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20 December 2017, Volume 44 Issue 12
Cloning and Bioinformatics Analysis of Inhibin α-subunit Gene of Arctic Fox and Wusuli Raccoon Dog
ZHANG Yu-fei, LI Xiao-xia, WANG Shi-yong, CAO Xin-yan, DIAO Yun-fei, YANG Yi-feng, CHEN Xiu-min, ZHAO Wei-gang, ZHAO Meng, WEI Hai-jun, XU Bao-zeng
2017, 44(12):  3391-3400.  doi:10.16431/j.cnki.1671-7236.2017.12.001
Abstract ( 148 )   PDF (7247KB) ( 228 )  
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This study was aimed to clone the inhibin α(INHα) subunits gene of arctic fox and Wusuli raccoon dog and analyze its genetic structure with bioinformatics. According to the published sequence of INHα subunit gene (GenBank accession No. XM_545660.5), one pair of specific PCR primers were designed. The INHα subunit gene of arctic fox and Wusuli raccoon dog were amplified by RT-PCR. The target gene was linked into cloning vector to be sequenced and systemically analyzed by bioinformatics. The results showed that INHα subunit gene fragment of arctic fox and Wusuli raccoon dog included an 1 107 bp whole length CDS (encoding 369 amino acids). The INHα subunit gene of arctic fox and Wusuli raccoon dog shared 97.9% and 97.6% of similar nucleotide sequence with INHα subunit gene of dog, respectively. Phylogenetic analysis showed that the INHα subunit gene of arctic fox and Wusuli raccoon dog were genetically close to dog, and it was highly conserved in different species and evolutionary processes. The prediction of INHα subunit tertiary structure showed that the cysteine knot directs members of the INHα subunit to adopt a "butterfly-shaped" or "open hand" configuration, with the α-helix defining the "wrist" and β-sheets forming the "fingers" of the molecule. The INHα subunit gene of arctic fox and Wusuli raccoon dog were cloned successfully and analyzed by bioinformatics software, which would help to research the biological function of inhibin in the simultaneous development of oocyte-granulosa cells.

Cloning and Tissue Expression Analysis of PDK4 and FGF10 Genes in Guizhou Congjiang Xiang Pig
YANG Yang, XU Hou-qiang, CHEN Wei, ZHOU Di, XU Min, ZHANG Qing-qing, ZHAO Huan-ping, SUN Cheng-juan, WANG Yuan-yuan, ZHANG Ming, YANG Tao
2017, 44(12):  3401-3409.  doi:10.16431/j.cnki.1671-7236.2017.12.002
Abstract ( 150 )   PDF (2103KB) ( 198 )  
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The objective of this study was to clone PDK4 and FGF10 genes, and investigate the expression level of PDK4 and FGF10 genes mRNA in different tissues of Large White pig and Congjiang Xiang pig. The PDK4 and FGF10 genes were cloned by RT-PCR and analyzed by bioinformatics, the relative expression of PDK4 and FGF10 genes were detected by Real-time PCR. The results showed that the coding region of PDK4 gene was 1 224 bp, encoding 407 amino acids; The coding region of FGF10 gene was 636 bp and encoded 211 amino acids. The homologies of nucleotide sequences of PDK4 gene with sheep, horse and human were 93%, 92% and 91%,respectively. The homologies of nucleotide sequences of FGF10 gene with sheep, cattle, human and mouse were 94%,93%, 93% and 90%, respectively. The phylogenetic tree of PDK4 gene showed that the genetic relationship of Congjiang Xiang pig, cattle and sheep were very close, the phylogenetic tree of FGF10 gene indicated that the genetic relationship of Congjiang Xiang pig, cattle, sheep, human and macaque were very close, but the genetic relationship of Congjiang Xiang pig, rat and chicken were far away. Real-time PCR results showed that, in different tissues of Congjiang Xiang pig,PDK4 gene expression in kidney tissue was higher than other tissues, with a higher expression in stomach and adipose as well,FGF10 gene expression in stomach tissue was higher than other tissues, with a higher expression in kidney and adipose as well, but both of PDK4 and FGF10 genes expression were the lowest in longissimus dorsi. In different tissues of Large White pig, both of PDK4 and FGF10 genes were expressed the highest in adipose than other tissues, PDK4 gene expression in longissimus dorsi was the lowest, while the FGF10 gene expression the lowest in heart. This study successfully cloned the PDK4 and FGF10 genes of Large White pig and Congjiang Xiang pig,and detected the relative expression of PDK4 and FGF10 genes in different tissues of Large White pig and Congjiang Xiang pig, and also provided scientific basis for further study on regulation of PDK4 and FGF10 genes on lipid metabolism and deposition.

Prokaryotic Expression and Preparation Polyclonal Antibody of Wool Follicle KAP7 Gene of Hetian Sheep and Karakul Sheep in Southern Xinjiang
LI Zhi-gang, WANG Li, LI Shu-wei
2017, 44(12):  3410-3417.  doi:10.16431/j.cnki.1671-7236.2017.12.003
Abstract ( 192 )   PDF (1746KB) ( 151 )  
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This study was aimed to prepare polyclonal antibodies of wool follicle keratin associated protein 7 (KAP7) gene of three local variety (strain) sheep of Plain-type Hetain sheep, Mountain-type Hetain sheep and Karakul sheep in Southern Xinjiang, to provide the basis for exploring influences of follicle KAP7 gene to coarse wool quantity and quality. This study took the skin wool follicle samples from three local variety (strain) sheep in Southern Xinjiang, extracted total RNA from the skin wool follicle samples, obtained cDNA through reverse transcription, then amplified the wool follicle KAP7 gene fragments by PCR. We constructed the pMD19-T-KAP7 recombination cloning plasmids, and tested the correction of the plasmids by PCR assay, enzyme digest and sequence analysis, then constructed the prokaryotic expression recombination plasmids of pET-28a(+)-KAP7,expressed them in E.coli BL21 (DE3) cells. SDS-PAGE electrophoresis was employed to detect the prokaryotic expression wool follicle KAP7 protein. We obtained the target proteins by extraction, purification and reclaim. The polyclonal antibodies serum were got from the rabbits which were immunifaciented with the purification KAP7 protien. The antibodies titer were detected by indirect ELISA. The results showed that there were 99% homology among the Merino sheep with the mountain-type and plain-type Hetian sheep, but there was a mutation at the 22nd base pair of KAP7 gene of plain-type Hetian sheep, in which it conversed from G to C, and this made the 8th amino acid of KAP7 protein to converse from Gly to Arg. For the mountain-type Hetian sheep, there was a mutation from A to C at the 70th base pair, this changed the 24th amino acid of KAP7 protein from Thr to Ala. The homology of wool follicle KAP7 gene was 100% between Karakul sheep and Merino sheep, so there was no differences on the amino acids sequences between them. The prokaryotic expression plasmids pET-28a(+)-KAP7 of wool follicle KAP7 gene could be expressed in E.coli BL21 cells, and the wool follicle KAP7 proteins were about 10 ku. The positive polyclonal antibodies serum of the sheep wool follicle KAP7 proteins, which were obtained from immunized rabbits, had immunological activity and specificity, and the antibodies titer could be up to 1:10 000.

Effects of Aflatoxin B1 Individual,Binary or Tertiary Mixed with Ochratoxin A and Zearalenone on Intestinal Microorganism of Dairy Goat
FAN Cai-yun, SU Di, LI Xiao-jiao, HUANG Shuai, ZHANG Yang-dong, ZHENG Nan, LIU Guang-lei, LIU Zhen, CHENG Jian-bo
2017, 44(12):  3418-3425.  doi:10.16431/j.cnki.1671-7236.2017.12.004
Abstract ( 202 )   PDF (2540KB) ( 225 )  
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This experiment was conducted to investigate the effects of aflatoxin B1 (AFB1) individual,binary or tertiary mixed with ochratoxin A (OTA) and zearalenone (ZEN) on the bacterial composition and diversity of dairy goat intestinal tract. Twenty lactating Laoshan dairy goats with similar conditions were randomly assigned to five treatments (n=4). The dairy goats in control group (group A) were fed basal diet,while that of groups B,C,D and E were fed basal diet supplementation 50 μg/kg AFB1+500 μg/kg ZEN,50 μg/kg AFB1+100 μg/kg OTA,50 μg/kg AFB1, 50 μg/kg AFB1+100 μg/kg OTA+500 μg/kg ZEN,respectively. The experimental period consisted of 7 days for adaptation and 14 days for feeding mycotoxins. Faeces samples were collected on the 11th of training. The total DNA of feces was extracted using genomic DNA extraction kit,then 16S rRNA was amplified,and the 16S rRNA gene cloning library of dairy goat intestinal microorganism was constructed and data analysis was performed. The results of the dilution curves and the diversity index showed that single AFB1 or mixed with other toxins had a tendency to reduce the dairy goat intestinal microorganism,but there was no significant difference (P>0.05).However,the total number of intestinal flora decreased significantly after AFB1 mixed with other toxins treatement (P<0.05). The Firmicutes,Bacteroidetes and Tenericutes were found as the main phylum in the intestines of dairy goats using the species annotation of the obtained OTUs representative sequence. According to LEfSe analysis,the combination of AFB1,OTA and ZEN could significantly increase the abundance of Succinivibrionaceae,Phascolarctobacterium and Aeromonadales (P<0.05).The abundance of Holdemania and BF311 bacillus in control group were significantly higher than those of other groups (P<0.05). These results indicated that AFB1 mixed with ZEN and OTA could influence the gut microbial community structure of dairy goat,and the effects of different mycotoxin combinations complied with the following pattern:AFB1+OTA+ZEN > AFB1+OTA > AFB1+ZEN > AFB1.

