimental period was 63 days.The results showed:1 to 21 days,there was no significant difference in growth performance and plasma immune parameters among all treatments (P > 0.05).Bursa of Fabricius index of oligomeric chitosan group was higher than control and antibiotic groups (P > 0.05).The ileum pH in probiotics and sodium butyrate groups were significantly lower than antibiotic group (P < 0.05).22 to 42 days,there was no significant difference in growth performance among all treatments (P > 0.05).Spleen index of antibiotic-free combination group 3 was significantly higher than control group(P < 0.05).The jejunum pH in probiotics group was lower than antibiotic group (P > 0.05).Plasma IgG content in sodium butyrate,antibiotic-free combination groups 1,2,and 3 were significantly lower than control and antibiotic groups (P < 0.05).IgA content in antibiotic-free combination group 2 was significantly lower than control and antibiotic groups,and IgM content was significantly lower than antibiotic group (P < 0.05).Plasma IgM content in antibiotic-free combination group 1 was significantly higher than control and antibiotic groups (P < 0.05).IgA and IgG contents in probiotics group were higher than control and antibiotic group (P > 0.05),and IgM was significantly higher than control group (P < 0.05).43 to 63 days,ADG of antibiotic-free combination group 3 was significantly higher than antibiotic group (P < 0.05).The duodenum pH in probiotics group was significantly lower than antibiotics group (P < 0.05).Abdominal fat rate of antibiotic-free combination group 1 was significantly lower than control and antibiotic groups (P < 0.05).There was no significant difference in meat quality and plasma immune parameters among all treatments (P > 0.05).According to the results of growth performance,intestinal pH,immune performance and carcass performance in three stages,it was recommended to add 500 mg/kg Bacillus subtilis and 300 mg/kg sodium butyrate for improving growth performance,add 500 mg/kg Bacillus subtilis and 30 mg/kg oligomeric chitosan for improving carcass performance,and add 500 mg/kg Bacillus subtilis alone for improving intestinal health of rapidly growing Lingnan Yellow-feathered male chicken aged from 1 to 63 day.
Effects of Zinc Methionine Instead of Zinc Sulfate on Growth Performance, Meat Quality and Antioxidant Status of Broilers
WU Liang, ZHAO Xu, ZOU Tianhao, ZHANG Qiuhua, YANG Zaibin
2020, 47(4):  1092-1098.  doi:10.16431/j.cnki.1671-7236.2020.04.014
Abstract ( 339 )   PDF (754KB) ( 94 )  
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To investigate the effects of zinc methionine instead of zinc sulfate on growth performance,meat quality and antioxidant status of broilers,200 one-day-old healthy male Arbor Acres (AA) broiler chicks with similar body weight (BW) were randomly allocated into 2 treatments with 5 replicates of 20 broilers per replicate in a completely randomized design.Broiler chickens in both treatments were fed a basal diet and the level of zinc was set at 40 mg/kg.In the treatment one,zinc was added in the form of zinc sulfate,and in the treatment two,zinc was added in the form of zinc methionine.The experiment lasted for 42 days.Results showed as follows:① Compared with zinc sulfate,zinc methionine significantly reduced the feed conversion rate of broilers in the grower phase or the entire period of the experiment (P<0.05),but did not affect the average daily feed intake,average daily gain,body weight at both 21 and 42 days of age of broilers (P>0.05).② Compared with zinc sulfate,zinc methionine significantly reduced the percentage of eviscerated yield (P<0.05),increased the breast muscle weight,abdominal fat weight and percentage of abdominal fat (P<0.05),but did not affect the slaughter weight,dressed weight,eviscerated weight,leg muscle weight,carcass yield,proportion of breast and thigh at 42 days of age (P>0.05).③ No significant differences were found in meat color,drip losses and boiling losses of breast muscle between zinc methionine and zinc sulfate treatments (P>0.05).④ Compared with zinc sulfate,zinc methionine significantly increased CuZn-superoxide dismutase (CuZn-SOD) activity in the serum at both 21 and 42 days of age and total antioxidant capacity (T-AOC) in the serum at 42 days of age (P<0.05),reduced malondialdehyde (MDA)concentration in the serum at 21 days of age (P<0.05),but did not affect the glutathion peroxidase (GSH-Px) activity (P>0.05).In conclusion,zinc methionine instead of zinc sulfate could positively affect growth performance and antioxidant status of broilers.
Effect of Yeast Polysaccharide on Growth Performance,Bacterial Load and Endogenous Antimicrobial Peptides in Rabbits Infected with Campylobacter jejuni Expression
ZHENG Jiangping, LIU Ning, BAI Xuerui, ZHANG Feike
2020, 47(4):  1099-1108.  doi:10.16431/j.cnki.1671-7236.2020.04.015
Abstract ( 246 )   PDF (1110KB) ( 70 )  
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This study was aimed to investigate the effect of yeast polysaccharide (YPS) supplementation on growth performance,Campylobacter jejuni(C.jejuni) carriage and antimicrobial peptides in the epithelial tissue of cecum and skin in Rex rabbits.Five treatments included control and C.jejuni challenge with the addition of YPS at 0,200,400 or 600 mg/kg of diet.The trial lasted for 35 days and C.jejuni challenge was occurred at first day of feeding trial.The results showed that C.Jejuni challenge worsened final body weight,feed intake,body weight gain and feed efficiency (P < 0.05),whereas YPS supplementation compensated body weight gain and feed efficiency (P < 0.05).C.jejuni populations in the cecal digesta,skin,liver and spleen were decreased in the treatments containing YPS (P < 0.05). C.jejuni induced mRNA upregulation of Cathelicidin,Galectin-3 and LEAP2 gene (P < 0.05),but had no effect on DEFA4 gene,and addition of YPS further upregulated the gene expression (P < 0.05). Linear and quadratic trends of the three doses of YPS were found on final body weight and body weight gain (P<0.05),linear trends on C.jejuni carriage in tissues of skin,spleen and liver (P<0.05),linear trends on Cathelicidin and Galectin-3 in tissues of cecum and skin (P<0.05),and quadratic trends on Galectin-3,LEAP2 and DEFA4 gene in the cecal epithelium (P<0.05).It was concluded that YPS could effectively control C.jejuni infection by decreasing C.Jejuni carriage and activing endogenous epithelial antimicrobial peptides,and had no negative effect on the growth performance of rabbits.
