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    Interference Efficiency of Piwi Gene Expression in the Chicken Germ Stem Cells
    LI Zhi-teng, CHANG Guo-bin, XU Lu, MA Teng, CHEN Jing, CHEN Rong, WANG Hong-zhi, LIU Lu, XU Qi, CHEN Guo-hong
    Scientia Agricultura Sinica    2016, 49 (3): 563-472.   DOI: 10.3864/j.issn.0578-1752.2016.03.014
    Abstract345)      PDF (3741KB)(456)       Save
    【Objective】Solving the low transfection efficiency and interference efficiency in poultry stem cell is a big challenge so far. This study was designed to compare the interference effects on Piwi gene expression in chicken primordial germ cells (PGCs) using different ways, aiming to explore effective methods to interfere the poultry stem cells, which is a fundamental work for the study of the self-renewal of stem cell, RNA silencing and post-transcriptional regulation. 【Method】According to the techniques of the separation of primary germ cell, the authors selected 10th and 4th days fertilized eggs post incubation to separate the chicken embryo fibroblast cells(CEF) and germinal ridge, respectively; Herein, the CEF cells were taken as a feeder layer and the PGCs cells were treated by Trypsin-EDTA. The PGCs cells were further identified by the morphology, chemical method and immunology assay. According to the mRNA sequence of chicken Piwi gene published by Genbank (NM_001098852), three positive siRNAs were chemically synthesized and the BLOCK-iT™ Alexa Fluor® Red Fluorescent Oligo was as a negative control. Simultaneously, three interfering vectors of shRNA were constructed using the free carrier of pRNA-U6.1, and an empty vector with GFP fluorophore was as a negative control. Afterwards, the transfection efficiency was detected by the siRNA and shRNA negative control using different types of transfection reagent to optimize the condition, and extracted the RNA of PGCs cells at 24, 48, 72 and 144 h post transfection and analyzed differential expressions of Piwi gene with RT-PCR techniques. All data were analyzed by the SPSS software.【Result】With the premise of well identification of the PGCs cells, siRNA and shRNA were successfully transfected into the cells under the optimized conditions with 50 pmol siRNA and 2 μL Lipofectamine TM 2000 for siRNA transfection group and 500 ng shRNA and 1.5 μL X-Treme GENE HP DNA Transfection Reagent for shRNA transfection group. Compared to the blank group, the Piwi expressions of the negative control had no significant difference at the four time points (24,48,72 and 144 h) in siRNA and shRNA intervention group (P>0.05); Compared to the negative control and the blank group, Piwi gene expressions were significantly down-regulated in experimental groups at the first three time points in siRNA intervention group, while were significantly down-regulated at all of time points in the shRNA intervention group (P<0.05).【Conclusion】Collectively, the interference effects on Piwi gene expression in chicken PGCs were compared by siRNA and shRNA RNAi technology, and indicated that the shRNA vector had higher and more stable interference efficiency, which help to provide some basic information for future research of RNAi technology.
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    Investigation and Analysis of Main AFN in Soybean Meal and Fermented Soybean Meal
    YANG Yu-juan, YAO Yi-sha, QIN Yu-chang, QIU Jing, LI Jun-guo, LI Jun, GU Xu
    Scientia Agricultura Sinica    2016, 49 (3): 573-580.   DOI: 10.3864/j.issn.0578-1752.2016.03.015
    Abstract700)      PDF (388KB)(1525)       Save
    【Objective】Soybean meal is the main raw material of feed. However, various anti-nutritional factors (ANF) in soybean meal hinder the digestion, absorption and utilization for nutrients, which would have negative effects on animal growth and health. Studies have shown that soybean meal fermented by microorganism could decrease the undesirable effect of ANF. While, fermentation strains, soybean species, and manufacturers may have an effect in fermentation process. Moreover, seldom reports referred to the levels of ANF in soybean meal and fermented soybean meal. Based on this, 65 batches of available soybean meal and 54 batches of fermented soybean meal were collected in this study, followed by analysis of 6 kinds of ANF, including glycinin, β-conglycinin, trypsin inhibitors, raffinose, stachyose and urease, in order to investigate the actual levels of ANF in soybean meal and fermented soybean meal.【Method】In this study, glycinin, β-conglycinin and trypsin inhibitors were analyzed by Enzyme-link Immunosorbent Assay (ELISA) method. Experimental procedures were conducted according to instructions which were sample pretreatment, plus, washing, adding enzymatic reagent, color reaction, and termination. The analysis method of raffinose and stachyose was HPLC after extracted by microwave. Analysis of urease refers to GB/T 8622-2006, and titration was utilized for analysis of urease activity.【Result】The average concentration of glycinin in soybean meal (129.3 mg·g-1) is 57.7% higher than fermented soybean meal (54.7 mg·g-1). The percentile method was used for analysis. The normal values in soybean meal and fermented soybean were 58.9-177.3 mg·g-1 and ND (Not Detected)-109.4 mg·g-1, respectively. Moreover, the fermented soybean meal (37.6 mg·g-1) is 63.2% lower than the concentration of β-conglycinin in soybean meal (102.2 mg·g-1). The normal values in soybean meal and fermented soybean were 42.8-147.2 mg·g-1 and ND-61.8 mg·g-1, respectively. The average concentration of trypsin inhibitors in soybean meal (18.4 mg·g-1) is 59.1% higher than fermented soybean meal (7.5 mg·g-1). In addition, the normal values of trypsin inhibitor in soybean meal and fermented soybean meal were ND-28.9 mg·g-1 and ND-9.9 mg·g-1. The average concentration of raffinose in soybean meal and fermented soybean meal were 11.02 mg·g-1 and 1.93 mg·g-1, with the former is 80% higher. The normal values of raffinose in soybean meal and fermented soybean meal were ND-13.79 mg·g-1 and ND-4.65 mg·g-1. The average value of stachyose average in soybean meal (29.70 mg·g-1) is higher than in fermented soybean meal (5.19 mg·g-1). The normal values of stachyose are ND-33.29 mg·g-1, ND-11.58 mg·g-1, respectively. The normal values of urease in soybean meal was in the range of ND to 0.40 U·g-1, while the activity value of urease of fermented soybean meal was under the detection limit.  It was concluded that the concentrations of ANF in fermented soybean meal have decreased differently compared with soybean meal.【Conclusion】This research provided data support for further optimization of feed processing, and could act as a guide for breeding companies to choose higher quality of soybean meal and fermented soybean meal as feed raw materials.