Screening of the Reference Genes for the Study of Peripheral Blood Mononuclear Cells by Quantitative Real-time PCR in Min Pigs
MA Hong, LIU Yu, WANG Liang, WANG Wen-tao, WU Sai-hui, HE Xin-miao, LIU Di
2017, 44(12):  3426-3433.  doi:10.16431/j.cnki.1671-7236.2017.12.005
Abstract ( 173 )   PDF (1184KB) ( 193 )  
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Min pig is a local pig breed in Northeast China. It has been well-adapted to the local cold weather,but few genes related to its environmental adaptation have been studied. For studies about environmental adaptation of Min pig on molecular level,it is important to have proper reference genes for quantification of gene expression by quantitative Real-time PCR. In this study,12 reference genes (B2M,ACTB,RPL11,RPL4,YWHAZ,GAPDH,HPRT1,SDHA,HMBS,IDH3B,TUBB2B and TBP1) were evaluated for their potential as the reference gene in Min pig peripheral blood mononuclear cells under different temperatures. Blood samples were collected from 3 Min pigs which were under -25,5,10 and 30℃,respectively. Mononuclear cells were separated using density gradient centrifugation. Statistical algorithms including geNorm,Normfinder and BestKeeper were employed to assess the stabilities of these genes. Analysis of geNorm and Normfinder revealed that all these 12 genes were highly stable. However,ACTB,GAPDH,SDHA,HPRT1,TBP1 and YWHAZ genes (SD<1) were found to be more stable than other six genes (SD>1),of which TBP1 was the most stable one using BestKeeper program. To summarize,ACTB,GAPDH,SDHA,HPRT1, TBP1 and YWHAZ genes were suitable to be the reference genes,with TBP1 was the best one.

Establishment of Real-time Recombinase Polymerase Amplification for Detection of Porcine Epidemic Diarrhoea Virus
LV Ji-zhou, FAN Yan-ru, FENG Chun-yan, YUAN Xiang-fen, WU Shao-qiang
2017, 44(12):  3434-3439.  doi:10.16431/j.cnki.1671-7236.2017.12.006
Abstract ( 222 )   PDF (1298KB) ( 220 )  
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To develop a precise and rapid diagnosis method for detecting porcine epidemic diarrhoea virus (PEDV), a series of recombinase polymerase amplification (RPA) primers and exo-probes were established based on the highly conserved M gene of PEDV. Then a Real-time RPA assay was developed to detect PEDV using pUC57 plasmid carrying M gene fragment of PEDV as template, and the membrane or nucleotide capsid proteins from TGEV, PRRSV, PCV2 and CSFV were utilized as control. Then the sensitivity and specificity of this Real-time RPA assay was evaluated. The results showed that the Real-time reaction could detect PEDV specifically at 39℃ within 20 min with the detection limit of 10 copies/μL of plasmid DNA, and there was no cross-reaction with other control viral pathogens. Besides, the established Real-time PRA method could successfully detecte the PEDV M gene in the plasma and plasma protein power. The Real-time established in this study was simple, rapid and sensitive, which could be a novel and reliable method for diagnosing and control of PED.

Establishment and Rudimentary Application of the nano-dPCR for Detection of PCV2 and PCV3
LIANG Lin, PANG Chun-hua, LUO Ya-kun, ZHOU Ling, WANG Jing, LIU Chang, LIU Qi, CUI Shang-jin
2017, 44(12):  3440-3445.  doi:10.16431/j.cnki.1671-7236.2017.12.007
Abstract ( 268 )   PDF (1055KB) ( 246 )  
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This study was aimed to establish a duplex nanoparticle-assisted PCR (nano-dPCR) method for the simultaneous detection of porcine circovirus type 2 (PCV2) and PCV3, and to apply this method in the detection of PCV2 and PCV3. Primers specific to PCV2 and PCV3 were designed by reference to the respective gene sequences in GenBank. The reaction conditions of nano-dPCR were also optimized. An evaluation of the specificity and sensitivity of the established nano-dPCR protocol proved the method to be both specific and sensitive, with the lower detection limit for the amount of nucleic acid in PCV2 and PCV3 to be 93.2 and 91.6 copies/μL, respectively. This sensitivity was 100 times higher than that of conventional PCR. The method was applied to inspect 265 clinical samples sent for testing, and the results showed that PCV2 and PCV3 infection in pig was rather common, with 16.6% positive for PCV3, 14.7% positive for PCV2, and 6.8% for mixed infection. The detection results on clinical samples supported the newly established nano-dPCR method as a rapid and sensitive differential diagnosis for the early infection of PCV2 and PCV3.

Effects of Compound Chinese Herbal Medicinal Polysaccharide Extraction on NF-κB,TNF-α and IL-6 mRNA Expression in Different MHC B-Lβ Ⅱ Genotype of Chickens Lymphocyte
MA Zhao, ZHU Xiao-qing, YANG Li, LIU Xiao-ting, ZHANG Bao-jun, GU Xin-li, SHANG Yun-xia
2017, 44(12):  3446-3452.  doi:10.16431/j.cnki.1671-7236.2017.12.008
Abstract ( 221 )   PDF (967KB) ( 209 )  
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In order to explore the effect of compound Chinese herbal medicines polysaccharides(cCHMPS) on immunomodulatory in different MHC B-Lβ Ⅱ genotype chickens,200 White feather broiler were chosen and PCR-SSCP technique was applied to analyze the polymorphism of MHC B-Lβ Ⅱ gene. The peripheral blood were collected according to the different MHC B-Lβ Ⅱ genotype,and the lymphocytes were isolated from peripheral blood and the cCHMPS were added with a final concentration of 100,75,50 and 0 μg/mL for co-culturing 24 h.Then the expression of NF-κB,TNF-α,IL-6 mRNA in lymphocyte using Real-time PCR method were detected. The results showed that:Compared with the control group,different does of cCHMPS could significantly improve NF-κB,TNF-α,IL-6 mRNA expression levels in chicken with different MHC B-Lβ Ⅱ genotypes (P<0.05), and when the cCHMPS concentration was 50 μg/mL,the NF-κB,TNF-α,IL-6 mRNA expression levels in lymphocyte of AB and AA genotype chicken were significantly higher than that of other groups (P<0.05).The NF-κB,IL-6 mRNA expression levels of AC genotype chicken were significantly higher than the other groups (P<0.05). The TNF-α mRNA expression levels of AC genotype chicken were significantly higher than the other groups when cCHMPS was 100 μg/mL (P<0.05).There results indicated that the cCHMPS could stimulate NF-κB,TNF-α,IL-6 mRNA expression in different MHC B-Lβ Ⅱ genotype chickens,and the optimum immunomodulatory does were different in each MHC B-Lβ Ⅱ genotype chicken.

Construction and Expression of the Heavy Chain Tetramer Precursor Chain of SLA-1 in Hebao Pig
GAO Hua, GUO Yang, ZHAI Xiao-xin, ZHANG Zong-hui, GAO Feng-shan
2017, 44(12):  3453-3458.  doi:10.16431/j.cnki.1671-7236.2017.12.009
Abstract ( 198 )   PDF (1097KB) ( 173 )  
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To construct tetramer precursor chain of swine lymphocyte antigen 1 (SLA-1) heavy chain in Hebao pig and study its protein expression in the pET-21a (+) vector,the SLA-1 complete genome sequence was referenced with the characteristics of the expression vector,and a pair of primers was designed to integrate the BirA substrate peptide (BSP) sequence at the C-terminus of the SLA-1-HB01 and the SLA-1-HB01-BSP was amplified by PCR. Then,the products were cloned into the pEASY T1 vector and the positive clones of SLA-1-HB01-BSP/pEASY T1 was selected. After the double digestion,the interest of the gene in positive clone was further ligated into the pET-21a (+) expression vector and transformed into E.coli BL21 to construct the recombinant strain of pET-21a (+)/SLA-1-HB01-BSP. After induction with IPTG,the target protein were detected by SDS-PAGE. Finally,the inclusion body of the SLA-1-HB01-BSP was isolated and detected to evaluate its purity. The PCR results showed that the length of SLA-1-HB01-BSP was about 898 bp,which was consistent with the theoretical value. The amplified fragment was successfully cloned into pEASY T1 vector, and the positive clones were identified by Nde Ⅰ and Xho Ⅰ digestion. The size of inserted fragment was 876 bp. The interest of gene was also inserted into pET-21a (+) and transformed into E.coil BL21 successfully. After induction,SDS-PAGE detection results showed that the target protein was 31.4 ku. Further detection showed that the target protein was mainly expressed as inclusion bodies,and the purity of the protein was about 80%. In this study,the recombinant tetramer precursor of SLA-1-HB01 heavy chain was constructed in pET-21a (+) expression line successfully, which would lay a foundation to detect the tetramer of SLA class Ⅰ molecular.