Genetics and Breeding
Analysis of Microsatellite Genetic Diversity in Argali and Its Hybrid Offspring
CHI Haobin, LANG Xia, WANG Cailian, LIU Lishan
2020, 47(4):  1109-1120.  doi:10.16431/j.cnki.1671-7236.2020.04.016
Abstract ( 203 )   PDF (1056KB) ( 62 )  
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In order to better understand the genetic information of Argali sheep and its hybrid offspring (Argali sheep ♂×Oula sheep ♀),and provide a theoretical basis for the evaluation genetic resources of Argali sheep and the rejuvenation and improvement of Oula sheep,24 microsatellite loci were used to analyze the genetic diversity by PCR amplification and capillary electrophoresis in 24 Argali sheep and 24 hybrid sheep.The results showed that,167 alleles were detected at 24 microsatellite loci in 24 Argali sheep,the average number of alleles was 6.9583,the average effective allele was 3.3665,the average observed heterozygosity was 0.4497,the average expected heterozygosity was 0.6779,and the average polymorphic information content was 0.6265,MAF70 locus had the highest numbers of alleles,OarFCB193 locus had the highest numbers of effective alleles,expected heterozygosity and polymorphism information content,ILSTS11 locus had the highest observe heterozygosity.In 24 hybrid sheep,154 alleles were found in 24 microsatellite loci,the average number of alleles was 6.4166,the average effective allele was 3.4061,the average observed heterozygosity was 0.4566,the average expected heterozygosity was 0.6751,and the average polymorphic information content was 0.6265.MAF70 locus had the highest numbers of alleles,numbers of effective alleles,expected heterozygosity and polymorphism information content,OarJMP58 locus had the highest observe heterozygosity.It was found that the genetic indicators of Argali sheep and hybrid offspring were similar in genetics,and the data could be used for evaluate the genetic resources of Argali sheep and the rejuvenation and improvement of Oula sheep.
Effects of Carnitine on Total Antioxidant Capacity and Expression of Antioxidant Enzyme and Apoptosis Related Genes of Frozen-thawed Boar Sperm
LYU Yanqiu, XU Man, XU Yichao, CHENG Mimi, JIN Yi
2020, 47(4):  1121-1129.  doi:10.16431/j.cnki.1671-7236.2020.04.017
Abstract ( 194 )   PDF (1954KB) ( 89 )  
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This study was aimed to investigate the effects of carnitine on frozen-thawed boar sperm quality,anti-oxidation,anti-apoptosis and sperm-egg fertilization ability in vitro.The sperm was divided into a fresh sperm group and 4 frozen-thawed sperm groups.The frozen-thawed sperm groups were treated with different concentrations of carnitine (0,0.025,0.05 and 0.075 mg/mL).The quality,total antioxidant capacity,sperm-egg binding ability,mRNA expression of antioxidant enzyme related and apoptosis-related genes of sperm were investigated.The results showed that in frozen-thawed groups,carnitine treatment significantly increased sperm motility,plasma membrane integrity rate and acrosome membrane integrity rate compared with 0 mg/mL treatment (P < 0.05),meanwhile treatment with 0.05 mg/mL carnitine showed the highest improvement.The concentration of MDA in sperm was significantly increased after frozen-thaw treatment (P < 0.05),among them,a significant decrease was only measured in 0.05 mg/mL treatment group.Compared with fresh sperm group,the total antioxidant capacity of freeze-thaw group sperm decreased significantly (P < 0.05),0.05 mg/mL carnitine treatment group showed the highest total antioxidant capacity in frozen-thawed groups.Furthermore,compared with fresh sperm group,the relative expression of apoptosis-related genes Caspase-3 and Bax increased significantly (P < 0.05),the relative expression of anti-apoptotic gene Bcl-2 decreased significantly (P < 0.05),and the expression of antioxidant enzyme-related genes SOD2,CAT and GPx decreased significantly in 0 mg/mL carnitine treatment (P < 0.05).Compared with fresh sperm group,the ability of sperm-egg binding in every treatment group showed a decrease,0.05 mg/mL carnitine treatment also showed the highest improvement (P < 0.05).The results indicated that the addition of carnitine could improve the quality,anti-oxidation ability,anti-apoptosis ability and sperm-egg binding ability of frozen-thawed boar sperm.
Research Progress on Diversity of Hair-coat Types in Cashmere goats
LI Xiaokai, FAN Yixing, QIAO Xian, ZHANG Lei, WANG Fenghong, WANG Zhiying, WANG Ruijun, ZHANG Yanjun, LIU Zhihong, WANG Zhixin, HE Libing, LI Jinquan, SU Rui, ZHANG Jiaxin
2020, 47(4):  1130-1139.  doi:10.16431/j.cnki.1671-7236.2020.04.018
Abstract ( 281 )   PDF (1084KB) ( 105 )  
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Cashmere goats could be further divided into diverse distinction phenotypes,with their characteristics of wool fiber,growth time and color,as long hair and short hair,coarse hair and fine hair,mosaic type,curvature cashmere morphology and natural cashmere morphology,perennial and seasonal growth,white,grey and black cashmere.Compared with other classification,the diversity of coarse hair traits was more abundant.According to the study of their coat types,it was found that it had intermediate or low phenotypic correlation and genetic correlation with other economic traits,and had a certain reference value for indirect selection for cashmere economic traits.Based on the relevant research data of cashmere goats recently,this paper discussed the diversity of cashmere goat hair-coat types and the differences in their study criteria.The prospect and thinking for the future research of different hair-coat types in cashmere goats were put forward to provide reference and theoretical basis for the future molecular biology research of hair-coat types and the breeding of new strains.