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    Cited: Baidu(1)
    POMC Expression and Localization in Sheep Skin with Different Coat Color
    MA Shu-hui, LI Ya-nan, HE Xiao-yan, WANG Hai-dong, YANG Lei, DONG Chang-sheng
    Scientia Agricultura Sinica    2015, 48 (9): 1818-1824.   DOI: 10.3864/j.issn.0578-1752.2015.09.15
    Abstract519)   HTML1)    PDF (855KB)(680)       Save
    【Objective】The objective of this study is to identify the regulation effect of POMC on coat pigment formation via analyzing the differences of POMC expression and location between different coat colors of sheep skin, in order to further enrich the theoretical knowledge about the formation mechanism of animal coat color. 【Method】 Three black adult sheep and three white sheep with matched age were selected, then three pieces of skin tissue from the buttocks of sheep were collected, respectively. One of them was used for immunohistochemistry assay to investigate the expression location of POMC between different coat color sheep after tissue embedded, the others were hold in liquid nitrogen for the extraction of total RNA and protein. The POMC-specific primers were designed by blasting in the NCBI database. After the reverse transcription of total RNA, QRT-PCR assay was proceeded to detect POMC mRNA expression of different coat color sheep. Then the protein expression level of POMC between different color sheep was evaluated by using Western blotting. All data were analyzed by SPSS19.0 to explore the mediate effect of POMC on coat color formation mechanism. 【Result】Immunohistochemistry showed that POMC was expressed in both black and white sheep skin, and the expression sites were located around the keratinocytes of hair follicle root sheath and epidermal. QRT-PCR showed that the relative expression of POMC mRNA in black sheep skin was 2.5004±0.2577**, while in white was 1.0032±0.0350, the quantity expression of POMC mRNA level of black sheep was 2.50 times as that of white sheep (P<0.01). Western blotting result showed that the expression of POMC protein in black and white sheep were 1.5253±0.04026* and 1.0005±0.0180, respectively, the expression of POMC protein in black sheep was 1.55 times as that in white sheep (P<0.05). 【Conclusion】POMC expressed normally in black and white sheep skin, and its expression was located in consistent position, the expression of POMC in black and white sheep skin have differences, which may involve in formation of coat color.
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    Expression and Identification of the σ3 Gene of Avian Reovirus in Transgenic Tobacco
    WANG Sheng, XIE Zhi-xun, HUANG Li, XIE Li-ji, DENG Xian-wen, XIE Zhi-qin, LIU Jia-bo, LUO Si-si, ZENG Ting-ting
    Scientia Agricultura Sinica    2015, 48 (9): 1836-1844.   DOI: 10.3864/j.issn.0578-1752.2015.09.17
    Abstract405)   HTML1)    PDF (874KB)(479)       Save
    【Objective】 An experiment was carried out in laboratory to study on expression of Avian reovirus σ3 gene in tobacco and its genicity reactions of the expression products. 【Method】 A pair of primers was designed according to the major antigen region of the σ3 gene derived from GenBank. A plant vector of pBI121-σ3 constitutively expressed σ3 gene was constructed. The pBI121-σ3 vector was transferred into the Agrobacterium strain EHA105 by thermal activation method and a recombinant Agrobacterium strain EHA105 containing pBI121-σ3 was obtained. After transformation of tobacco plants via Agrobacterium tumefaciens, the resistant plants were selected with kanamycin. The resistant plants were firstly analyzed by PCR, using σ3 gene specific primers, and then real-time fluorescence quantitative PCR was used to further estimate the copy number of the σ3 gene in positive plants, and endogenous RNR2 gene in tobacco was used as a reference gene. With a serial of dilutions, the standard curves of the cycle threshold (CT) relative to the log of each initial template copy of σ3 and RNR2 genes were obtained. The transgenic copy number was obtained by comparing the initial template copy of σ3 gene with that of RNR2. Western blot analysis was used to examine the σ3 protein expression in transgenic tobacco. 【Result】 The recombinant plasmid was identified by restriction endonuclease analysis and by gene sequencing. It was confirmed by DNA sequencing that the recombinant plasmid pBI121-σ3 contains a complete σ3 gene, a correct insertion site and expected open reading frame (ORF). The plant nuclear vectors expressed the σ3 gene by using CaMV 35S promoter and NOS terminator, and a kanamycin resistance marker (NPT II). The σ3 gene may be expressed as a 61.6 ku green fluorescent fusion protein(GFP) in tobacco.  MS medium containing 350 mg cefotaxime·L-1 inhibited the growth of Agrobacteria and 100 mg kanamycin·L-1 can provide enough selective pressures. Eight resistant plants containing σ3 gene were obtained by PCR screening. The standard curve of RNR2 gene was expressed as y=-3.5352x+15.143, the standard curve of σ3 gene was expressed as y=-3.5366x+1.8265. Among the five putative transgenic lines, five had four copy numbers, whereas the negative control had none. Western blotting results showed that the 61.6 ku recombinant σ3 protein was successfully expressed, and the protein was specifically recognized by anti ARV positive serum. 【Conclusion】 A plant expression vector of pBI121-σ3 was successfully constructed and the regenerated transgenic σ3 gene tobacco plants were obtained. Recombinant σ3 protein has a good reactivity with anti ARV positive serum. The research findings provide a basis for further analysis on plants as bioreactors development and the production of σ3 oral vaccines.
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    Impact of Putrescine and Proline on Suckling Piglet Jejunum Villus - crypt Axis Epithelial Polyamine Metabolism and Wnt Signal Pathway
    WANG Xiao-cheng, XIONG Xia, YANG Huan-sheng, GAO Wei, GONG Min, YIN Yu-long
    Scientia Agricultura Sinica    2015, 48 (8): 1609-1615.   DOI: 10.3864/j.issn.0578-1752.2015.08.15
    Abstract389)   HTML10)    PDF (360KB)(539)       Save
    【Objective】The aim of this paper is to study the effects of putrescine and proline on polyamines (putrescine, spermidine and spermine) metabolites, relative mRNA expression levels of Wnt signaling molecules along the villus-crypt axis in the jejunum of sucking piglet. 【Method】 Eighteen 0-day old newborn crossbred (Landrace × Yorkshire × Duroc) piglets were randomly divided into three groups: control group, putrescine group and proline group. The piglets in control group were administered an equal volume of saline, and that in other two groups received putrescine (5 mg·kg-1 body weight ) and proline (25 mg·kg-1 body weight ), respectively. Piglets were weaned at 14-day old and slaughtered on 3 days postweaning. The jejuna epithelial cells along the crypt-villus axis were isolated by Krebs Henseleit (KH) solution and yield 3 “cell fractions” (Fraction 1, 2 and 3). The concentration of polyamine in F1, F2 and F3 was determined by high performance liquid chromatography, and relative mRNA expression of related genes of polyamine metabolic and wnt signaling pathways were measured by real time PCR. 【Result】 The concentration of putrescine, spermine and spermidine in F1 were significantly increased (P<0.05) in proline group compared to control group, while there was no significant difference in putrescine group (P>0.05). The concentrations of spermine and spermidine in F2 of proline or putrescine groups were significantly higher than that of control group (P<0.05). the concentrations of putrescine, spermine and spermidine in F3 of three group were not changed significantly (P>0.05). The expression of ornithine decarboxylase (ODC) gene in F1 of putrescine group was significantly higher than that of proline group and control group (P<0.05). The expression of arginase in F2 of proline group was significantly higher than that of putrescine group and control group (P<0.05). The expression of SFRP3 in F2 of proline group was significantly higher than that of putrescine group (P<0.05); the expression of sFRP4 in F3 of putrescine group is significantly higher than that of control group and proline group (P<0.05). 【Conclusion】 Exogenous putrescine and proline increased the concentration of polyamine in differentiated epithelial cells of jejunum in sucking piglet and promoted the polyamine metabolism, however, no statistically significant effect was observed on crypt cell. In addition, exogenous putrescine and proline promote differentiation in the intestinal epithelial cells by Wnt signaling pathways.