Analysis of TLR1,TLR2,TLR4 and TLR5 Expression in Different Tissues of Jinding Duck
XU Wen-juan, TAO Zhi-yun, ZHU Chun-hong, SONG Wei-tao, LIU Hong-xiang, ZHANG Shuang-jie, LI Hui-fang
2017, 44(12):  3459-3465.  doi:10.16431/j.cnki.1671-7236.2017.12.010
Abstract ( 195 )   PDF (1962KB) ( 190 )  
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The aim of this study was to explore the expression profiles of Toll-like receptor (TLR) in different tissues,and to lay the foundation of TLRs research for ducks.Ten male Jinding ducks of 300 days of age were slaughtered,and the blood sample and 14 different kinds of tissues (spleen, liver, testise, lung,hypothalamus,pituitary gland,skin,leg muscle,heart,kidney, chest muscle,caecum,small intestine and thymus) were collected. The Primer Premier 5.0 software was used to design specific primers and the quantitative Real-time PCR method was used to detect the relative expression levels of TLR1,TLR2,TLR4 and TLR5 mRNA. The results showed that there were only one specific peak on the melting curve of each gene amplification products,indicating that the specificity of the primers were strong. The amplification efficiency of the target genes was 101.4% to 105.0% and the correlation coefficient (R2) was 0.98 to 1.000. The four kinds of TLRs expressed in all of the 14 tissues and blood with varying abundance in different tissues for each of the 4 kinds of TLRs.The expression levels of TLR1 was lowest in hypothalamus,and highest in chest muscle. The expression levels of TLR2 was lowest in small intestine,and highest in lung. The lowest expressed tissues for TLR4 and TLR5 were testis, and the highest expressed tissues were skin.The study indicated that TLRs were widely expressed in various tissues of duck,which laid the foundation for further study on the role mechanism of duck TLRs in the process of pathogen infection.

Research Advances in Pathogenicity and Detection Techniques of Porcine Kobuvirus
HUANG Xiao-xing, WANG Xian, JIN Wen-jie
2017, 44(12):  3466-3472.  doi:10.16431/j.cnki.1671-7236.2017.12.011
Abstract ( 281 )   PDF (705KB) ( 179 )  
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Porcine kobuvirus (PKV) is a member of the newly detected in the genus Kobuvirus, family Picornaviridae in healthy pigs and diarrhea pigs. It may cause diarrhea of pigs and cause significant economic losses to pig industry. At present, the studies find that, PKV is widely distributed in pigs and has been detected in diarrhea and clinically healthy pigs, and the positive rate of the virus varies from 3.9% to 100.0%. A typical porcine kobuvirus virion is 30 nm in diameter, and its genome is 8 120 bp in length and contains a single open reading frame (ORF) encoding a protein of 2 488 amino acids. The genome organization is typical for picornaviruses:A 5' UTR, a leader (L) protein, structural proteins P1 (VP0, VP3 and VP1), non-structural proteins P2 (2A, 2B and 2C) and P3 (3A, 3B, 3C and 3D), a 3' UTR and a Poly (A) tail. There are 30 amino acid deletions in the 2B coding region of the porcine kobuvirus, VP1 protein is the most frequent variant of the small RNA virus, structural protein, which contains the main epitope, can promote the body to produce neutralizing antibodies. At present, the methods of detecting PKV are reverse transcription polymerase chain reaction (RT-PCR), TaqMan probe Real-time RT-PCR and reverse transcription-loop mediated amplification (RT-LAMP). In this paper, a brief overview of the taxonomy, epidemiological profile, genome structure, genetic characteristics and detection techniques of PKV was made, so as to provide references for the further study and understand PKV.

Regulation Effects and Mchanism of Amino Acids on Lipid Metabolism in Mammals
XIAO Kai-li, YIN Yu-long, ZHANG Lin, DENG Bai-chuan, DENG Jin-ping, TAN Cheng-quan
2017, 44(12):  3473-3481.  doi:10.16431/j.cnki.1671-7236.2017.12.012
Abstract ( 198 )   PDF (790KB) ( 227 )  
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Lipid metabolism not only affects human health but also is vital to livestock and poultry production. Therefore, it is necessary to study the nutrition regulation strategy of lipid metabolism. This paper mainly introduces the four amino acids which are important for the regulation of lipid metabolism, including methionine (Met),leucine (Leu),arginine (Arg) and glutamate (Glu), and summarizes the regulation effects and mechanism on lipid metabolism of the four amino acids at the physiological level, cellular level and molecular level. This paper introduces these four amino acids in the role of nutritional research, physiological effects in animal experiments,the mechanism of regulation effect on lipid metabolism and the combination effect. Meanwhile,the regulation and mechanism of amino acid on lipid metabolism in adipose,liver and muscle tissue were emphatically discussed. This paper reviews the progress of four amino acids in basic research and livestock production application, and further analyze the potential directions in the future research on the deep mechanism of lipid metabolism to clarify the regulation effects and potential mechanism of the amino acids on mammals' lipid metabolism, which was aimed to provide a reference for the accuracy using of amino acids.

Comparative Study on the Beef Traits Between Jiaxian Red Cattle and Crossbred Beef Cattle
ZHANG Hua-ju, XU Ya-bo, WANG Lin-lin, SUN Bin-bin, LI Zhi-gang, DING Ya-jun, SUN Hong-xia
2017, 44(12):  3482-3490.  doi:10.16431/j.cnki.1671-7236.2017.12.013
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In order to study the beef traits and beef quality of Jiaxian Red bull with crossbred beef cattle, 5 purebred Jiaxian Red bull, 5 Red Angus cattle×Jiaxian Red bull F1 generation, 5 Simmental cattle×Jiaxian Red bull F1 generation and 5 Charolais cattle×Jiaxian Red bull F1 generation were chosen and assigned into groups A,B,C and D for continual finishing for 3 months of age under the same feeding and management conditions and slaughtered at the end of the trial. The results showed that there were no significant difference in body height, body length, chest girth and cannon circumference between Red Angus cattle×Jiaxian Red bull F1 generation and purebred Jiaxian Red bull, the same as in Charolais cattle×Jiaxian Red bull F1 generation (P>0.05). The weight of head, skin, fore hooves and tail, the skin thick, esophagus, trachea, liver, spleen, lung, kidney, and carcass meat percentage, loin eye area and the meat bone ratio in crossbred beef cattle groups were not significant differences compared with purebred Jiaxian Red bull (P>0.05), but its hind legs wide in carcass was significantly higher than that of purebred Jiaxian Red bull (P<0.05). The high-grade meat weight and high quality meat weight in Red Angus cattle×Jiaxian Red bull F1 generation were 70.22 and 105.22 kg, respectively, and was the highest in four groups,but the high-grade beef percentage among four groups did not show significant differences (P>0.05). The high quality meat percentage in Red Angus cattle×Jiaxian Red bull F1 reached 34.43%, was significantly higher than that of Jiaxian Red bull and Simmental cattle×Jiaxian Red bull F1 generation (P<0.05), and was higher than that of Charolais cattle×Jiaxian Red bull F1 generation (P>0.05). In terms of meat quality, the content of inosinic acid that related with the meat flavor in Red Angus cattle×Jiaxian Red bull F1 was the highest in four groups, while the content of unsaturated fatty acid and the flavor related amino acid, such as aspartic acid, glutamic acid, glycine and alanine, in Charolais cattle×Jiaxian Red bull F1 were the highest in four groups,and their values were 53.85% and 8.04%, respectively. To sum up, the results suggested that there was no significant difference in body development and beef traits using Red Angus cattle, Simmental cattle and Charolais cattle to improve Jiaxian Red bull, while Red Angus cattle crossbred with Jiaxian Red bull could moderately improve the weight of high-grade beef and high-quality beef, and Charolais cattle could moderately improve the beef quality of Jiaxian Red bull.