Preliminary Study of Conditional Cell Depletion in Uterus
WU Linlin, ZHANG Heng, LI Shijie, MA Xinghong
2020, 47(4):  1140-1147.  doi:10.16431/j.cnki.1671-7236.2020.04.019
Abstract ( 236 )   PDF (3548KB) ( 142 )  
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In order to study the function of cells and the interaction between various cells in vivo,a new method of conditional cell depletion which could specifically eliminate specific cells was studied.Uterine-specific diphtheria toxin receptor transgenic mice were obtained by mating PRCre/+ tool mice with pCAG-loxp-STOP-loxp-DTR-2A-EGFP transgenic mice (DTR mice).The genes were identified by PCR and agarose gel electrophoresis and PRCre/+/pCAG-loxp-STOP-loxp-DTR-2A-EGFP female mice (PR-DTR mice)were screened out.The DTR mice and wild type mice (WT mice)were used as experimental control groups.Immunohistochemistry and Western blotting were used to detect the expression of GFP protein in the uterus.1 ng/d diphtheria toxin was injected intraperitoneally to observe the differences in external morphology of the uterus of PR-DTR, DTR and wild type mice.The internal morphology of the uterus was observed by HE staining.Immunohistochemistry was used to detect the expression of FOXA2 protein in PR-DTR, DTR and WT female mice after injecting diphtheria toxin.It was found that epithelium,stromal and muscle cells of mouse expressed GFP protein.The uterus of PR-DTR mice was redness and swelling after injection of diphtheria toxin,and there were no significant changes in the uterus of DTR mice and WT mice.Abnormal cavities in the uterus of PR-DTR mice were observed by HE staining.There were no significant differences in the uterus of DTR and WT mice,the detection of FOXA2 expression in the uterus of PR-DTR mice injected with diphtheria toxin showed that these abnormal cavities were not glands,indicating that diphtheria toxin caused cell death.It was confirmed that cells expressing progesterone receptors in mouse uterus expressed DTR.Experiments showed that diphtheria toxin played a role in the uterus of PR-DTR mice,and could conditionally eliminate cells expressing progesterone receptors in the uterus.It provided a new method for studying the function of uterine cells and the interaction between uterine cells.
Preventive Veterinary Medicine
Effect of UBC13 on the Polarization of Th2 and Th17 Cells in Asthma Model Mice
GUO Yin, MENG Weijin, SUN Jiankang, JIANG Junbing
2020, 47(4):  1148-1155.  doi:10.16431/j.cnki.1671-7236.2020.04.020
Abstract ( 241 )   PDF (5163KB) ( 105 )  
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The aim of this study was to investigate the effect of UBC13 protein on the polarization of Th2 and Th17 cells in asthma model mice.18 BALB/c mice were randomly divided into PBS,asthma model (OVA) and UBC13 protein (UBC13) groups.The OVA and UBC13 groups were sensitized and aerosolized using ovalbumin (OVA) and lipopolysaccharide (LPS) to establish asthma model.Then 0.5 h before each atomization,UBC13 group was intraperitoneally injected with 100 μg UBC13.After 24 h of the last atomization,all mice were killed and samples were taken.HE and PAS staining were used to observe the pathological changes of lung sections and the secretion of airway mucus,respectively.The IgE concentration in serum and the levels of IL-4,IL-5 and IL-17A in bronchoalveolar lavage fluid (BALF) were detected by ELISA,the relative expression of Th2 (IL-4,IL-5,IL-13 and IFN-γ) and Th17 (IL-1β,IL-6,IL-17A and IL-23) cytokines were detected by Real-time PCR.The results showed that the asthma model was successfully established.Compared with PBS group,the IgE content in OVA group increased extremely significantly (P < 0.01),and the lung also occured the pathological changes.The relative expression of IL-1β,IL-4,IL-6,IL-13 and IL-17A mRNA increased extremely significantly in lung (P < 0.01),the relative expression of IL-5 and IL-23 mRNA increased significantly (P < 0.05),the relative expression of IFN-γ mRNA declined extremely significantly (P < 0.01),and the levels of IL-4,IL-5 and IL-17A in BALF increased extremely significantly (P < 0.01).In addition,compared with OVA group,the serum IgE content declined significantly in UBC13 group (P < 0.05).HE and PAS stainings results showed that inflammatory cell infiltration and mucus secretion decreased in pathological sections.The relative expression of IL-1β,IL-4,IL-17A,IL-13 and IL-23 mRNA declined significantly in lung (P < 0.05),the relative expression of IFN-γ mRNA increased significantly in lung (P<0.05),the relative expression of IL-6 mRNA declined significantly (P < 0.01).The level of IL-4 and IL-5 in BALF decreased significantly (P < 0.01;P < 0.05),but the level of IL-17A was not significant difference (P > 0.05).It indicated that UBC13 protein could downregulate the expression of Th2 and Th17 cytokines and inhibit the polarization of Th2 and Th17 cells in asthma model.
Immunogenicity of Recombinant Salmonella Typhimurium Expressing Protective Antigen Gene F1I2 of Pseudomonas aeruginosa in Mink
ZHANG Mingliang, SUN Changjiang, GU Jingmin, CUI Ziyin, HAN Wenyu
2020, 47(4):  1156-1162.  doi:10.16431/j.cnki.1671-7236.2020.04.021
Abstract ( 205 )   PDF (940KB) ( 64 )  
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The purpose of this study was to investigate the immunogenicity of recombinant Salmonella Typhimurium strain Δasdlh430(pYA-F1I2) carrying Pseudomonas aeruginosa protective antigen genes F190-342(F1) and I21-83(I2) in mink.Nine 7-month-old healthy minks were randomly divided into three groups,three in each group:Control group,hypodermic injection of sterile normal saline;LH430 strain immunization group,hypodermic injection of 2.0×108 CFU per mink;Δasdlh430(pYA-F1I2) strain immunization group,hypodermic injection of 2.0×108 CFU per mink.Every group was immunized once on the 15th day after the first immunization.On the 15th,30th and 45th day after the first immunization,the blood of mink was collected and the level of IgG in serum was measured.Peripheral blood monocytes were isolated on the 45th day after the first immunization.The number of F1I2 specific antibody secreting cells and Salmonella specific antibody secreting cells were detected by Eli-Spot.At the same time,lymphocytes were isolated from the spleen,and the specific lymphocyte proliferation of F1I2 and Salmonella was detected by MTT method.The results showed that the titers of F1I2 specific IgG antibody and Salmonella specific IgG antibody in serum of Δasdlh430(pYA-F1I2) group increased gradually at the sampling point,and reached the maximum value at the 45th day.On the 45th day after immunization,the number of F1I2 specific IgG secretory cells in Δasdlh430(pYA-F1I2) group was extremely significantly higher than that in control and LH430 groups (P < 0.01),and the number of Salmonella specific antibody secretory cells was extremely significantly higher than that of control group (P < 0.01),but there was no significant difference with LH430 group (P > 0.05).At the same time,F1I2 antigen and Salmonella antigen stimulated the proliferation of mink spleen lymphocytes significantly (P < 0.01).This study could provide a theoretical basis for the development of a new bivalent genetic engineering vaccine against mink hemorrhagic pneumonia and Salmonella infection.