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    Study on the Association Between the Expression of Candidate Genes in Different Waves and Hair Follicle Characteristics
    NI Rong, SUN Wei, YIN Jing-feng, Lü Xiao-yang, WANG Qing-zeng, SU Rui, CHEN Ling, WU Wen-zhong, XU Hou-sheng, LI Yong, CHEN Jia-zhen, LIU Wei-zhong
    Scientia Agricultura Sinica    2015, 48 (8): 1616-1623.   DOI: 10.3864/j.issn.0578-1752.2015.08.16
    Abstract372)   HTML1)    PDF (1221KB)(431)       Save
     【Objective】The article aims to study the relevance between the expression of 5 candidate genes in different patterns and hair follicle characteristics, in order to learn more about the correlation between candidate genes and hair follicle characteristics and screen the genes for later functional verification. 【Method】The article detected the expression of 5 genes between different groups by RT-PCR, combined histology and microscopic observation techniques, analyzed the correlation between 5 genes and hair follicles. 【Result】 The results showed that: Hu-sheep hair follicle was distributed in groups, and it was mainly three follicles in a group, the larger diameter was the primary hair follicles, and the relatively small diameter was primary follicles.The diameter of primary follicles in large waves was significant differences with medium and small waves (P<0.01) , and there was no significant differences between small and medium waves(P>0.05). The diameter of secondary follicles in medium waves was significantly differences with large and small waves(P<0.01), but there were no significant differences between small and large waves(P>0.05).The number of primary follicles was no significant differences among large, medium and small waves(P>0.05), but in the same vision,the number of primary follicles in large waves was more than that of medium and small waves.The number of hair follicles in medium waves was significant differences with large and small waves(P<0.01), but there were no significant differences between large and small waves(P>0.05);The expression of MMP2 among medium waves was highly significant differences with large and small waves(P<0.01) and showed no significant differences between large and small waves(P>0.05). The expression of BMP7 and SFXN1 among small waves was significant differences with large and medium waves(P<0.05) and showed no significant differences between large and medium waves; The expression of remaining genes was not significant differences among large, medium and small waves(P>0.05). The relative expression of MMP2 had a significant positive correlation with secondary follicle in large waves(P<0.05). The relative expression of BMP7 was significantly correlated with primary follicle diameter in small waves, with the number of small waves’ secondary follicles showed a highly significant positive correlation(P<0.01), and negatively correlated with medium waves’ primary follicle diameter, the number of secondary follicle(P; The relative expression of SFXN1 was negatively correlated with large waves’ primary follicles(P<0.05), showed significantly positively related and highly significant positive correlation with small waves’ primary and secondary follicle diameter, and in medium waves’ primary follicles showed significant negative correlation. Under the condition of the same expression of BMP7 gene, the primary follicles’diameter was more than medium waves, the number of secondary follicles was more than medium waves, it was consistent with biopsy results. With the same expression of MMP2 gene , the number of large waves was more than small waves and medium was the least. It was consistent with biopsy. In addition, with the same expression of SFXN1 gene, the diameter of small waves was the largest and medium was the least. It was not match with the biopsy results, but the number of secondary follicles was slightly difference, the trend was the same, the results were basically consistent. Although other genes associated with hair follicles, their expression in large, medium, small waves were not significant.【Conclusion】 BMP7、MMP2、SFXN1 can be used as an important candidate gene for early breeding.<0.05)
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    Cited: Baidu(2)
    Effect of HOPX Gene Overexpression on Chicken Preadipocyte Proliferation
    SHI Hong-yan, HE Qi, CHENG Min, SUN Ying-ning, LI Hui, WANG Ning
    Scientia Agricultura Sinica    2015, 48 (8): 1624-1631.   DOI: 10.3864/j.issn.0578-1752.2015.08.17
    Abstract440)   HTML2)    PDF (984KB)(618)       Save
    【Objective】The objective of this study was to construct the eukaryotic expression vector of chicken full-length HOPX gene and investigate the effect of HOPX gene overexpression on chicken preadipocytes proliferation.【Method】Using Primer Premier 5.0 software, a pair of primers was designed to amplify the full-length coding sequence (CDS) of chicken HOPX. The full-length coding sequence (CDS) of chicken HOPX gene was PCR amplified from the cDNA from the abdominal fat tissues of AA broiler chickens and cloned into pCMV-HA vector. Chicken preadipocytes were isolated from the abdominal fat tissues of 12-day-old AA broiler chicken by collagenase digestion, cultured and transfected with pCMV-HA-HOPX and pCMV-HA empty vector, respectively. Cell proliferation was assayed by microscopic examination and Cell Counting Kit-8 (CCK-8). Gene expression was measured by Western blotting and quantitative Real-time RT-PCR. 【Result】 The sequencing results showed that the full-length coding sequence of chicken HOPX gene is222bp and identical with NCBI reference sequence (NM_204556). Western blotting analysis showed that pCMV-HA-HOPX could correctly express the HA-tagged HOPX. The microscopic examination showed that the numbers of the preadipocytes transfected with pCMV-HA-HOPX were less than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h and 48 h after of cell adhesion. CCK-8 analysis showed that the OD values of the preadipocytes transfected with pCMV-HA-HOPX were significantly lower than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h, 48 h and 72 h after of cell adhesion (P0.01). Consistently, at 24 h after of cell adhesion, the mRNA expression of CyclinD1 was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.05); at 48 h after of cell adhesion, the mRNA expression of PCNA was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.05); at 72 h after of cell adhension, the mRNA expression of CyclinD1 and PCNA were extremely significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.01) 【Conclusion】 HOPX gene overexpression in vitro inhibits chicken preadipocyte proliferation.
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    Cited: Baidu(1)
    Effect of Dietary Fat Sources and Dosages on Growth, Health, Serum Lipid and Liver Cholesterol Metabolism of Mice
    HUANG Yang, LI Ping-hua, HE Li-chun, WANG Han, NIU Qing, SHI Lei,ZHOU Bo, HUANG Rui-hua
    Scientia Agricultura Sinica    2015, 48 (5): 966-975.   DOI: 10.3864/j.issn.0578-1752.2015.05.15
    Abstract507)   HTML4)    PDF (413KB)(624)       Save
    【Objective】This experiment was conducted to investigate the effect of dietary fat source and dosages on growth performance, health status, serum lipid indicators and liver cholesterol metabolism-related gene mRNA expression level and to explore the mechanism of dietary fats source and dosage on hepatic cholesterol metabolism of mice. Results will contribute to select a suitable amount and type of oil for mammal.【Method】 Forty eight 3-week-old healthy KM mice whose body weights were 16-19 g were randomly assigned into four groups with 4 replicates per group and 3 mice each. Mice were fed: normal diet (control group); 4% bean oil diet (group B); 4% emulsified coconut powder diet (group L); 8% emulsified coconut powder diet (group H)for fourteen days, respectively. During the whole experiment, daily feeding times, feed quantity and remaining amount of feed of each time were recorded. All animals were fed and watered ad libitum. According to the recorded data, body weight and average daily feed intake (ADFI), the average daily gain (ADG), feed gain ratio (F/G) were calculated. Blood distribution, health index and liver weight of mice were measured. The concentrations of triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) in serum were determined. The expression of mRNA of 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), cholesterol 7 alpha-hydroxylase (CYP7A1) and low density lipoprotein-receptor (LDLR) in liver were determined by real time PCR.【Result】Supplementation of 4% bean oil significantly increased body weight, ADFI and liver index of mice compared with the control group (P<0.05), but had no significant effects on ADG, F/G or liver weight (P>0.05). Supplementation of 4% emulsified coconut powder failed to significantly change growth performance of mice compared with the control group (P>0.05). Supplementation of 8% emulsified coconut powder significantly increased ADFI of mice compared with the control group (P<0.05), but had no significant effects on other growth performance (P>0.05). Healthy status: Fat had no significant effects on blood distribution and health index (P>0.05). Compared with the control group, TC and HDL-C levels in the serum significantly decreased in 4% bean oil group (P<0.05), TG and LDL-C levels have no significant difference. Supplementation of 4% emulsified coconut powder had no significant effects on serum lipid index of mice (P>0.05). Supplementation of 8% emulsified coconut powder significantly decreased serum TC levels (P<0.05), but had no significant effects on TG, HDL-C and LDL-C levels (P>0.05). Supplementation of 8% emulsified coconut powder significantly increased the expression of mRNA of CYP7A1 of mice compared with the control group (P<0.05). Four percent bean oil diet and 4% emulsified coconut powder diet had no significant effects on the expression of mRNA of CYP7A1 (P>0.05). There were no significant differences in the expression of mRNA of HMGCR and LDLR among four groups (P>0.05). 【Conclusion】 Supplementation of bean oil increased the growth performance on ADFI and body weight of mice, decreased serum total cholesterol and high-density lipoprotein cholesterol. Supplementation of high doses emulsified coconut powder improved ADFI and decreased serum total cholesterol, and increased CYP7A1 mRNA expression in liver of mice. Compared to the high dosage of emulsified coconut powder, low dosage had no significant effects on improving lipid metabolism of mice. Fat in the diet probably affected liver cholesterol metabolism by regulating the gene expression levels of cholesterol metabolism-related enzymes in the liver to maintain cholesterol metabolism homeostasis in mice.