Effects of Rice Husk Powder on Performance, Body Size Traits, Internal Organs Development and Economic Benefit of Young Turkey
DAI Hang, LU Man, YANG Hai-ming, YU Jun, JU Geng-yue, LIAO Zheng-jun
2017, 44(12):  3491-3496.  doi:10.16431/j.cnki.1671-7236.2017.12.014
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The purpose of this experiment was to study the effect of adding rice husk powder on the growth performance, body size traits, development of internal organs and economic benefits of young turkey. 180 young turkeys with similar body weight were selected and randomly divided into two groups, with each group having 5 replicates, each replicate of 10 birds. The control group was fed with basal diet, the experimental group was fed with basal diet added rice husk powder. The experiment lasted 8 weeks. The results showed that there was no significant difference in avarage daily feed intake (ADFI) between control group and experimental group (P>0.05).Compared with control group, the body weight and avarage daily weight gain (ADG) of the birds in experimental group increased extremely significantly (P<0.01), while the ratio of feed intake and daily weight gain (F/G) decreased extremely significantly (P<0.01); The heart weight of the birds tended to increase (P=0.09), whereas the proventriculus weight significantly increased (P<0.05), and the gizzard weight extremely significantly increased (P<0.01) in experimental group. There were no significant differences in body size traits, organ index, intestinal weight and length of the birds between control group and experimental group (P>0.05). The economic benefit of each bird in experimental group at 56 days of age was increased by 39.4% compared with control group (P<0.05). In summary, adding rice husk powder in young turkey's diets could improve the growth performance and the economic benefits of turkey.

Evaluation of Associative Effects of Alfalfa Meal and Ammoniated Corn Straw by Gas Production Technique in vitro
YU Man-man, JIANG Yu-xuan, ZHANG Mei-mei, XIE Xiao-lai, WANG Chen-xi
2017, 44(12):  3497-3504.  doi:10.16431/j.cnki.1671-7236.2017.12.015
Abstract ( 163 )   PDF (848KB) ( 152 )  
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This experiment was conducted to determine the best roughage combination of alfalfa meal (AM) and ammoniated corn straw (ACS),so as to improve the efficiency of roughage utilization and reduce the cost of feeding. The AM was mixed with ACS in the proportion of 100:0,80:20,60:40,50:50,40:60,20:80 and 0:100 with 3 replicates per group. The accumulated gas production (GP) at fermentation for 3,6,12,24,48 h,and the changes of pH,dry matter disappearance rate (DMD),ammonia nitrogen (NH3-N) concentration, microbial protein (MCP) and volatile fat acids (VFA) concentration,and their single factor associative effects index (SFAEI) and multiply factors associative effects index (MFAEI) were determined by gas production technique in vitro after fermentation for 48 h. The results showed as follows:The accumulated gas production of AM20:ACS80 group was higher than that of the other groups,and the DMD of AM0:ACS100 group was significantly lower than other groups (P<0.05).The NH3-N concentration of AM20:ACS80 and AM0:ACS100 groups was significantly higher than the other groups (P<0.05).The MCP concentration was highest in AM20:ACS80 group,and the concentration of acetic acid and TVFA in AM20:ACS80 group was the highest (P<0.05),and the acetic acid/propionic acid ratio in all groups was more than 3,which indicating that it belonged to the acetic acid fermentation type.The pH during the fermentation ranged from 6.69 to 6.85.The results of MFAEI and SFAEI of each combination indicated that only the AM20:ACS80 group showed the positive associative effects. In conclusion,the 20:80 combination ratio for AM and ACS showed the best associative effects.

Effect of Fagopyrum dibotrys (D.Don) Hara on Growth Performance, Immune Function and Intestinal Structure of Broilers
TAN Lu-lin, ZHANG Ding-hong, ZHANG Jie, XU Zhong-hui, DENG Rong
2017, 44(12):  3505-3511.  doi:10.16431/j.cnki.1671-7236.2017.12.016
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This experiment was conducted to investigate the effects of Fagopyrum dibotrys (D.Don) Hara on growth performance,immune function and intestinal structure of broilers. A total of 270 one-day-old healthy Tiejiaoma broilers were chosen and randomly divided into 3 groups with 6 replicates per group and 15 broilers per replicate.Control group was fed with the basal diet,group Ⅱ was fed with basal diet adding 8% flavomycin 60 mg/kg +15% aureomycin 270 mg/kg and group Ⅲ was fed with basal diet adding 1% Fagopyrum dibotrys (D.Don) Hara. The trial period lasted for 21 days. The results showed as follow:① At 21 days of age,the average daily feed intake of group Ⅲ was significantly lower than that of groups Ⅰ and Ⅱ (P<0.05).However,there was no significant difference in the average daily gain and F/G among the three groups (P>0.05).②At 21 days of age,the thymus and bursa of Fabricius indexes of groups Ⅱ and Ⅲ were significantly higher than that of group Ⅰ (P<0.05);But the spleen index were not significantly different among the three groups (P>0.05).③ At 21 days of age,the content of IgA and IgM in serum of groups Ⅲ were significantly higher than that of group Ⅰ (P<0.05),and the content of IgG in serum of groups Ⅱ and Ⅲ were significantly higher than that of group Ⅰ (P<0.05).④At 21 days of age,the content of IL-2,TNF-α and sIgA in intestinal mucosa of groups Ⅱ and Ⅲ were significantly higher than that of group Ⅰ (P<0.05),and the content of IL-6 in intestinal mucosa of group Ⅲ were significantly higher than that of groups Ⅰ and Ⅱ (P<0.05).⑤At 21 days of age,the villus height of jejunum in groups Ⅱ and Ⅲ were significantly higher than that of group Ⅰ (P<0.05),the villus height/crypt depth of jejunum in group Ⅱ was significantly higher than that of groups Ⅰ and Ⅲ (P<0.05). However,the crypt depth was not significantly different among the three groups (P>0.05).In conclusion,that Fagopyrum dibotrys (D.Don) Hara could not affect the growth performance of broilers,but it could improve the development of immune organs,increase the secretion of serum immunoglobulin, promote the immune function of intestinal mucosa and intestinal morphology of jejunum.

Effects of Fermented Green Juice and Cellulase on the Fermentation Quality of Rice Straw Silage and Rice Straw with Sugarcane Tip Mixed Silage
SONG Ge, ZHU Xiao-qing, ZHANG Shi, ZHUANG Yi-fen
2017, 44(12):  3512-3518.  doi:10.16431/j.cnki.1671-7236.2017.12.017
Abstract ( 211 )   PDF (811KB) ( 177 )  
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Rice straw and sugarcane tip were used as the silage materials in this research,and there were two groups:Rice straw silage and mixed silage of rice straw and sugarcane tip with the mixed ratio 6:4(m/m).Four treatment groups were set up in each silage:Control group (CON) and three treatment groups which added 2 mL/kg fermented green juice (FGJ),3 mL/kg cellulase (CEL) and 2 mL/kg FGJ+3 mL/kg CEL (MIX),and treatment was repeated 3 times. The materials were ensiled at room temperature for 60 days,and the fermentation quality and chemical compositions were tested. The result showed that for the two kinds of raw silage materials,the contents of WSC and CP in rice straw were lower than those in sugarcane tip,while the contents of NDF and ADF in rice straw were higher than those of sugarcane tip.Compared with the CON group,the NDF,ADF,pH and NH3-N in FGJ group were extremely significantly decreased when rice straw was silaged alone (P<0.01),while WSC was extremely significantly increased (P<0.01),and DMR was increased. The NDF,ADF,pH,GLR and NH3-N in MIX group were extremely significantly decreased (P<0.01),while lactic acid,WSC and DMR were increased. In CEL group, the ADF was significantly decreased (P<0.05), and other indicators also had improved in different degrees. Compared with rice straw silage,the NDF,HC,pH and NH3-N of mixed silage were lower,while DMR and WSC were obviously increased. In conclusion,the fermented green juice and it compound with cellulase had the similar effects on improving the fermentation quality,and the effects were both better than that of cellulase. The fermentation quality of rice straw with sugarcane tip mixed silage was better than rice straw silage.

Research Progress on Propionate Application in Ruminant
CAO Na, ZHANG Ya-wei, WU Hao, MENG Qing-xiang, ZHOU Zhen-ming
2017, 44(12):  3519-3524.  doi:10.16431/j.cnki.1671-7236.2017.12.018
Abstract ( 271 )   PDF (747KB) ( 846 )  
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Propionate is a kind of new,efficient and safe additive with the functions of anti-mildew,anticorrosion and treatment of diseases,which is widely used in feed processing industry. Propionate is decomposed into propionic acid and mineral elements in the rumen, and propionic acid is the main active ingredient. Propionate can promote rumen development,regulate blood glucose,maintain energy balance,therefore,it has been used to treat ketosis in practice production. At present,the propionate has been studied mostly at the rumen infusion stage,and in vitro experiments are focused on the liver gluconeogenesis pathway,which revolves around energy metabolism. But there are a few researches about the effects of propionate on production performance and fermentation. With the development of study of short chain fatty acids in the medical field,it brings a new opportunity for its development in husbandry. Whether propionate has the ability to promote rumen development, alleviate inflammation and enhance immunity needed to be further explored and verified. This paper mainly introduces the metabolic pathway and the mechanism of propionate and its application value in ruminant animal nutrition,combined with the production performance and maintain health,in order to reveal its potential value and provides scientific basis for its application in ruminant.