Prokaryotic Expression of EP153R Protein of African Swine Fever Virus and Preparation of Its Polyclonal Antibodies
WANG Zhaoyang, LIN Weidong, LIU Xueting, JIANG Yajun, XIN Ting, HOU Shaohua, GUO Xiaoyu, ZHU Hongfei, JIA Hong
2020, 47(4):  1163-1171.  doi:10.16431/j.cnki.1671-7236.2020.04.022
Abstract ( 253 )   PDF (2033KB) ( 138 )  
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The aim of this study was to construct the prokaryotic expression system of EP153R gene of African swine fever virus (ASFV),express the recombinant protein EP153R (rEP153R),and prepare the polyclonal antibody against rEP153R.EP153R gene sequence was synthesized according to EP153R gene sequence of ASFV Georgia 2007/1 (GenBank accession No.:FR682468.1),and inserted into pET-28a vector to construct pET-28a-EP153R recombinant plasmid.After identification by double enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,which were induced by IPTG at 16 and 37 ℃ for 12 h respectively.The recombinant protein was expressed at the optimum temperature and identified by SDS-PAGE and Western blotting.The 25 ku bands of SDS-PAGE protein gel were cut,and the expression of recombinant protein was identified by liquid chromatography-mass spectrometry LC-MS.The polyclonal antibody against rEP153R was prepared by immunizing mice with purified rEP153R.The antibody titer was detected by indirect ELISA,and the specificity was identified by Western blotting.The results showed that the expression level of the recombinant bacteria was high at 16 ℃,and it was expressed in the form of inclusion body,and the product appeared obvious bands at 25 ku,which was consistent with the expected protein size,indicating that rEP153R was successfully expressed.The results of LC-MS showed that the specific peptides of YIGLINK、NESVLLR and KYIGLINK were matched,and the protein was confirmed to be rEP153R.The titer of polyclonal antibody was 1∶128 000 by indirect ELISA.Western blotting results showed that the antibody had good specificity.The above results showed that rEP153R was successfully expressed and its specific antibody was prepared,which provided technical support and material for further study of biological function of the protein and identification of live carrier vaccine of African swine fever.
Molecular Epidemiological Investigation on ORF5 Gene of Porcine Reproductive and Respiratory Syndrome Virus in Sichuan in 2018
LIU Hanyang, ZHANG Yi, XIAO Ran, CHEN Bin, WU Yongsheng, SU Zhipeng, LI Yupeng, PEI Chaoxin, XU Zhenying, ZHU Jiawen
2020, 47(4):  1172-1182.  doi:10.16431/j.cnki.1671-7236.2020.04.023
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In order to study the epidemic and evolution trend of porcine reproductive and respiratory syndrome virus (PRRSV) in Sichuan,this study took GP5 protein as the research object,detected 378 suspected PRRSV samples collected in different districts,cities and counties of Sichuan province in 2018 by Real-time PCR,and analyzed the nucleotide sequence and deduced amino acid sequence of ORF5 gene of some strains and predicted the N-glycosylation site.69 positive samples were detected by Real-time PCR,the positive rate was 18.25%.The ORF5 gene sequence analysis of 23 PRRSV strains showed that the nucleotide homology of ORF5 gene was 81.6%-100%,and the deduced amino acid homology was 81.4%-100%.All of them belonged to America type.Among them,1 strain belonged to subtype Ⅰ represented by the classic strain VR2332,17 strains belonged to subtype Ⅱ represented by the highly pathogenic strain JXA1,and 5 strains belonged to subtype Ⅲ represented by the recombinant strain NADC30,which indicated that there was genetic diversity of PRRSV epidemic strains in Sichuan province in 2018,HP-PRRSV was still the main epidemic strain,but there were still evolving classic strains.At the same time,recombinant strains which were similar to NADC30 and QYYZ began to appear.Through the analysis of amino acid mutation sites and N-glycosylation sites,it was found that 23 PRRSV strains had some mutations in two important epitope related areas and virulence related sites of GP5.It was speculated that these mutations might affect the virus’s escape from immune system and virus virulence,suggesting that in PRRSV prevention and control,genetic evolution analysis should be strengthened,and selected targeted vaccines and control strategies according to different types of strains.
Epidemiological Investigation Analysis of Porcine Circovirus Type 3 in Guangxi
DUAN Qunpeng, ZHAO Shuo, QIN Yibin, LU Bingxia, HE Ying, CHEN Zhongwei, LIU Lei, ZHOU Yingning, LI Bin, JIANG Dongfu, LIANG Jiaxing, ZHAO Wu
2020, 47(4):  1183-1190.  doi:10.16431/j.cnki.1671-7236.2020.04.024
Abstract ( 252 )   PDF (1228KB) ( 104 )  
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The purpose of this study was to investigate the infection situation and epidemic characteristics of porcine circovirus 3 (PCV3) in the pig population of Guangxi,so as to provide scientific basis for effective prevention and control.PCR method was used to detect PCV3 in 917 pig tissue samples with pathological changes from 482 large-scale pig farms in Guangxi from September 2017 to March 2019,and the difference of positive detection rate of PCV3 in different regions,years,seasons and age groups of pigs were considered.145 PCV3 positive samples were simultaneously tested for CSFV,PRRSV,PCV2,PRV and PEDV.The results showed that except for Baise and Fangchenggang,which were not detected,PCV3 had different degrees of infection in other 12 cities in Guangxi.From 2017 to 2019,the positive rate of PCV3 infection and the positive rate of PCV3 infected pig farms in Guangxi showed a trend of gradual increase.The infection rate of PCV3 was the lowest in summer,which was significantly lower than that in spring,autumn and winter.The infection rate of PCV3 was the highest in finishing pigs (21.70%),followed by aborted sows,protected pigs and aborted fetuses (20.00%,19.05% and 11.02%,respectively),and the lowest in suckling pigs (8.21%).PCV3 had the highest single infection rate (30.34%).Mixed infection of PCV3 with other pathogens was also relatively common,even with quadruple infection.The investigation results showed that PCV3 infection was more common in Guangxi,and increased in recent years,and might be seasonal,it had a certain degree of harm to pig groups in all growth stages,especially in finishing pigs,and it also had a great harm to sows and conservation pigs.The single positive rate of PCV3 was higher,but it was mainly mixed infection,especially with PRRSV and PCV2,which suggested that there might be synergism among the three pathogens in the process of infection.