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    Cited: Baidu(1)
    PCV2 Virus Like Particles Vaccine Produced with Recombinant Cap Protein Expressed in E.coli
    ZHAO Xiao-yun, QIAO Xu-wen, CHEN Jin, LI Peng-cheng, YU Xiao-ming, ZHU Guo-qiang, ZHENG Qi-sheng, HOU Ji-bo
    Scientia Agricultura Sinica    2015, 48 (5): 976-986.   DOI: 10.3864/j.issn.0578-1752.2015.05.16
    Abstract662)   HTML3)    PDF (4782KB)(1294)       Save
    【Objective】The objective of this study is to develop recombinant virus like particles vaccine for Porcine circovirus type 2 through soluble prokaryotic expression of ORF2 gene. 【Method】 The optimized ORF2 gene of PCV2 NJ strain was synthesized according to codon usage of E.coli, and then the optimized ORF2 gene was cloned into pQZ1 to get the recombinant prokaryotic expression vector named pQZ-Cap. The target gene was expressed with 1.0 mmol·L-1 IPTG induction for 24 h under 15℃ on the prokaryotic expression platform in authors’ laboratory following the recombinant plasmid pQZ-Cap transformed into host E.coli BL21. Firstly, SDS-PAGE and Western blotting were used to identify the expression and solubility of the recombinant protein. Secondly, assemble for recombinant Cap VLP was evaluated through electron microscope analysis technology. Furthermore, swines were inoculated with recombinant Cap protein emulisified in SPPEIC 206 adjuvant, and the immunogenicity of recombinant Cap protein vaccine was evaluated by PCV2 specific antibody detection and virus attack.【Result】SDS-PAGE and Western blotting results indicated that the optimized ORF2 gene of PCV2 NJ strain could be efficiently expressed in E.coli BL21 in form of completely soluble. Abundant PCV2 VLP with diameter about 17 nm could be observed through electron microscope, exist in supernatant of the induced E.coli after ultrasonication. VLP vaccine composed of recombinant Cap protein possessed satisfactory immunity. Swine immunized with this VLP vaccine generated perfect antibody response with 100% qualified at 21 d post one sole vaccination and could be completely protected from pathogenic virus attack.【Conclusion】To summarize, optimized ORF2 gene of PCV2 NJ strain was successfully expressed in E.coli completely soluble and recombinant Cap protein can auto-assemble into VLP particles after ultrasonication. The VLP particles pose perfect immunity and could be used as subunit vaccines to prevent PCV2 infection.
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    Gene Cloning and Expression Analysis of 5-HT Receptors in Silkworm (Bombyx mori)
    LI Hai-yin, LI Yan, CHEN Xi, CHEN Peng, CHEN Ping
    Scientia Agricultura Sinica    2015, 48 (5): 987-1001.   DOI: 10.3864/j.issn.0578-1752.2015.05.17
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    【Objective】The objective of this study is to clone four kinds of 5-HT receptor genes and investigate their expression in different tissues of Bombyx mori, and to provide basic knowledge for further functional studies of these 5-HT receptor genes.【Method】Four 5-HT receptors were cloned based on genome database and using RT-PCR techniques. The bioinformatic method was used to analyze 5-HT receptor genes of B. mori and homology between species. The expression profiles of these genes in different tissues of larvae and adults were investigated by using semi-quantitative real-time PCR.【Result】Based on the predicted gene sequences, special primers were designed to clone the 5-HT receptor genes. Four 5-HT receptor genes 5-HT1ABm, 5-HT1BBm, 5-HT2Bm and 5-HT7Bm (GenBank accession number: KM236100-KM236103) were cloned. The open reading frame (ORF) of 5-HT1ABm, 5-HT1BBm, 5-HT2Bm and 5-HT7Bm was 1 395, 1 341, 1 881 and 1 497 bp, which encoded a polypeptide of 464, 446, 626 and 498 amino acids, respectively. They were typical of G protein-coupled receptors with seven transmembrane protein domains. The sequences were aligned and the phylogenetic tree of four 5-HT receptors was analyzed with other insects and vertebrates. Similarity of the amino acid sequence of 5-HT1ABm, 5-HT1BBm, 5-HT2Bm and 5-HT7Bm was only 30.4%. While, the similarity of 5-HT1A, 5-HT1B, 5-HT2 and 5-HT7 were 45.4%, 61.4%, 48.4%, and 54.1%, respectively in insects. In addition, 5-HT receptors had high homology and more conservative transmembrane region than non-transmembrane region in insects and vertebrates. The phylogenetic tree indicated that the same type receptors from different species got together, then the same receptor formed branch as species genetic relationships. The evolutionary relationships of 5-HT receptors in silkworm were close to Manduca sexta. The results of semi-quantitative real-time PCR showed that 5-HT1Abm, 5-HT1BBm expressed in all tissues in larvae, 5-HT2Bm only had expression in head, ventral chain and testis in larvae, 5-HT7Bm had expression in head, ventral chain, midgut, fat body, testis and ovary in larvae. 5-HT1ABm, 5-HT1BBm,and 5-HT7Bm expressedin other tissues except head in male of adults, whereas 5-HT7Bm in the reproductive system expression of male adults was significantly higher than other tissues. 5-HT2Bm did not express in adult.【Conclusion】Four 5-HT receptor genes were cloned in silkworm. They have more conservative in insects and vertebrates, and homologous relationships are close to M. sexta. The results of semi-quantitative real-time PCR showed that the tissue expression patterns were diverse.