Effect of Chinese Herbal Medicine Additives on Milk Production of Rex Rabbit
YE Xiang-yang, XIE Bao-cai, REN Zhan-jun, BAI Jing
2017, 44(12):  3525-3529.  doi:10.16431/j.cnki.1671-7236.2017.12.019
Abstract ( 213 )   PDF (728KB) ( 202 )  
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This study was designed to explore the effect of Chinese herbal medicine additives on milk production and reproduction performance of rabbit does. Total 99 does were separated into 3 treatments (33 does in each treatment) and fed with the basal diet adding 0, 1%, 2% Chinese herbal medicine additives,respectively. Pre-feeding period was 4 d and test period was 26 d (from the first day of postpartum to the last day before next mating). Daily milk production of does was detected after delivery 0, 7, 14, 21 and 28 d. Both litter size and the number of weaned rabbits were recorded. The results showed that milk production of does fed with 2% Chinese herbal medicine additives were increased extremely significantly (P<0.01) or significantly (P<0.05) compared to control group, and there was no significant difference between control group and 1% Chinese herbal medicine additives group (P>0.05). Survival rate of weaned rabbits increased with the increasing dosage of the additives, but did not differ significantly (P>0.05). In conclusion, the Chinese herbal medicine additives could improve milk production of rabbit does and survival rate of weaned rabbits, and the optimal dosage should be 2%.

Effect of Different Alfalfa and Rice Straw Proportion on Associative Effects of Sheep's Diets
LIU Hui-hui, YUAN Jiu, PING Li-ying, ZHU Bao-zhen, WANG Jun-jun, ZHAO Peng-fei, HE Tian-le, YU Hai-shan, WAN Xin-jie
2017, 44(12):  3530-3535.  doi:10.16431/j.cnki.1671-7236.2017.12.020
Abstract ( 285 )   PDF (747KB) ( 256 )  
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This experiment was conducted to investigate the effects of the proportion of alfalfa and rice straw on associative effects (AE) of diets in vitro. Rice straw:alfalfa were 60:0, 50:10, 40:20, 30:30, 20:40,10:50,0:60 when concentrate-roughage ratio (C:R) was 40:60, and rice straw:alfalfa were 70:0, 60:10,50:20, 40:30,30:40,20:50,10:60,0:70 when C:R was 30:70. Gas production (GP) was recorded at 2, 4, 6, 9, 12, 24, 36, 48, 72 and 96 h. The AE was defined as the difference between the observed in vitro GP24 h and the predicted value from individual feed fermented alone. The results showed as follows:① GP24 h of the group 50:10 was extremely significantly greater than the group 10:50 (P<0.01), and GP24 h of the groups 40:20 and 60:0 were significantly greater than the groups 10:50 (P<0.05) when C:R was 40:60. The AE of all groups were positive and there were no significant difference among them (P>0.05). When C:R was 40:60, the GP24 h,b,a+b of group 50:10 were the greatest and it's AE was the best.②GP24 h of the groups 60:10, 70:0 and 50:20 were significantly greater than the group of 0:70 (P<0.05) and groups 40:30 and 20:50 were greater than the group 0:70 (P=0.063, P=0.064) when C:R was 30:70. The AE of all groups had no significant difference (P>0.05). When C:R was 30:70, the GP24 h,b,a+b of group 40:30 was the greatest and it's AE was the best, followed by the groups 20:50 and 10:60. It is concluded that the AE of the groups of 50:10 (C:R was 40:60), 40:30, 20:50 and 10:60 (C:R was 30:70) were greater.

Effects of Compound Probiotics on Fermentation Quality of Total Mixed Ration
ZHANG Zhi-guo, WANG Dan, GAO Yang, LI Jun, YANG Lian-yu, ZHANG Ji-ze
2017, 44(12):  3536-3542.  doi:10.16431/j.cnki.1671-7236.2017.12.021
Abstract ( 197 )   PDF (740KB) ( 182 )  
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The study was aimed to investigate the influences of compound microbial on the quality of fermented TMR. There were 3 treatments:Control group (basal TMR),experimental group 1 (TMR with 0.5% compound microbial supplement (Lactobacillus, Bacillus subtilis, Bacillus cereus and yeast)), experimental group 2 (TMR with 0.5% supplement (Lactobacillus, Bacillus subtilis, Bacillus cereus and cellulolytic bacteria)), with 6 replicates in each group and 2 tankers per replicate. The experiment lasted 30 d. Water content of the materials in all the tankers were controlled at 50% and sealed airtightly for 30 d. Samples were cllected at 0,3,5,7,15 and 30 d. Mycotoxins, pH, ammonium nitrogen, aerobic stability and nutrient content were determined and analyzed. The results showed as follow:①During 0~30 d, the content of mycotoxins showed an upward trend in all treatments. On the 30th day, aflatoxin B1 content and ochratoxin-A content in the control group were exceeded than the national standard. However, three kinds of mycotoxins were not exceeded in experimental groups 1 and 2 during the 30 d fermentation.② Aerobic stability and storage time were positively correlated, the aerobic stability of experimental group 1 was better than other treatments. ③ With the last of storage time, pH in 3 groups showed downward trend and ammonium nitrogen content showed an upward trend. pH value and ammonium nitrogen content in experimental group 1 was significantly lower than the control group during 7-30 d (P<0.05). ④ On the 30th day, DM content in control group was significantly lower than the other treatments (P<0.05);During 7-30 d, compared with the control group, CP content in experimental groups 1 and 2 were significantly decreased (P<0.05). The contents of NDF and ADF were negatively correlated with storage time, moreover, During 7~30 d, the levels of NDF and ADF in two experimental groups with compound microbial significantly decreased (P<0.05). In conclusion, the compound microbial supplement could prolong the storage time of TMR and strengthen its aerobic stability. In addition, the degradation of feed nutrient was limited. The compound probiotics, which was composed of Lactobacillus, Bacillus subtilis, Bacillus cereus and yeast was more effective.

Polymorphisms and Its Genetic Variation Analysis of DQB2 Gene Exon 2 in Kazakh Sheep
WANG Yuan-yuan, YAN Guo, LUO Cheng, QI Jiang-jiao, ZHOU Guang-pu, TAN Jun, GAO Jian-feng
2017, 44(12):  3543-3553.  doi:10.16431/j.cnki.1671-7236.2017.12.022
Abstract ( 213 )   PDF (5685KB) ( 163 )  
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This study was aimed to investigate the association between the polymorphism of DQB2 gene exon 2 and the susceptibility to Kazakh sheep brucellosis. The DQB2 gene exon 2 of Kazakh sheep lymphocyte antigen was amplified by PCR-SSCP method from 146 healthy and 28 infected with Brucella Kazakh sheep, and the single nucleotide polymorphisms (SNP) was analyzed, then the different alleles were selected for cloning and sequencing. In order to analyze its correlation with brucellosis susceptibility, the differences in gene frequency and genotype frequency of each SNP locus were analyzed by Chi-square test. Bioinformatics softwares were used to analyze the secondary structure of mRNA, the secondary structure, tertiary structure and epitope of protein. The sequencing result showed that 33 SNPs were detected in 270 bp DNA sequence, the gene frequencies of C9G and A180G were extremely significantly different in case group and control group (P<0.01), and its genotype frequencies presented significantly difference (P<0.05). Similarly, A13T and C133G loci were significant difference in case group and control group (P<0.05). Further analysis result showed that the minimum free energy of the A180G mutation site was the lowest and its mRNA secondary structure was the most stable; Both A13T and C133G mutation sites caused the changes of mRNA secondary structure, protein secondary, tertiary structure and antigenic epitope of protein, respectively. The results showed that the polymorphism of DQB2 gene exon 2 might be significantly correlated with brucellosis susceptibility in Kazakh sheep.

Polymorphism Analysis of PIS Region Sequence in Guanzhong Dairy Goat
HOU Ce, SU Xiao-ping, WANG Qi-tian, CUI Kui-qing, SHI De-shun, LIU Qing-you
2017, 44(12):  3554-3562.  doi:10.16431/j.cnki.1671-7236.2017.12.023
Abstract ( 206 )   PDF (6929KB) ( 202 )  
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Polled trait of goat is important economic trait in livestock production,goat polled intersex syndrome (PIS) influenced polled phenotype and intersex phenotype of goats. PIS sequence locats in a approximately 11.7 kb nucleotide sequence in chromosome 1. This experiment was aimed to study the effect of PIS sequence on polled phenotype in Guanzhong dairy goats (3 horned goats, 3 polled goats). The PIS whole sequence in Guanzhong dairy goats were amplified using four pair of primers (PIS1-1, PIS1-2, PIS2 and PIS3), the whole absence of PIS sequence was detected using the fifth primer (PIS whole). The sequencing result was assembled, analyzed and annotated. The result showed that the PIS sequence full length was 12.814 kb, and the BLAST alignment result was correct. PIS whole primer didn't amplify any srtipes, which showed that there was no a total deletion of 11.7 kb in Guanzhong dairy goats. SeqMan software analysis result showed that there was a total of 30 polymorphic sites, wihch were likely to connect with polled/horned phenotype in Guanzhong dairy goats. The annotation result of concatenation sequence showed that there were one tRNA encoding locus, two microsatellites, three microRNA encoding loci, L1-EN conserved domain, RT-nLTR-like conserved domain, also find a CpG island, one pair of inverted repeats, and two tandem repeats. This experiment result had a significant reference value for studying the molecular mechanism of polled phenotype in Guanzhong dairy goats.