Development of a Time-resolved Fluorescence Immunochromatography Assay Kit for Detection of Bovine Serum Amyloid A
WU Meng, WEI Zhijing, FAN Yiming, CUI Chenguang, LI Shuang, DONG Weiya
2020, 47(4):  1191-1198.  doi:10.16431/j.cnki.1671-7236.2020.04.025
Abstract ( 252 )   PDF (1009KB) ( 87 )  
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The purpose of this study was to develop a time-resolved fluorescence immunochromatography kit for clinical rapid detection of bovine serum amyloid A (SAA) content in milk.By using double antibody sandwich method and fluorescence immunochromatography detection technology,the immunochromatography detection kit was manufactured.The SAA monoclonal antibody used for labeling was labeled with fluorescent microspheres,and chicken IgY was labeled with fluorescent microspheres,too.Then the above two kinds of fluorescent microspheres labeled antibody mixture was fixed on the binding pad.The SAA monoclonal antibody used for coating was coated on the detection area of nitrocellulose membrane,and goat anti-chicken IgY was coated on the quality control area of nitrocellulose membrane.The standard curve of the kit was drawn after screening of antibody pairing materials and optimization of the process of fluorescent microsphere labeled antibody.Besides,the blank limit,precision,stability and sample testing performance of the kit were preliminarily evaluated.The results showed that,the Medix SAA-2 monoclonal antibody as coating antibody and YBX SAA-3 monoclonal antibody as labeling antibody was the optimum antibody pairing materials.In the process of labeling antibody with fluorescent microsphere,the optimal results were that the mass ratio of fluorescent microspheres to antibodies was 40∶1,and the carboxyl molar ratio of coupling agent to fluorescent microspheres was 2∶1.The four-parameter fitting curve equation of the standard curve of the kit was y=(1.03947-0.00182)/[1+(x/12.08222)×(-0.84692)]+0.00182,the linear correlation coefficient was R2=0.9997.The blank limit of the developed bovine SAA detection kit was 0.052 mg/L.Precision test result showed that,the intra-assay coefficient of variation was < 15%,and the inter-assay coefficient of variation was < 20%.The room temperature stability test showed that,the fluorescence T/C value of the sealed kit stored at room temperature for 6 months decreased by about 15%.The correlation coefficient R2 of self-made kit and Shanghai Blue Gene kit was 0.97.In summary,the developed kit had the advantages of simple operation,high sensitivity and low cost,which could meet the needs of clinical determination,and provided a new type of fast and accurate detection method for bovine SAA detection.
Research Progress of Genetic Engineering Recombinant Lactic Acid Bacteria on Epidemic Prevention and Control of Animal Diseases
LIU Naizhi, QI Xiuye, GUO Yangli, CHENG Fuliang, XU Haiyan, GU Wei, SHAN Baolong, WANG Chunfeng
2020, 47(4):  1199-1208.  doi:10.16431/j.cnki.1671-7236.2020.04.026
Abstract ( 234 )   PDF (1073KB) ( 130 )  
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Most infectious pathogens enter the host through the mucosal surface.Therefore,besides the traditional vaccines that induce systemic immunity,mucosal vaccination should also be developed to establish the first line of defense at the mucosal invasion site to eliminate pathogens.Lactic acid bacteria are common probiotics in the intestines of humans and most animals,and have the functions of nutrition,immunity and anti-infection.Since 1990s,lactic acid bacteria have been used to express exogenous genes as food-grade carriers.The ability of lactic acid bacteria to elicit an immune response against expressed foreign antigens has led to their function as potential mucosal vaccine candidates.Lactic acid bacteria as vaccine vectors have many attractive advantages:Simple,noninvasive administration (usually oral or intranasal),the acceptance and stability of genetic modifications,expression of protein without purification,relatively low cost,and the highest level of safety possible,long-term survival in the body and continuous expression of foreign proteins,a single vaccination can produce a lasting immune response,and so on.At present,lactic acid bacteria as the normal bacterial group are selected as the carriers,and the recombinant lactic acid bacteria have been rapidly developed in many fields,such as,bacteria,viruses,parasites,etc.,and they have achieved significant effects in the corresponding disease models.This paper reviewed the preparation of genetically engineered recombinant lactic acid bacteria and the research progress in the prevention and control of animal diseases in the past five years.
Molecular Identification of Hydatid Cysts from Sheep and Yaks of Tibet
XIA Chenyang, SHI Bin, TANG Wenqiang, Danqulamu, JIA Wanzhong, Bai Ji, WU Yantao, YAN Hongbin, LIU Jianzhi
2020, 47(4):  1209-1215.  doi:10.16431/j.cnki.1671-7236.2020.04.027
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The purpose of this study was to identify the hydatid pathogen in yaks and sheep in Naqu and Lhasa of Tibet Autonomous Region and analyze its genetic variation.5 hydatid cysts were isolated from 5 sheep and 18 hydatid cysts from 15 yaks collected in Dangxiong county of Lhasa,and Jiali county of Naqu at the end of November 2016.Genomic DNA was extracted,nad1 gene was amplified by PCR,and nad1 full gene sequence was obtained by sequencing.DNAStar MegAlign software was used to analyze the sequence homology.Taking nad1 gene sequence of Echinococcus published in GenBank as comparison object,the phylogenetic tree was constructed by ML.The results showed that the nad1 gene sequence of 23 hydatid pathogens from yaks and sheep was highly homologous with that of the gene sequence of Echinococcus granulosus (G1 genotype),the homology was 99.6% to 99.8%,and the genetic distance of 23 nad1 genes was 0 to 0.0022447.The base variation rate of homologous genes was 0.2% to 0.4%,and that of homologous genes of Echinococcus was 14.9% to 19.8%.5 samples of nad1 gene had base mutation at different sites,and the mutation sites had sequence conversion.The results showed that the Echinococcus of yaks and sheep collected in this study were G1 genotype of Echinococcus granulosus,its nad1 gene had small variation and high sequence consistency.