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    The Disaster, Ecological Distribution and Control of Poisonous Weeds in Natural Grasslands of Xinjiang Uygur Autonomous Region
    YAN Du-jian, ZHOU Qi-wu, LU Hao, WU Chen-chen, ZHAO Bao-yu, CAO Dan-dan, MA Feng, LIU Xiao-xue
    Scientia Agricultura Sinica    2015, 48 (3): 565-582.   DOI: 10.3864/j.issn.0578-1752.2015.03.16
    Abstract583)   HTML4)    PDF (1064KB)(922)       Save
    Natural grassland is the material basis for the development of husbandry in the minority area, and it is also a very important component to maintain the balance of natural ecological environment in Xinjiang. For a long time, because of drought, severe overgrazing, blind reclamation, population increase and other natural and man-made factors, as well as inadequate infrastructure construction and lagging management of the grass, high-quality forage is decreasing year by year and poisonous weeds are fast spreading, leading to grasslands reverse succession and contagious poisonous weeds, widespread desertification, destroying the balance of natural ecological environment, and a large number of livestock poisoning even death due to grazed poisonous weeds mistakenly. Especially in recent decades, the rapid spreading of poisonous weeds have caused the reduction of grassland vegetation and biodiversity, the simplification of vegetation, decline of grass yield, and the degeneration of natural grassland with edible pasture grass, instead of more different levels of degraded grasslands even severe contagious poisonous weeds. In some regions, the grassland poisoned has led to the poisonous grass disaster take place frequently, even broke out, which have increased from the basic ecological problem to a social problem, seriously affected the local social stability and grassland ecological security. At present, more than 85% available natural grasslands are degenerating to a greater or lesser extent and 37.50% of them are severely degraded, with an poisonous weeds disaster area of nearly 700 million hm2, which has accounted for 20.42% of the total hazard area of natural grassland of the country, and the annual death number of animals with poisoning is more than 3000 in average . Xinjiang natural grassland contains common poisonous plants of 81 species, 24 families and 54 genera. Among them, the Aconitum carmichaeli, Aconitum soongaricum, Achnatherum inebrians, Oxytropis glabra, Astragalus variabilis, Anabasis aphylla, Pedicularis, Ligularia sibirica and Cucuta virosa are more serious harmful poisonous weeds to grassland animal husbandry, which accounted for approximately more than 80% of the total poisonous weeds hazard area. Therefore, a comprehensive understanding of poisonous weeds disaster situation in natural grassland of Xinjiang, weed species and geographic distribution, the effective prevention and control, the solution of the poisonous weeds disaster, is of great significance to improve the grassland productivity and forage quality, promote the sustainable development of animal husbandry in pastoral areas and farmers’ income, flourishing frontier minority economy and stabilize the ecological environment balance. According to the analysis and summary of data reported and results of the project group actually investigated in Xinjiang natural grasslands, the disaster situation, species of poisonous weeds and geographical distribution, prevention and control measures and reasonable utilization are reviewed. The disaster condition of regional grassland ecological and animal husbandry production caused by the poisonous grass, main poisonous grass species, geographic distribution and damage area of natural grassland in Xinjiang is summarized, respectively. At the same time, on the basis of the project team dedicated to the prevention and control of the China’s western poisonous grass research for many years, the existing problems and reasons of poisonous grass varieties, severe disasters, difficulty of prevention and treatment in natural grassland of Xinjiang were analyzed. The authors put forward some thoughts that from the angle of the ecology, the traditional control idea should be changed to strengthen the research of poisonous plant biology, ecology, toxicology characteristics, prevention and control of poisoning disease as well as comprehensive utilization etc. Thus it is helpful for us to understand the scientific poisonous weeds. Based on the principle of “change harm into good, make waste profitable”, place equal stress on ecological protection and development and utilization, study and learn from the successful technologies in prevention and control of poisonous plants in developed countries such as USA, take comprehensive prevention and control technology that ecological control means combined with other multiple methods to prevent poisonous weeds disaster, thus providing people with the basic information to know and research of the main poisonous weeds in natural grasslands in Xinjiang.
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    Impact of Concentrations and Immune-stimulating Times of Poly I:C on Gene Expression of Porcine Peripheral Blood Mononuclear Cells
    WANG Ji-ying, WANG Yan-ping, WANG Huai-zhong, WANG Hai-fei, LIU Jian-feng, GUO Jian-feng
    Scientia Agricultura Sinica    2015, 48 (3): 583-593.   DOI: 10.3864/j.issn.0578-1752.2015.03.17
    Abstract377)   HTML2)    PDF (422KB)(573)       Save
    【Objective】 Polyinosinic:polycytidylic acid (Poly I:C) is a synthetic double-stranded polyribonucleotide. As an analogue of viral double-stranded RNA, Poly I:C is widely used as immunologic stimulant to study the immune regulation and inflammatory reaction of an organism to the RNA viruses. So far, no studies were reported on determination of the optional concentration and stimulating time of porcine peripheral blood mononuclear cells (PBMC) in response to Poly I:C stimulation. In the present study, we systematically analyzed the impact of Poly I:C concentrations and stimulating times on the expression of several cytokines and pattern recognition receptors and the optional concentration and stimulating time of Poly I:C in the PBMC were determined, which would provide an experimental foundation for the future use of Poly I:C and porcine PBMC to study the immune response of pigs to RNA viruses. 【Method】 Using isolated PBMC and 1:5 EDTA-anticoagulated diluted blood of three Landrace piglets, the in vitro immune stimulating experiment was performed with a series of Poly I:C concentrations (0, 10, 20 and 40 μg·mL-1) and immune-stimulating culture times (4 h, 8 h, 12 h and 24 h). The expression of several cytokines (IL6, IL8, TNFα, IL10, IRF3, IFNα and IFNγ) and pattern recognition receptors (TLR3 and TLR4) were determined by qPCR. According to the relative expression trends of these cytokines and receptors, it was analyzed to achieve the optimal Poly I:C concentration and immune-stimulating culture time. 【Result】 The expression of the cytokines and receptors were affected by Poly I:C concentrations as well as immune-stimulating times. Each gene had its characteristic expression trend, and varied in Poly I:C concentration and immune-stimulating time to attain the highest expression. For the two interferon genes, IFNα and IFNγ, the highest expressions were observed when the stimulating time was 4 h, and their expression decreased gradually with the stimulating time. For the other five cytokines (IL6, IL8, TNFα, IL10 and IRF3) and two receptor genes (TLR3 and TLR4), the expressions were gradually increased with the Poly I:C concentrations and stimulation times, and reached the highest when Poly I:C concentrations and stimulation times were 20-40 μg·mL-1 and 12-24 h, respectively. However, for the genes whose expressions were highest when Poly I:C concentration and stimulation time were 20 μg·mL-1 and 12 h, their expressions showed small decline when Poly I:C concentration and stimulation time were 40 μg·mL-1 and 24 h. Furthermore, in the study, it was also tested the expressions of the above genes of 1:5 EDTA-anticoagulated diluted blood in response to various Poly I:C concentrations and stimulation times. The results indicated that the whole expressions of these genes were lower than those in PBMC, especially for two interleukin genes, IL6 and IL8, whose expression in diluted whole blood was not only low but the variation trends with Poly I:C concentrations and stimulation time were reversed with those in PBMC. Compared with PBMC, though diluted blood preserved the integrity of the immune state of the pig’s blood, the major drawback is that the anticoagulant is kept in it. The anticoagulant used in the study was EDTAK2, which can combine with calcium ion to form chelate, so as to prevent blood clotting. However, calcium ion is an important signal molecule in cells, and the combination of calcium ion with EDTA may have a certain impact on the subsequent cell function. In addition, some of the compounds and plasma proteins in whole blood may be also involved in the regulation of immune cells to Poly I:C immune stimulation so as to reduce immune response. 【Conclusion】 Comprehensive analysis of the expression trends of the cytokines and receptors were detected, and it was concluded that, in the porcine PBMC stimulation experiment using Poly I:C, the optimal concentration and immune-stimulating time are 20 μg·mL-1 and 24 hours, respectively. EDTA- anticoagulated diluted blood has less immune response to Poly I:C and different variation trends with Poly I:C concentrations and stimulation times.