Effects of UCHL1 Inhibition on Porcine Oocyte Matuation in vitro, Zona Pellucida Ubiquitination and Polyspermy
SHI Wen-shu, JIN Yi
2017, 44(12):  3563-3569.  doi:10.16431/j.cnki.1671-7236.2017.12.024
Abstract ( 184 )   PDF (2124KB) ( 242 )  
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This study was aimed to examine the effects of UCHL1 inhibition on porcine oocyte maturation in vitro, zona pellucida (ZP) ubiquitination and polyspermy. DAPI staining, Hoechst staining and SDS-PAGE methods were used to detect the matuation rate of porcine oocytes in vitro, the level of ubiquitination of zona pellucida (ZP) and polyspermy. The results showed that after different concentrations UCHL1 inhibitor (10, 20, 25 and 30 μmol/L, DMSO and control group) were added into maturation medium for culturing 46 h in vitro, the mature rate of control group was 86.22%, while the maturation rate of 30 μmol/L group was 15.30%, and the maturation rate of every treatment group had significant difference (P<0.05). Western blotting result showed that every group generated ubiquitin markers of ZP were about 61, 80 and 106 ku in different degree. According to the analysis of gray value, the result had significant difference (P<0.05). Conducting fertilization in vitro, the number of sperm adhered on oocyte ZP in control group was the most, the number of sperm running into oocyte was fewer, the number of sperm adhered on oocyte ZP with 30 μmol/L UCHL1 inhibitor was the fewest, and there was almost no sperm running into the oocyte. The results showed that UCHL1 inhibitor had an impact on maturation of porcine oocytes in vitro. With the higher concentration of UCHL1, the lower degree of ZP protein ubiquitinated, UCHL1 could regulate sperm attachment and polyspermy.

Cloning and Phylogenetic Analysis of IFNβ1 Gene of Reindeer
ZHAI Jian-cheng, WANG Qiang-hui, XIA Yan-ling, LI He-ping
2017, 44(12):  3570-3577.  doi:10.16431/j.cnki.1671-7236.2017.12.025
Abstract ( 159 )   PDF (4378KB) ( 139 )  
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This study was aimed to clone the full-length sequence of interferon beta 1 (IFNβ1) gene from the top of reindeer antler, and analyze its molecular characteristics, to lay a foundation for its biological activity and practical application. One pair of primers was designed based on the conserved sequence of IFNβ1 gene of cattle and sheep published in GenBank. IFNβ1 gene of reindeer was amplified by PCR from genome DNA extracted from mesenchyme of reindeer antler tip, and the product was cloned and sequenced. The result showed that the full length sequence of reindeer IFNβ1 gene was 561 bp, encoded 186 amino acids and contained 3 glycosylation sites. The encoded protein contained 4 alpha helixes, 4 beta fold zones and 7 beta turn angles. Compared the amino acids of IFNβ1 gene with other mammal species, the homology was 45.1% to 92.0%. The results of sequence and phylogenetic tree analysis showed that IFNβ1 gene had species diversity, the relationship was proportional to homology. This result laid the foundation for further research on the expression, biological activity and application of reindeer IFNβ1 gene.

Comparison of Production Performance of Different Mating Combination Between Castor Rex Rabbit and Plicae Rex Rabbit
ZHAO Yue-ping, YANG Cui-jun, SUN Quan-wen, WU Shu-qin, WU Zhan-fu, HAO Rong-chao, LU Jing, MA Meng-pan, REN Ling-ling
2017, 44(12):  3578-3584.  doi:10.16431/j.cnki.1671-7236.2017.12.026
Abstract ( 178 )   PDF (748KB) ( 168 )  
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In order to compare the production performance of different mating combination between Castor Rex rabbit and Plicae Rex rabbit,40 Castor Rex rabbit (10 ♂ and 30 ♀) and 40 Plicae Rex rabbit (10 ♂ and 30 ♀) with 6 to 7 month old were selected.Hybrid testing was conducted using Plicate Rex rabbit and Castor Rex rabbit as crossing parents which were Castor Rex rabbit pure breeding (Castor Rex rabbit ♂×Castor Rex rabbit ♀),Plicate Rex rabbit pure breeding (Plicae Rex rabbit ♂×Plicae Rex rabbit ♀),orthogonal combination (Castor Rex rabbit ♂×Plicae Rex rabbit ♀)and reciprocal combination (Plicae Rex rabbit ♂×Castor Rex rabbit ♀).The reproductive performance of rabbits does and the growth performance,the slaughter performance and wool quality of their offsprings were measured respectively. The results showed that the neonatal weight of litter,litter size,lactescence,survival rate,weaning weight of litter,F/G,slaughter rate,skin area,hair density,hair fineness and coarse wool rate of orthogonal combination were 320.71 g,6.71,1 727.43 g,83.91%,5 304.14 g,4.06,62.43%,1 160.20 cm2,15 951.70/cm2,16.32 μm and 6.83%,respectively,while that of reciprocal combination were 315.14 g,6.52,1 710.00 g,80.19%,4 814.82 g,4.30,62.93%,1 172.20 cm2,16 092.01/cm2,16.43 μm and 6.78%,respectively,and the differences between reciprocal combination and orthogonal combination were not significant (P>0.05).The F/G,slaughter rate and coarse wool rate of F1 in Castor Rex rabbit pure breeding were higher than that of Plicate Rex rabbit pure breeding,while the other indexes were all lower than Plicate Rex rabbit pure breeding,of which the differences between two pure breeding, except F/G,slaughter rate,hair fineness and coarse wool rate, were significantly different (P<0.05).In conclusion,the reproductive performance of parental generation and growth and slaughter performance of F1 in orthogonal combination was slightly better than that of reciprocal combination,while the wool quality of F1 in reciprocal combination was better than orthogonal combination.

Prokaryotic Expression and Bioinformatics Analysis of VP2 Gene of Infectious Bursal Disease Virus C4 Strain
WANG Jie-qiong, ZHAO Yu-jie, ZHOU Yun-fei, HUANG Zong-mei, CHEN Pan-pan, ZHOU Wei-fan, LIU Lin, LI Xin-sheng
2017, 44(12):  3585-3591.  doi:10.16431/j.cnki.1671-7236.2017.12.027
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This study was aimed to investigate the relationship between the virulence characteristics of infectious bursal disease virus(IBDV) C4 strain and its VP2 amino acid sequence. The RNA of IBDV C4 strain was extracted,and its VP2 gene was amplified by RT-PCR.VP2 nucleotide sequences and deduced amino acids of different virulent IBDV strains were compared. At the same time, prokaryotic expression vector pET-32a(+) was used to express the VP2 gene. The expression of recombinant VP2 protein was detected by SDS-PAGE and Western blotting. The results showed that the VP2 gene of IBDV C4 strain belonged to the very virulent infectious bursal disease virus (vvIBDV) in evolutionary relationship, the VP2 nucleotides homology between IBDV C4 strain and other vvIBDV strains were 98.1% to 98.7%, and there were no mutations in S-W-S-A-S-G-S (326-332 amino acids) and 222(A), 256(I), 294(I) and 299(S). The VP2 amino acid sequence of IBDV C4 strain was consistent with the characteristics of other vvIBDV strains. However, there were three differences amino acids sites at 201(D/G), 281(G/R) and 313(V/A) between the amino acids of the C4 strain and the very virulent strain UK661. And the change of 281(R) was in the small hydrophilic region of 279 to 290, which was related to the antigenicity of the virus; The recombinant VP2 protein molecular weight expressed in Escherichia coli BL21 was about 67 ku. This study provided a basis for further research on antigenic changes resulting from amino acid variation of 201(G), 281 (R) and 313(A). These results indicated that the VP2 gene of the IBDV C4 strain was consistent with the major characteristics of the vvIBDV strain VP2 gene. The difference of three amino acid sites in the vvIBDV strain C4 might be related to the evolution of virulence of IBDV strain in China.

Investigation of Intestinal Parasites in Dairy Cattle in Parts of Hebei Province and Tianjin
HU Su-hui, ZHANG Zhen-jie, LIU Zhen-zhen, CHEN Kai-li, ZHANG Gui-ling, LI Jun-qiang, ZHANG Long-xian, WANG Rong-jun
2017, 44(12):  3592-3597.  doi:10.16431/j.cnki.1671-7236.2017.12.028
Abstract ( 254 )   PDF (1535KB) ( 251 )  
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In order to understand the prevalence of intestinal parasites in dairy cattle in Hebei and Tianjin near to Bohai area, the centrifugal precipitation, Sheather's sugar flotation, and Lugol's iodine staining techniques were used to examine 1 040 fecal samples from 12 dairy cattle farms in Hebei and Tianjin areas. The overall infection rate was 61.63%, with the prevalence being 59.78% and 66.12% in Hebei and Tianjin, respectively. Eight parasite species were identified, including Coccidium spp., Giardia duodenalis, Cryptosporidium spp., amoeba, Strongylus spp., Trichuris spp., cestode and trematode, and the infection rates were 40.96%, 1.63%, 8.46%, 33.27%, 2.02%, 3.56%, 0.96% and 0.96%, respectively. Coccidia spp. and amoeba were the predominant parasites. The present study indicated the prevalence of intestinal parasites were universal in dairy cattle in Hebei and Tianjin areas. Thus, the farms should take necessary prevention and control measures.