Epidemiological Investigation and VP1 Gene Sequence Analysis of Porcine Enterovirus Type G in Guangxi
YANG Chunjie, LIU Xueting, MI Xue, CHEN Ying, WEI Zuzhang, HUANG Weijian, OUYANG Kang
2020, 47(4):  1216-1224.  doi:10.16431/j.cnki.1671-7236.2020.04.028
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To analyze the prevalence and molecular genetic evolution of porcime enterovirus G (EV-G) in Guangxi,222 clinical diarrhea samples were collected in Guangxi from 2017 to 2018 in this study,EV-G detection and epidemiological investigation were conducted,as well as VP1 gene amplification,cloning and sequence analysis.According to the results of epidemiological survey,the positive rate of EV-G samples in Guangxi from 2017 to 2018 was 6.76% (15/222),while the positive rate of pig farms was as high as 16.98% (9/53).It was noteworthy that the positive rate of EV-G samples in Guangxi had increased from 4.55% in 2017 to 10.00% in 2018,which showed a significant increase.At the same time,the detection rate of this virus was higher in winter and spring.Sequence homology analysis showed that the nucleotide homology between the two EV-G VP1 genes obtained in this study was 99.2% and the amino acid homology was 98.8%.Compared with domestic and foreign strains,the nucleotide and amino acid identity was 62.4% to 80.1% and 45.7% to 71.7%,respectively.Genetic evolution analysis showed that the two EV-G VP1 sequences obtained in this study both belonged to the G1 subtype in the same branch,and were closely related to the American isolate 13-03212. The results of amino acid sequence comparison showed that the two strains of EV-G VP1 had great variation at multiple sites.Antigen index analysis showed that the antigenicity of EV-G in Guangxi had a large variation.The results of this study indicated that there was a certain degree of EV-G infection in Guangxi during 2017 to 2018.This study provided a reference for the EV-G epidemic profile and the molecular characteristics of EV-G.
Effects of RFRP-3 on Morphological Structure and Immune Function of Spleen in Rats
ZHANG Duoni, HUO Konglin, LI Changle, ZHANG Xin, SONG Xingxing, HU Wen, LUO Rongrong, XU Wenhao, CHEN Lei, CHEN Xinyi, LI Xun, WANG Xiaoye
2020, 47(4):  1225-1232.  doi:10.16431/j.cnki.1671-7236.2020.04.029
Abstract ( 196 )   PDF (3793KB) ( 162 )  
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The purpose of this study was to investigate the effects of different RFRP-3 treatment on morphological structure and immune function of spleen in SD rats.The experiment was comprised of two parts:10 SD rats were chosen for the study about the distribution of RFRP-3 and GPR147.In the other part,30 SD rats were randomly assigned into 3 groups.They were control group (0.9% NaCl),low-dose group and high-dose group (1 and 10 μg/100 μL,total 200 μL of RFRP-3).Intraperitoneal injection was continued for 14 days.The expression of RFRP-3 and its receptor (GPR147) were investigated by using semi-quantitative RT-PCR and Western blotting.The distribution of RFRP-3 and GPR147 were determined by using immunohistochemistry.Blood was collected and serum was isolated to detect immune-related factors by using a protein chip array after anesthesia.Spleen length was measured and spleen index was calculated after the autopsy.The effect of RFRP-3 on morphology of spleen was detected by HE staining.The expression of IL-β and TNF-α mRNA in spleen was detected by Real-time quantitative PCR.Results showed that no RFRP-3 mRNA expression was observed in spleen,GPR147 mRNA was moderately expressed.The results of Western blotting showed that both RFRP-3 and GPR147 were detected in spleen.RFRP-3 was mainly distributed in red pulp,while GPR147 was mainly distributed in white pulp of spleen.The concentrations of IL-1α,IL-1β,MCP-1 and TNF-α were significantly increased in the rats treated with RFRP-3.The length of spleen increased and spleen index increased,too.The results of HE showed that the white pulp area expanded with more splenic sinus and the red blood cell in red pulp were evidently increased.The results of Real-time quantitative PCR showed that the expression of IL-1β and TNF-α mRNA in spleen of SD rats were extremely significantly or significantly increased (P<0.01;P<0.05).The above results showed that RFRP-3 might act on the spleen via GPR147 through the paracrine manner and promote immune response.
Basic Veterinary Medicine
Study on Forming Process and Scale-up of Zhongjiefeng Sanqing Granule
DENG Ruihan, PENG Jianbo, WU Wenjian, TANG Tingchong, TAO Qing, LIAO Xiaoguang, WANG Chunyuan, HAO Zhihui, YANG Shanzhong, HE Jiakang
2020, 47(4):  1233-1240.  doi:10.16431/j.cnki.1671-7236.2020.04.030
Abstract ( 180 )   PDF (887KB) ( 120 )  
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In order to optimize the forming process and to examine the rationality of the scale up effect of Zhongjiefeng Sanqing granule,the forming process and scale-up process were performed.In the optimal forming process,orthogonal design was used to optimize three factors,namely the ratio of extract powder to auxiliary materials,the ratio of sucrose to dextrin and the concentration of ethanol,which the molding rate,melting rate and moisture absorption rate were used as the indexes of the process parameters.For the scale up design,the extraction yield,product yield and the content of active ingredient as the indexes to investigate the feasibility of the scale up process.The results showed that the preferred Zhongjiefeng Sanqing granule optimal molding process was that the ratio of sucrose to dextrin was 3∶2,the ethanol concentration was 65%,and the ratio of extract powder to excipient was 1∶6,the critical relative humidity of the granule was 73.49%.The extraction yields of the three batches of Zhongjiefeng Sanqing extract (intermediate) were 8.58%,8.58% and 8.63%,respectively,and the product yields of the three batches of Zhongjiefeng Sanqing granule (finished products) were 93.40%,92.13% and 93.53%,respectively.The content of the active ingredients,namely isoxazidine and rosmarinic acid in the intermediates and finished products,were stable and controllable.It was shown that the molding process of Zhongjiefeng Sanqing granule was reasonable,and the scale up production was stable and controllable,which was suitable for mass production.