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    Rethinking the Withdrawal of Antimicrobial Growth Promotants in Animal Feed
    HAO Hai-hong, CHENG Gu-yue, DAI Meng-hong, WANG Xu, WANG Yu-lian, HUANG Ling-li, LIU Zhen-li, YUAN Zong-hui
    Scientia Agricultura Sinica    2015, 48 (3): 594-603.   DOI: 10.3864/j.issn.0578-1752.2015.03.18
    Abstract399)   HTML4)    PDF (403KB)(1276)       Save
    Withdrawal of antimicrobial growth promotants (AGPs) in animal feed issued by European Union (EU) countries caused widespread controversy in the international community. This paper comprehensively reviewed the antimicrobial resistance monitoring data from animal original bacteria and the risk assessment results of veterinary usage. After profoundly rethinking the ban of AGPs in animal feed, the results showed that (1) the risk of some AGPs (e.g. macrolide AGP of tylosin and streptogramin AGP of virginiamycin) seemed to be overstated. The risk of the usage of macrolide AGPs in food animals to emergence of macrolide resistant Campylobacter in human is negligible, and the use of virginiamycin as AGPs could hardly affect the treatment of human infections caused by Enterococcus; (2)There is a lack of scientific evidence for supporting the proposition of transmission of antimicrobial resistance from farm to dining table. Although there are some evidence that antimicrobial resistant bacteria could directly transmit from food animal to those persons who closely contacted with animals, there is no direct and sufficient evidents to support the transfer of antimicrobial resistant pathogens through food chain to persons; (3) Withdrawal of AGPs did not change the epidemiology of resistant pathogens, especially for the avoparcin in glycopeptides, enrofloxacin in fluoroquinolones and chlorotetracycline in tetracyclines. After ban of these three classes of AGPs, the number of resistant bacteria from both animal and human continued to increase. The reason may be attributed to the enhanced fitness of fluoroquinolone resistance in Campylobacter and the increase of the consumption of therapeutic tetracycline agents; (4) Withdrawal of the AGPs may brought a certain loss for the animal breeding industry. For example, it may increase the incidence of necrotizing enteritis caused by Clostridium, increase therapeutic use of antimicrobial agents in the farmed animals, and increase breeding materials and corresponding venues, etc.; (5) Withdrawal of the AGPs may cause a certain influence on animal food safety and human public health. For instance, it may affect the fermentation of animal intestinal flora and thereby increase the emissions of harmful gas. It may increase the chances of bacteria contamination during food processing and therefore increase the incidence of foodborne pathogens in human. Conclusively, the political decision must be developed based on the balance of pros and cons and the adjustment of spatial and temporal condition. It also need to comprehensively consider the function of withdrawal of AGPs on control of antimicrobial resistance and the influence of withdrawal of AGPs on the animal breeding industry and human public health. Face the development of animal breeding industry in China and the world food sustainable needs, it is necessary to conduct in-depth research and systemic risk assessment to find new and rational strategy to control resistance. It is also essential to strengthen the government supervision to avoid drug abuse and promote the rational use of antimicrobial agents in food animals.
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    Association of GHSR and GHRL Gene Genetic Variation with Growth Traits in Two Guizhou Goat Breeds
    SONG Tao-wei, CAI Hui-fen, LUO Wei-xing, LIU Ruo-yu, ZHANG Yi-yu, SUN Yan-yan, LIU Bin
    Scientia Agricultura Sinica    2015, 48 (1): 140-153.   DOI: 10.3864/j.issn.0578-1752.2015.01.14
    Abstract565)   HTML3)    PDF (3896KB)(615)       Save
    【Objective】 The aim of this study was to explore the relationships between GHSR and GHRL gene and body weight & size traits, and search the molecular genetic markers related to goat growth traits.【Method】Guizhou white goat and Guizhou black goat were chosen as subjects, the DNA pooling was constructed and the technology of direct sequencing of PCR products and PCR-SSCP were used to detect the single nucleotide polymorphism of GHSR and GHRL genes, and its association of polymorphisms and different genotypes and combined genotypes with growth traits was analyzed by SPSS(18.0). Meanwhile, the bioinformatics analysis of GHSR gene was conducted by online software.【Result】The result showed that two SNPs (G200A and T628C) were detected in exon 2 and 3′UTR but no SNPs in exon1 and 5′UTR of GHSR gene, respectively. G200A was a sense mutation site and devided into three genotypes GG, GA and AA by PCR-SSCP. Genotype GG was the dominant genotype and allele G was dominant allele with the frequencies of 0.6731 and 0.5243 in Guizhou white goat and Guizhou black goat (♀) groups instead of allele A in Guizhou black goat (♂). All populations were moderate polymorphic in G200A site (0.25<PIC<0.50). G141A was found in intron 2 and two sense mutation sites (T78C and C14T) were identified in exon2 and exon4 of GHRL gene, respectively. There were two genotypes (CT and CC) in C14T site, genotype CC was the dominant genotype, which the frequency were 0.7692, 0.9417 and 0.9390, allele C was dominant allele with the frequency of 0.8846, 0.9709 and 0.9695, respectively. These populations were low polymorphic (PIC<0.25). χ2 test indicated that SNPs(G200A and C14T) fit with Hardy-Weinberg equilibrium in the population (P>0.05). Correlation analysis revealed that G200A site was significantly associated with body weight, body height, body length and hucklebone width (P<0.05), GG genotype was superior genotype. Additionally, body weight, body height and chest girth of individuals of Guizhou black goat (♂)with genotype CC was significantly higher than those with genotype CT(P<0.05) in C14T site. Meanwhile, the combined genotype was significantly associated with body height and chest girth, the individuals with CCGG genotype had significantly better body height than that of genotype CCAA (P<0.05). Bioinformatics analysis indicated that no core promoter regions and CpG island were found but a CAAT-box and several important transcription factors in 5′UTR region of GHSR gene. G200A had led to the change of secondary structure of mRNA. The secondary structure of protein was mainly α-helix (48.90%). Tertiary structure and transmembrane helix prediction showed that GHSR protein was a membrane protein.【Conclusion】The results revealed that SNPs and combined genotype of GHSR and GHRL gene had effect on growth traits in goat, G200A and C14T sites could be used as effective genetic markers for goat molecular breeding.