Effect of Different Mulberry Leaf Extracts on Spleen Lymphocyte Proliferation in Mice
CHEN Xiao-lan, YANG Hai-feng, QU Jing-wen, LI Ran, JIANG Chun-mao, LU Guang-fu, BO Ruo-nan
2017, 44(12):  3598-3604.  doi:10.16431/j.cnki.1671-7236.2017.12.029
Abstract ( 193 )   PDF (1023KB) ( 231 )  
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This experiment was aimed to compare the effects of different mulberry leaf extract on spleen lymphocyte proliferation in mice. Mulberry leaf polysaccharides (MLP-1, MLP-2, MLP-3, MLP-4 and MLP-5) were prepared with mulberry leaf aqueous extract (MLAE) by different concentration of alcohol deposit, polysaccharides concentration of all extracts were determined by phenol sulfuric acid method. Five different MLPs and MLAE were as the experimental drug, when polysaccharide concentration was 15.625, 31.25, 62.5, 125 and 250 μg/mL, the changes of spleen B and T lymphocytes proliferation in mice which were stimulated by MLPs and MLAE in single or synergistical stimulation of drugs with LPS and PHA were determined by MTT method. The results showed that when polysaccharide concentration was in the range of 15.625 to 250 μg/mL, MLPs and MLAE in single or synergistical stimulation with LPS and PHA could stimulate the spleen B and T lymphocytes proliferation, MLP-1 at low concentration could stimulate significantly spleen B lymphocyte proliferation remarkably (P<0.05), and MLP-3 and MLP-5 at low concentrations showed strong stimulation ability of T lymphocyte proliferation. When MLAE was in synergistical stimulation with LPS at most concentrations points, the spleen B lymphocytes proliferation in mice were significantly higher than those of the cell control and LPS control groups, which revealed that MLAE could significantly improve the humoral immune function (P<0.05).

Prokaryotic Expression of PEB1A Protein of Campylobacter jejuni and Establishment of an Indirect ELISA
WANG Li, SHA Zhou, LI Shi-yu, LIANG Jia-ming, WANG Xing-long
2017, 44(12):  3605-3611.  doi:10.16431/j.cnki.1671-7236.2017.12.030
Abstract ( 175 )   PDF (1079KB) ( 185 )  
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In order to develop an indirect ELISA method to detect Campylobacter jejuni antibody, PEB1A gene of Campylobacter jejuni was cloned and amplified by PCR, the prokaryotic expression vector pET32a-PEBIA was constructed, and then transferred into the expression strain E.coli BL21 (DE3), and obtained about 47 ku of soluble protein. Western blotting result showed that the expressed recombinant protein had good biological activity, an indirect ELISA method for detecting antibody against Campylobacter jejuni was developed using expressed PEB1A protein as coating antigen,and detected its specificity, sensitivity, repeatability, respectively. The results showed that the established method for detection of Campylobacter jejuni antibody critical value was 0.3424. This method only specifically reacts with Campylobacter jejuni positive sera, and had no cross-reactivity with other antiserum and strong specificity. In addition, the coefficients of variations in both inter-and intra-assay were less than 5% indicating that it had good repeatability and stability. The establishment of this method could be applied to the rapid detection of Campylobacter jejuni in serum, and provided basis for further prevention and control of Campylobacter jejuni diarrhea.

Study on Expression and Preliminary Immunocompetence of CIC Protein Containing Molecular Adjuvants
ZHANG Shao-duo, LAI Yi-jun, JIANG Hui, CHENG Xin-ran, YE Yu-ting, HE Xi-yu, XU Le, SONG Bai-fen, YU Li-quan, CUI Yu-dong, MA Jin-zhu
2017, 44(12):  3612-3617.  doi:10.16431/j.cnki.1671-7236.2017.12.031
Abstract ( 181 )   PDF (1261KB) ( 166 )  
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To express CTB-IsdBid-Clfais(CIC) protein and evaluate its immunogenicity, CTB (as a molecular adjuvant) could be tandem linked with IsdBid-Clfais gene by the overlapping PCR method, then CTB-IsdBid-Clfais(CIC) was inserted into pET-32a(+) vector to construct recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais. The recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais were transformed into Escherichia coli (E.coli) BL21 to express the CTB-IsdBid-Clfa(CIC) protein, the CIC expression protein and its immune activity were detected by Western blotting and ELISA,respectively. The results showed that the length of CIC gene were 2 072 bp, and CIC was correctly inserted into the pET-32a(+) plasmids, the pET-32a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed. Western blotting result confirmed that the molecular weight of CIC proteins was 95.9 ku, which were correctly expressed by E.coli BL21 with pET-32a (+)-CTB-IsdBid-Clfais plasmids. ELISA results showed that there was no significant difference among the CIC, IsdBid and Clfais protein groups (P>0.05), and there was extremely significant difference between CIC and BSA protein groups (P<0.01). In conclusion, the pET-32a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed, CIC proteins were correctly expressed, and were able to react with serum from mice immunized with IsdBid and Clfais,respectively,therefore, CIC proteins had strong immune activity.

Research Advances on Calf Pathogenic Diarrhea and Vaccine
GUO Shen-tao, YU Hui, LIN Xu-ye, LI Hua
2017, 44(12):  3618-3624.  doi:10.16431/j.cnki.1671-7236.2017.12.032
Abstract ( 227 )   PDF (877KB) ( 165 )  
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Calf diarrhea is a kind of digestive tract disease caused by a variety of pathogenies, which has a great impact on the survival, growth and development of calves. Escherichia coli, bovine rotavirus, bovine coronavirus and other pathogens often cause diarrhea in calves, in addition, the secondary infection and mixed infection also occurre in different pathogens, which lead to great difficulty for prevention of calf diarrhea. A variety of vaccines have been developed for the prevention and treatment of calf diarrhea at home and abroad. Now, except only minor commercial calf diarrhea vaccines have been found in abroad, there is no one multivalent commercial calf diarrhea vaccine in China. Based on previous study, the progress of pathogens and vaccines for calf diarrhea were elaborated in the study, which could provide exploitation references for the commercial calf diarrhea vaccine in China.

Isolation, Identification and Drug Resistance Analysis of Interstinal Pathogenic Escherichia coli and Salmonella Isolated from Diarrhea Piglets
LI Chen, ZHANG Shuang-xiang, ZHOU Bi-jun, CHENG Zhen-tao, WEN Ming, MA Guang-qiang, WANG Wei, HU Xing-yi, ZHANG Hai, DING Zun-e, WANG Kai-gong
2017, 44(12):  3625-3633.  doi:10.16431/j.cnki.1671-7236.2017.12.033
Abstract ( 182 )   PDF (1502KB) ( 213 )  
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In order to find out the serotype, resistant phenotype and genotype of Escherichia coli (E. coli) and Salmonella in piglets, this study collected 128 samples of diarrhea piglets from seven large-scale pig farms in five cities in Guizhou province, and the E. coli and Salmonella were isolated and identified. The pathogenicity of the strain was identified by animal test. The drug resistance of the main pathogen was tested by drug susceptibility paper. The resistance gene of each pathogen was detected by PCR. The drug resistance and genotype correlation of the bacterial were analyzed. The results showed that 78 strains of pathogenic E. coli and 21 strains of Salmonella were isolated and identified in this study. The serotypes of pathogenic E. coli were predominantly O138 and O87. Salmonella Typhimurium and Salmonella Enteritidis were predominant serotypes. The susceptibility test showed that the resistant strains of 78 strains of E. coli were more than 80% resistant to β-lactams and more than 40% for other antibacterials. The resistance rate of 21 strains of Salmonella to aminoglycosides was more than 50% and more than 20% to other types of antibacterials; 12 and 10 kinds of drug resistance-related genes of E. coli and Salmonella were detected, respectively; The coincidence rate of resistant genotype and phenotype of two kinds of bacteria were above 60%, and both were multiple drug resistance. This study provided a theoretical basis for comprehensive prevention and control of piglets diarrhea.