Isolation and Identification of Porcine Contagious Actinobacillus pleuropneumoniae in Luoyang
LI Kaiming, DONG Faming, LIU Shouchuan, LIU Jie, LIANG Liqin, LI Kaiqiang
2020, 47(4):  1241-1249.  doi:10.16431/j.cnki.1671-7236.2020.04.031
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In order to determine the pathogenic species and serotypes of suspected infectious pleuropneumonia in a pig farm in Luoyang,and to provide a reference plan for its clinical scientific epidemic prevention and rational drug use,in this experiment,the diseased pig lungs and other diseased materials were collected aseptically,and the pathogenic bacteria were isolated and identified in laboratory by morphological observation,Gram staining,physiological and biochemical characteristics test,PCR diagnosis and other technologies.It was confirmed by gene sequencing and homology analysis.The pathogenicity and drug resistance of the isolated strains were analyzed by animal attack test and drug sensitivity test.Five pairs of primers were used for genotyping to determine the serotypes.The results showed that the colony morphology of 2007 was smooth,transparent,orderly,round and protuberant,Gram staining was negative and polytypic.It had the typical culture characteristics of Actinobacillus pleuropneumoniae (APP),and the biochemical results were consistent with the biochemical characteristics of Actinobacillus pleuropneumoniae.The results of homology analysis showed that the homology between the isolate 2007 and Actinobacillus pleuropneumoniae strain FJ848573.1 was 99.4%,indicating that the isolate 2007 was Actinobacillus pleuropneumoniae.The results showed that the isolate 2007 had strong pathogenicity to mice.The results of drug sensitivity test showed that the isolate 2007 was highly sensitive to temicillin,ampicillin and ceftiofur sodium,completely resistant to 20 kinds of antimicrobial agents,such as tilmicosin,erythromycin and florfenicol,and the resistance rate to 34 kinds of antibiotics was 76.5%,with strong multiple drug resistance.The results of the isolate 2007 genotyping showed that the target gene fragment was consistent with serotype 5.The results of this study provided specific prevention and treatment measures for the pig farms,and finally achieved good results,which provided some references for the establishment of clinical resistant fold points of Actinobacillus pleuropneumoniae.
Research Progress on Drugs for the Treatment of Toxoplasmosis
YAO Na, GONG Jingzhi, FAN Yimin, YANG Bin, TAO Jianping, HUANG Siyang
2020, 47(4):  1250-1257.  doi:10.16431/j.cnki.1671-7236.2020.04.032
Abstract ( 419 )   PDF (916KB) ( 195 )  
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Toxoplasmosis is a worldwide important zoonotic parasitic disease,which infects almost all warm-blooded animals,including human beings.At present,controlling this disease mainly depends on drugs.Clinically,sulfadiazine and pyrimethamine are used to treat the disease,which is seemed as the gold standard.However,this therapy has certain toxic and side effects,and is prone to drug resistance after long-term use.So development of anti-toxoplasmosis drug has always been a hotspot.Spiramycin,trimethoprim-sulfamethoxazole and so on are used as alternatives in treating toxoplasmosis,however,they all have different limitations.Therefore,the focus of research has been shifted to natural products.Plant extracts were used as the main component.Through screening the active molecules with anti-Toxoplasma gondii and modifying them properly,the anti-Toxoplasma gondii activity can be enhanced and the side effects can be reduced.Among animal extracts,the venom of some animals has anti-Toxoplasma gondii activity and can be good candidate drugs. With the progress of research methods,the new activities of some existing drugs have been continuously discovered.In the research direction of the new use of ‘old’ drugs,on the one hand,according to genetic relationship,the anti-Toxoplasma gondii activity of other anti-parasite drugs can be explored.On the other hand,depending on the drug action target,the anti-Toxoplasma gondii activity of drugs for the treatment of other diseases is being studied.To sum up,this article reviewed the clinical drug history of toxoplasmosis,the development of natural products and the new use of ‘old’ drugs,which provided new ideas for the development of anti-toxoplasmosis drugs.
Study on Anti-inflammatory Activity of Total Flavonoids from Viola yedoensis in Vitro
ZHANG Jing, SHAO Yongbin, GU Xinli, LUO Yan
2020, 47(4):  1258-1266.  doi:10.16431/j.cnki.1671-7236.2020.04.033
Abstract ( 197 )   PDF (1353KB) ( 59 )  
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To study the effects of total flavonoids from Viola yedoensis(TFV) on the content of inflammatory mediators and the expression of related genes in LPS-induced RAW264.7 cells.Screening of the optimum concentration of TFV for promoting RAW264.7 macrophage viability by MTT method.Then its effects on the release of NO,tumor necrosis factor α(TNF-α),IL-1β,IL-6,TNF-α,iNOS and COX-2 expression on RAW264.7 cells were investigated,thus to study and analyze the in vitro anti-inflammatory activity of TFV.Results showed that TFV could increase RAW264.7 cell viability in the concentration range of 5-50 μg/mL (P<0.05).Comparison with LPS model group,TFV could significantly reduce the production of NO,TNF-α,IL-6 and IL-1β (P<0.05),and could significantly reduce the expression of TNF-α,COX-2 and other inflammatory factors in RAW264.7 cells induced by LPS.TFV could down-regulate the expression of inflammatory factors such as IL-1β,IL-6 and TNF-α,and down-regulate the expression of TNF-α and COX-2,which may be one of the reason for the anti-inflammatory effect of Viola yedoensis.
Effects of Taimulin on Human CYP3A4,CYP1A2,CYP2E1 and CYP2D6 Activity
CHEN Fang, GAO Hong, LI Bo, ZUO Xiangyi, JIN Zhen, TANG Youzhi
2020, 47(4):  1267-1273.  doi:10.16431/j.cnki.1671-7236.2020.04.034
Abstract ( 256 )   PDF (894KB) ( 141 )  
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In this study,the in vitro CYP450 incubation assays were established using human liver microsomes (HLMs) to investigate the effects of Tiamulin on the metabolism of four substrate probes Testosterone,Phenacetin,Chlorzoxazone,and Dextromethorphan hydrobromide which reflect the effect of Tiamulin on CYP3A4,CYP1A2,CYP2E1 and CYP2D6 activity.Experiments were performed in 96-well plates with final volume of 100 μL in each well.The study consisted of three groups:a drug test group,a positive control group (without NADPH) and a negative control group (without CYP450 inhibitor).After incubation were terminated,the concentrations of probe substrates in each well were analyzed by LC-MS/MS internal standard method.The percentage of test drug metabolic was calculated from the ratio of the amount of drug metabolized of the drug test group and that of the negative control group.The percentage of test drug metabolic was plotted versus the logarithm of drugs concentration and the IC50 values of test drugs were obtained from the graph using Graphpad prism 6.0.Multiple incubation time points were set to investigate the effect of the incubation time on the IC50 value of Tiamulin against CYP3A4.The results showed that the IC50 value of Ketoconazole against CYP3A4 was 0.044 μmol/L,the IC50 value of α-Naphthoflavones against CYP1A2 was 0.030 μmol/L,the IC50 value of 4-Methylpyrazole against CYP2E1 was 0.022 μmol/L and the IC50 value of Quinidine against CYP2D6 was 0.096 μmol/L.The IC50 values of Tiamulin against CYP1A2 and CYP2D6 were higher than 50 μmol/L.The IC50 values of Tiamulin against CYP3A4 and CYP2E1 were 1.609 and 0.045 μmol/L,respectively.As the incubation time of Tiamulin and CYP3A4 prolonged from 10 min to 50 min,the IC50 value of Tiamulin against CYP3A4 was increased from 1.609 μmol/L to 8.657 μmol/L.For the IC50 values of the recognized inhibitors were consistent with the reference values,it could be concluded that the in vitro CYP450 incubation assays were constructed successfully.Tiamulin possessed no inhibition against CYP1A2 and CYP2D6,while existed a high inhibitory effect on CYP2E1 and CYP3A4.Tiamulin might be developed as a reversible inhibitor against CYP3A4 in the future.