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    Cited: Baidu(4)
    Effect of Different Nano-Zinc Levels in Dietary on Semen Quality, Activities of Antioxidant Enzyme and Expression of Copper Zinc Superoxide in Epididymis of Ram Lambs
    ZHANG Chun-xiang, QIN Xiao-wei, GUO Li-na, ZHANG Guo-lin, ZHANG Jian-xin, REN You-she
    Scientia Agricultura Sinica    2015, 48 (1): 154-164.   DOI: 10.3864/j.issn.0578-1752.2015.01.15
    Abstract623)   HTML10)    PDF (6402KB)(913)       Save

    【Objective】 The objectives of this study were to investigate the effects of nano-zinc supplementation in dietary on the semen quality parameters, the activities of antioxidant enzyme in seminal plasma and the expression of copper zinc superoxide dismutase in epididymis of ram lambs, and to analyze the correlation between semen quality parameters and the activities of antioxidant enzyme.【Method】Sixteen 9-month-old Jinzhong ram lambs with good health and approximate weight were randomly divided into 4 groups, fed with a basal diet with supplementation of 0, 50, 100 and 150 mg·kg-1 DM nano-zinc, respectively. The experimental period was 90 d. Semen was collected on 78 d and 79 d in consecutive two days, samples of 100 µL fresh semen was used to analyze semen quality parameters, the rest of fresh semen sample was centrifuged at 2 000 r/min for 10 min, the supernatant (seminal plasma) was collected for measurement of the activities of copper zinc superoxide dismutase, glutathione peroxidase and total anti-oxidation competence. All rams were castrated for collection of epididymis caput, corpus and cauda at the end of experiment. Expression of Cu-ZnSOD protein in epididymis was detected and located by immunohistochemistry. The mean optical density was analyzed with Image Pro Plus 7.0 software.【Result】The results showed that there was no significant difference in ejaculate volume between the treatment groups and control group (P>0.05). Compared with the control group, the groups with supplementation of 50 mg·kg-1 or 100 mg·kg-1 nano-zinc had higher sperm concentration, sperm motility and membrane integrity, and less percentage of abnormal sperm. The group with supplementation of 150 mg·kg-1 had higher membrane integrity than control group, while it had no significant difference in sperm concentration, sperm motility with control group. The rams in groups with supplementation of 50 mg·kg-1 or 100 mg·kg-1 nano-zinc also had higher activities of Cu-ZnSOD, GPxs and total anti-oxidation competence, and less MDA concentration in seminal plasma. The rams in group with supplementation of 150 mg·kg-1 had higher total anti-oxidation competence than the control group, while they had no significant difference in activities of Cu-ZnSOD, GPxs and MDA content with rams in the control group. The result of immunohistochemistry showed the positive signals were detected in pseudostratified columnar epithelium in epididymis caput and corpus, and simple columnar epithelium in epididymis cauda. The order of expression of Cu-ZnSOD protein in epididymis from rams in the treatment group was corpus>caput>cauda, while those in the control group was caput>corpus>cauda. The mean optical density of Cu-ZnSOD was higher in the group with supplementation of 50 mg·kg-1 or 100 mg·kg-1 nano-zinc in epididymis caput and corpus. The activity of Cu-ZnSOD in seminal plasma had a positive correlation with sperm concentration, sperm motility and membrane integrity, and a negative correlation with percentage of abnormal sperm.【Conclusion】The optimal supplementation of 50 mg·kg-1 or 100 mg·kg-1 nano-zinc in diet could improve semen quality and seminal plasma anti-oxidase activities, and the expression of Cu-ZnSOD in epididymis of rams. The activity of Cu-ZnSOD in seminal plasma could be regarded as index of evaluation of semen quality in sheep production. The further researchs need to be done in molecular mechanism of trace element zinc regulation on semen quality.

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    Cited: Baidu(7)
    Melanin Synthesis of Alpaca Melanocytes Regulated by miR-663 Through Targeting TGF-β1
    JIA Xiao-yun, JIN Lei-hao, MIAO Lian-juan, DING Na, FAN Rui-wen, DONG Chang-sheng
    Scientia Agricultura Sinica    2015, 48 (1): 165-173.   DOI: 10.3864/j.issn.0578-1752.2015.01.16
    Abstract374)   HTML2)    PDF (1154KB)(591)       Save
    【Objective】 The objective of the present study is to identify the target genes of miR-663 and investigate the role of miR-663 in melanin synthesis in alpaca melanocytes.【Method】 The potential targets and binding sites of TGF-β1 were predicted and analyzed by Targetscan, RNAhybrid and RNA22. The similarity of 3′UTR of TGF-β1 sequences from various species was analyzed by DNAMAN. The dual-luciferase construct of pmirGLO-TGF-β1-3′UTR was created by inserting partial TGF-β1 3′UTR into the pmirGLO vector by Sacand XbaⅠ restriction sites. The regulation of TGF-β1 by miR-663 was validated by co-transfecting pmirGLO-TGF-β1-3′UTR construct with miR-663 mimic into 293T cells. The over-expression of miR-663 was achieved by transfecting melanocytes with miR-663 mimic. The mRNA and protein levels of TGF-β1, Smad3, Smad4, Smad7 and β-catenin in melanocytes transfected with miR-663 mimic were analyzed by qRT-PCR or Western blotting, respectively. The effects of miR-663 on melanin synthesis were evaluated by measuring the melanin content of the cells.【Result】There are 68 potential targets for miR-663 predicted by bioinformatics, including 74 conserved binding sites and 44 less conserved binding sites. DNAMAN analysis showed that all 3′UTR sequences of TGF-β1 from analyzed species are highly conserved and enriched potential target sites. One of the potential targets of miR-663 is TGF-β1, which is involved in the development of hair follicle as well as melanin pigmentation. The alpaca 3′UTR sequence of TGF-β1 contains three miR-663 potential binding sites. To confirm the regulation of TGF-β1 by miR-663 through its 3′UTR, a dual-luciferase reporter vector pmirGLO-TGF-β1-3′UTR was successfully constructed and co-transfected into 293T cells with miR-663 mimic. The luciferase assay experiments showed that the luciferase activity was 31.01% lower in cells co-transfected with pmirGLO-TGF-β1-3′UTR and miR-663 mimic than that in control cells, suggesting TGF-β1 is a direct target of miR-663. When miR-663 mimic was transfected into melanocytes, the mRNA level of miR-663 increased to 345% but those of TGF-β1, β-catenin and Smad4 were reduced by 89%, 41%, and 34%, respectively. The protein level of TGF-β1 was reduced by 21%. The contents of melanin were significantly reduced by 42%.【Conclusion】TGF-β1 is a direct target gene of miR-663. Overexpression of miR-663 led to the decreased expression of TGF-β1 both at protein and mRNA levels. The miR-663 may influence/affect synthesis through regulation of TGF-β1 directly as well as TGF-β/Smad and Wnt signal pathways indirectly in skin melanocytes of Alpaca.