Research Progress on Main Pathogen Resistance of Dairy Cows and Control Technology
HUA Wei-yi, CHEN Xiao-jie, LIU Yi-ming, XU Fei, LI Xiu-bo
2017, 44(12):  3634-3641.  doi:10.16431/j.cnki.1671-7236.2017.12.034
Abstract ( 159 )   PDF (891KB) ( 266 )  
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At present, the usual and effective method of treating the cow disease caused by pathogens is still antimicrobials. The irrational use of antimicrobials makes bacteria produce resistance, and has become a global problem. The main pathogens of dairy cows include Staphylococcus aureus, Streptococcus, Escherichia coli and so on, which show a trend of multiple drug resistance. It not only brings great challenges for the clinical treatment of dairy cow, but also gives a threat to human health. Antimicrobial resistance is far more than the speed of people developing antimicrobials. Therefore, it is necessary to maintain the efficacy of existing antimicrobials for treating infections. On the one hand, we should master the bacterial resistance mechanism, such as bacterial molecular resistance mechanism, antibiotic efflux mechanism or reducing the intake mechanism, biofilm, so as to help us find the adequate therapeutics; On the other hand, taking different measures to reduce the emergence bacterial resistance, such as plasmid elimination, antibiotic substitutes, the development of efficient and safe antimicrobials, clinical rational drug (combined treatment) and so on. This article reviews dairy cow's main pathogen resistance, drug resistance mechanism, drug resistance control technology and make a prospect for this. It would provide reference for reducing the drug resistance, regulating the use of antimicrobials and improving the therapeutic effect.

Research Advance on Intestinal Microbial-epithelial Cell Barrier Interactions
WAN Hua-yun, HU Jun-yi, WANG Zi-xu, CHEN Yao-xing, CAO Jing, DONG Yan-jun, MA Bao-chen, DONG Yu-lan
2017, 44(12):  3642-3649.  doi:10.16431/j.cnki.1671-7236.2017.12.035
Abstract ( 315 )   PDF (832KB) ( 279 )  
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Intestinal epithelial cells (IECs) are the first line of defense against pathogenic microorganisms of animal organism, which are important component of mucosal mechanical barrier, immune barrier and chemical barrier, they have absorption and barrier double function. In the intestine, there are many kinds of microorganisms. According to its relationship with the host, it is divided into three types of commensal bacteria, conditional pathogenic bacteria and pathogenic bacteria, it plays an important role in the construction of intestinal barrier. Firstly, IECs identify the intestinal microbes by direct or indirect ways, and distinguish their own and non-self, it is immune tolerance to their own substances (such as, commensal bacteria), and produce specific immune response to non-self-substances (pathogenic bacteria). Both of IECs and intestinal commensal bacteria together against pathogens maintain intestinal health. When the pathogenic microorganisms invade the intestine, IECs defense pathogenic microorganisms mainly through extracellular secretions and cell surface mucus layer, and the former largely include mucin, antibacterial molecular and antimicrobial immunoglobulin. The intestinal symbiotic bacteria can resist the pathogenic microorganisms and maintain the normal intestinal mucosal barrier function through the competitive identification sites, the secretion of antimicrobial substances, the increase of mucus secretion, the induction of IECs renewal, proliferation and repair. In the process of resisting invasion of gut microbes, pathogenic microorganisms through their own movement, secretion of toxins and enzymes to destroy the intestinal epithelial barrier, and directly contact with IECs to damage them. So the interaction between IECs and intestinal bacteria maintain the intestinal homeostasis. In this paper, a review is made of the IECs and intestinal microbial structure and functional adaptations, and hope to elaborate the mechanism of intestinal microbial-epithelial cell barrier interaction.

Protective Effects of Rosmarinic Acid on Oxidative Lung Damage in Asthmatic Mice
LIANG Zheng-min, LIAO Xiao-guang, WEN Xue-mei, XU Yang-feng, DENG Xin, YAN Guo-qing, HE Jia-kang
2017, 44(12):  3650-3655.  doi:10.16431/j.cnki.1671-7236.2017.12.036
Abstract ( 194 )   PDF (2377KB) ( 311 )  
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In order to evaluate the protective effect of rosmarinic acid on oxidative lung damage in asthmatic mice, in this study, the asthma model was established by sensitizing and challenging female BALB/c mice with ovalbumin (OVA), and some mice were challenged by OVA and H2O2 as a positive control of oxidative lung damage. The BALF, lung tissues of mice were harvested 24 h after the final OVA challenge and the number of total cells and eosinophils were measured, ROS,SOD and GSH-Px levels were detected, and pathological changes of lung tissue were observed using HE staining. The results showed that rosmarinic acid significantly reduced the number of total cells and eosinophils in BALF, significantly inhibited the production of ROS in lung tissue and BALF, increased the levels of SOD and GSH-Px, and ameliorated pathological changes of lung tissue. The results suggested that rosmarinic acid could protect oxidative lung damage in asthmatic mice.

Advances in Modulation of Intestinal Innate Defense Responses by Probiotic Bacteria
ZHANG Man, JIN Xin, TIAN Qiao-zhen, LIU Jiao, WANG Yun-he, ZHANG Zhao-yi, YANG Yin-feng
2017, 44(12):  3656-3662.  doi:10.16431/j.cnki.1671-7236.2017.12.037
Abstract ( 138 )   PDF (825KB) ( 243 )  
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Probiotics are a kind of living microorganism which can be administered in feed and confer numerous health benefits to animals. Probiotics play a key role in maintaining the microbial balance of gastrointestinal, immunomodulation and pathogen defense, specific probiotics show certain degree of potential to reinforce the integrity of intestinal epithelium and regulate some immune components. The mechanism of probiotic action is an area of interest. Among all possible routes of modulation by probiotics of intestinal mediated defense responses, modulations of intestinal barrier function, innate and adaptive mucosal immune responses as well as signaling pathways are considered to play an important role in the intestinal defense responses against pathogenic bacteria. This review summarizes how probiotic bacteria interact with goblet cell-derived mucins, trefoil factors (TFFs),defensins, Toll-like receptors (TLRs), secretory immunoglobulin A (sIgA), heat shock proteins (HSPs) and P-glycoproteins (P-gp) in the gut to regulate the intestinal innate defense responses,which is regarded as the scientific basis for the future detailed study of benefits of probiotics towards the health of intestine and also dietary intervention against intestinal exposure to pathogens.

Isolation,Identification and Drug Resistance Analysis of Avian Salmonella in Anhui
SHEN Xue-huai, ZHANG Dan-jun, PAN Xiao-cheng, ZHAO Rui-hong, DAI Yin, HU Xiao-miao
2017, 44(12):  3663-3669.  doi:10.16431/j.cnki.1671-7236.2017.12.038
Abstract ( 147 )   PDF (1044KB) ( 449 )  
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In order to investigate the situation of predominant strain and antibiotic resistance of avian Salmonella in Anhui province, 42 strains Salmonella were isolated and identified from 116 suspected samples by culture characteristics, microscopic examination, biochemical test, PCR and serological identification,then drug resistance was analyzed by Kindy-Bauer method. There were 5 groups of 11 serotypes in 42 strains Salmonella, of which 30 strains belonged to B serogroup, 2 strains to A serogroup, 7 strains to D serogroup, 2 strains to C1 serogroup, 1 strain to C2 serogroup, and Salmonella Typhimurium of B serogroup were the predominant serotypes. All Salmonella isolates resistant rates to amikacin, levofloxacin and ciprofloxacin were less than 20%, and the resistance rates to ampicillin, streptomycin, furazolidone, spectinomycin, cotrimoxazole and doxycycline were more than 50%, while resistance rate to ampicillin was 97.62%, and 41 isolates had multiple drug resistance. The results showed that Salmonella Typhimurium was the predominant serotypes of avian Salmonella in Anhui, and isolates showed multiple drug resistance. This study would provide the data basis for the comprehensive prevention and treatment of clinical medicine of avian Salmonella.

Effect of Dexamethasone on Mucosa Morphology in Piglets
PAN Shu-lei, DENG Hong-yu, RUAN Guo-rong, WANG Quan-xi
2017, 44(12):  3670-3675.  doi:10.16431/j.cnki.1671-7236.2017.12.039
Abstract ( 166 )   PDF (998KB) ( 386 )  
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This study was aimed to investigate the effects of dexamethasone on the morphology of intestinal mucosa and intestinal immune barrier function, and study the feasibility of setting up the piglets intestinal damage model, which could provide a theoretical and scientific basis for the future research of the mechanism and prevention measures caused by other intestinal injury of glucocorticoids. 30 healthy piglets (1 week after weaning) were randomly divided into 6 groups, with 5 replicates in each group. The experimental groups Ⅰ, Ⅱ and Ⅲ were injected with dexamethasone 1, 2 and 3 mL, the control groups Ⅰ, Ⅱ and Ⅲ were injected with saline 1, 2 and 3 mL,respectively. HE staining,PAS staining and ELISA detection method were used to measure the intestinal villus length and crypt depth, villus length and crypt depth ratio, goblet cells and intraepithelial lymphocytes and sIgA. The results were as follows:Dexamethasone inhibited the growth of intestinal villus, the crypt depth became deeper, villus length and crypt depth ratio decreased,and the number of goblet cells, intraepithelial lymphocytes and sIgA levels were reduced in experimental groups, and the greater the dose, the more obvious the indicators changed. It suggested that dexamethasone could affect the growth of intestinal villus and the intestinal absorption of nutrients, damage mucosal immune barrier function in a dose dependent.