Clinical Veterinary Medicine
Effect of Apoptosis Protein after Eimeria tenella Infection in Chicken
WANG Lixia, SONG Licong, ZHANG Jianjun, AN Jian
2020, 47(4):  1274-1281.  doi:10.16431/j.cnki.1671-7236.2020.04.035
Abstract ( 218 )   PDF (1967KB) ( 75 )  
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In order to study the effect of Eimeria tenella infection on apoptosis related proteins in chicken serum,and explore the interaction between Eimeria tenella and its host.240 7-day-old Heilan Grey male chickens were divided into 4 groups,group A was non infection and non induction of apoptosis,group B was non infection and induction of apoptosis,group C was infection and non induction of apoptosis,group D was infection and induction of apoptosis.The treatment method of inducing apoptosis was to take 40% alcohol to induce apoptosis at 6.4 mL/kg body weight at 2.5 h before blood collection,while the treatment of not inducing apoptosis was replaced by 40% alcohol and other volumes of normal saline.At 24,48,72,96 and 120 h after Eimeria tenella infection (1×105 Eimeria tenella sporulated oocysts per chicken),10 chickens in each group were collected for blood collection.The concentrations of Cytochrome C (Cyto-C),Caspase (Cas)-9,Cas-8,Cas-3,Bcl-2,Bax,Bcl-XL,Bid were detected by ELISA.The results showed that the concentrations of Cyto-C,Cas-9,Cas-8 and Cas-3 in group D were lower than those in group B at 24,48,72 and 96 h after infection.The concentrations of Cyto-C,Cas-9,Cas-8 and Cas-3 in group D at 120 h were significant higher than those in group B (P<0.05).The value of Bcl-2/Bax and Bcl-XL/Bid in group D at 24,48,72 and 96 h after infection were higher than those in group B.The values of Bcl-2/Bax in group D at 120 h were lower than that in group B and the value of Bcl-XL/Bid in group D was higher than that in group B.The results showed that the apoptosis of host cells was inhibited by the increase of Bcl-2/Bax value,Cyto-C,Cas-9,Cas-8 and Cas-3 at the early stage of Eimeria tenella infection,and was promoted by the decrease of Bcl-2/Bax value,Cyto-C,Cas-9,Cas-8 and Cas-3 at the middle and late stage of infection.
Environmental Safety
Study on the Change Law of Milk Production Traits and Routine Blood Indicators of Holstein Dairy Cows in the North of Hebei Province in Autumn and Winter
WANG Yanan, FENG Man, ZHOU Yinghao, MAO Sen, QIU Dianrui, GUO Jianjun, LI Suxia, WANG Hongbin
2020, 47(4):  1282-1288.  doi:10.16431/j.cnki.1671-7236.2020.04.036
Abstract ( 199 )   PDF (800KB) ( 96 )  
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The purpose of the experiment was to explore the blood routine indexes of the dairy cows which were sensitive to climate change,provide the relevant data support and scientific basis for the research of low temperature stress of Holstein dairy cows,and provide the theoretical basis for the breeding and management for winter,and the selection of cold stress-resistant individuals in the area.In this study,the healthy Holstein lactating cows in a large-scale dairy farm in Weichang county,Chengde,Hebei province were taken as the research objects.The temperature and humidity index (THI) in autumn (October 24,2018 to October 30,2018),early winter (December 4,2018 to December 10,2018) and deep winter (December 25,2018 to December 31,2018) were monitored continuously.The blood samples of 15 Holstein dairy cows were collected for the measure of 9 blood routine indexes,and 8 milk production performance data of the experimental cows were collected.The changes of milk production performance and blood routine indexes of Holstein dairy cows at different time were analyzed,and the sensitive blood routine indexes of climate change were screened.The results showed that,in autumn,the average temperature of cowshed was 8.0 ℃,and the average THI was 49.38,indicating that the experimental cattle were in non-low temperature stress state in this period.In early winter,the average temperature of cowshed was -6.4 ℃,and the average THI was 23.56.In 168 h monitoring period,there was 92 h of 8≤THI < 25,76 h of 25≤THI < 39,the experimental cows were in moderate low temperature stress state in most time of this period.In the late winter,the average temperature of the cowshed was -7.59 ℃,the average THI was 21.86,and there was 43 h of 25≤THI < 39,117 h of 8≤THI < 25.The experimental cows were also in the state of moderate low temperature stress at most time of this period.With the decrease of temperature,rectal temperature and respiratory rate decreased significantly (P < 0.05).RBC,WBC,PLT,PCT,HGB,HCT,LY and MO increased significantly or extremely significantly in winter (P < 0.05;P < 0.01).Except HCT and WBC,the other indexes had no significant difference between early winter and deep winter (P > 0.05).MCV did not change significantly in different seasons (P > 0.05).Compared with autumn,the AMY decreased by 0.71 and 1.17 kg/d in early and deep winter,respectively (P < 0.01).FP,PP and F/P decreased significantly or extremely significantly in early winter (P < 0.05;P<0.01),and increased to autumn level in late winter (P > 0.05).SP decreased in early winter and increased in late winter significantly (P < 0.05);SCC in early winter and late winter were 1.84×105 and 1.63×105/mL higher than that in autumn (P < 0.05).There was no significant difference in LP and acidity among different periods (P > 0.05).In conclusion,the Holstein dairy cows were in non-stress state in autumn,were in moderate low temperature stress state in early and late winter,and the physiological indexes and milk production performance of cows were seriously affected by the climate.RBC,WBC,LY and MO were more sensitive blood routine indexes affected by climate.