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    The Biological Identification of FABP4 Transgenic Cattle
    ZHAO Jing-xian, LI Juan, YAN Xing-rong, NI He-min, CHEN Yan, ZHANG Lu-pei, GAO Hui-jiang, XU Shang-zhong, LI Jun-ya1, GAO Xue
    Scientia Agricultura Sinica    2014, 47 (24): 4895-4903.   DOI: 10.3864/j.issn.0578-1752.2014.24.013
    Abstract363)   HTML1)    PDF (1667KB)(540)       Save
    【Objective】 The objective of this study is to identify 3 transgenic beifers cattle by testing mRNA and protein expression of exogenous FABP4 gene, so as to provide a scientific basis for the further study of the safety of genetically modified cattle. 【Method】 The bovine FABP4 gene was modified, which was obtained from GenBank (accession No. NM_174314), to form an exogenous FABP4 gene by the degeneracy of synonymous codon. Then the pEGFP-C1-FABP4 eukaryotic expression vector was constructed and transferred into recipient cows by somatic cell nuclear transfer and embryo transfer technology. The No.1 and 2 transgenic animals were born on July 19, 2012 and No.3 individual was on August 1, 2012, but No.1 individual was dead on July 20, 2012. The tissue samples of No.1 transgenic individual and blood samples of No.2&3 transgenic individual and Bos Taurus at 8, 10, 12 and, 14 months were collected for identification. The RT-PCR was used to detect whether the transgenic individuals are positive or not. The fluorescence quantitative PCR was used to detect the mRNA expression of the exogenous FABP4 gene in different tissues of No.1 transgenic individual. Expression trend of exogenous, endogenous FABP4 gene of No.2&3 and Bos taurus individuals at 8, 10, 12 and, 14 months and difference of mRNA expression of FABP4 gene between transgenic and non-transgenic animals were analyzed. Moreover, the protein expression of exogenous FABP4 gene in transgenic cattle was detected by Western Blot.ResultThe results showed that three transgenic animals were all positive, because the production of exogenous FABP4 gene, which is 205 bp size, was detected in transgenic cattle, but pregnant cow and negative control didn′t have. The mRNA expressions of exogenous FABP4 gene were observed in different tissues of No.1 transgenic individual. The expressions of this gene in fat tissue and longissimus were significantly higher than other tissues, and it was the lowest in lung. With months changing, exogenous FABP4 gene expression of No.3 transgenic individual was higher than No.2′s and it presented an earlier increase and later decrease trend. The exogenous FABP4 gene expression level of No.2 individual rose from 8th to 12th month, and then it began to decline slightly. However, expression level of No.3 individual rose from 8th to 10th month, and then decreased gradually. The expression levels of endogenous FABP4 gene in transgenic and non-transgenic cattle displayed that it gradually declined by month in non-transgenic animals, but increased in transgenic one, and the expression level of transgenic cattle was lower than non-transgenic animal. It showed that the exogenous FABP4 gene had inhibition effect on the expression of endogenous FABP4 gene. The superposition expressions of exogenous and endogenous FABP4 gene demonstrated that entire FABP4 expression of transgenic individual was greatly higher than non-transgenic one. The result of protein expression was in close agreement with the result of mRNA expression. In addition to the lung, other tissues of No.1 individual all expressed FABP4 protein, and the expression of the longissimus was the highest. The protein expression level of No.3 individual was higher than No.2.【Conclusion】Three FABP4 transgenic animals were successfully acquired and exogenous FABP4 gene could be expressed normally in the transgenic animal′s body. The FABP4 expression of transgenic individual increased greatly than non-transgenic one. The exogenous FABP4 gene inhibits the expression of endogenous FABP4 gene in transgenic individual.
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    Cited: Baidu(1)
    Effects of Artemisinine on the Rumen Fermentation and Microbial Nitrogen Recycling Rate in Goats
    WANG Hong-rong, QIN Tao, WANG Chao
    Scientia Agricultura Sinica    2014, 47 (24): 4904-4914.   DOI: 10.3864/j.issn.0578-1752.2014.24.014
    Abstract388)   HTML8)    PDF (406KB)(466)       Save
    【Objective】 Artemisinine is a kind of plant(Artemisia apiacea) extracts which is a potential feed additive to manipulate the rumen fermentation pattern in the future. The main purpose of this study was to search a novel rumen regulating agent and reveal its consequences of manipulation in the rumen. Experiments were conducted to investigate the effect of artemisinine on the rumen fermentation parameters and nitrogen recycling rate between protozoa and bacteria in the rumen. 【Method】 Four Xuhui goats fitted with rumen cannula were used in the experiment. Animals were randomly assigned into four dietary treatments (adding 0, 0.2%, 0.4% and 0.6% artemisinine in diets, respectively) in a 4 × 4 Latin square design. All animals were kept in individual pen and fed with ad libitum diet consisted of grinded corn, soybean meal and Chinese ryegrass hay mainly and had free access to clean drinking water. Protozoa engulf bacteria rate and ruminal nitrogen recycling were determined by a novel fluorescence-labeled bacteria technique dyed with 5-([4,6-Dichlorotriazin-2-yl]amino) fluorescein hydrochloride.【Results】The results showed that the extent of pH ranged from 6.85 to 7.16. The addition of artemisinine could decrease the concentration of NH3-N concentration effectively; the consequences of ammonia concentration for 0.4% and 0.6% artemisinine treatments were significantly lower than that of the control group. It was shown that acetate, propionate as well as total volatile fatty acid (TVFA) concentration in the rumen were dramatically improved by adding artemisinine, and the ratio of acetate to propionate for artemisinine treatment groups was significantly lower than that of the control group(P<0.05). With the increasing addition of artemisinine, bacterial protein yields tended to increase, and bacterial protein yield of the highest 0.6% group was very significantly higher than that of the control group(P<0.01). Additionally, compared to the control group, no significant influence was detected in ruminal peptide nitrogen concentration. Additionally, artemisinine led to an increase of bacterial density, and a decrease of protozoal density. It was further observed that, the engulfing rate of rumen protozoa on bacteria was altered by artemisinine, and they were, 320.11cells/(cell h), 313.94cells/(cell h), 305.00cells/(cell h), 278.14cells/(cell h)for A, B, C, D, respectively. 【Conclusion】 It was concluded that artemisinine was more effective at 0.6% DM proportion of diet. It could decrease ammonia concentration and influence on ruminal fermentation pattern accordingly. The addition of artemisinine in diet could drop the total protozoal density and alter protozoa genus profile in the rumen, where the percentage of Entodinium was decreased, and that of Diplodinium and Isotricha were increased. With adding at 0.6% artemisinine, the rate of microbial protein recycling was increased by decreasing engulfing bacteria rate of protozoa in the rumen of goats and dietary protein efficiency was improved potentially.
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    Cited: Baidu(5)
    Effects of CZKJKL on Stress-Related Factors of Nerve - Endocrine - Immune System of Rats After Immunological Induction
    SUN Yao-gui, CHENG Jia, LI Hong-quan, WANG Jun-dong
    Scientia Agricultura Sinica    2014, 47 (23): 4718-4725.   DOI: 10.3864/j.issn.0578-1752.2014.23.016
    Abstract378)   HTML3)    PDF (384KB)(625)       Save
    【Objective】“Piglet early weaning stress syndrome” brought huge economic losses to the pig industry, immunological stress is one of the important factors. Chaizhukangji particles (CZKJKL) were prepared through modern pharmaceutical technology, total saikosaponins (TSS, the composition of the main therapeutic effects of Bupleuri Radix), polysaccharide of Atractyles macroceohala koidz (PAM, the composition of the main therapeutic effects of Atractylodis Macrocephalae Rhizoma), total glycosides of paeony (TGP, the composition of the main therapeutic effects of Paeoniae Radix Alba)were also selected, and proved that the preparation has a significant mitigation effect on stress of early weaned piglets and stress of early weaning piglets after immunological stress by lipopolysaccharide. The experiment was conducted to investigate the effects of CZKJKL on stress-related neurotransmitters, cytokines and hormones in HPA axis of nerve-endocrine-immune system of early weanling rats after LPS immunological stress, to further explore the anti-stress effects of CZKJKL.【Method】Total 72 early weanling wistar rats at the age of 18 days were randomly divided into 3 treatments of 24 rats each, with 6 replicates in each group and 4 rats in each replicate. The 3 treatments were group Ⅰ(control group), group Ⅱ (LPS group) and group Ⅲ (LPS+CZKJKL group). In the period of 18-24 days of age, the rats in group Ⅲ were fed with 5 000 mg·kg-1BW CZKJKL daily by gavage, the rats in group Ⅰ and group Ⅱ were fed with equivalent amount of sterile saline. At the age of 21 days, the rats in group Ⅱ and group Ⅲ were injected intraperitoneally with 4 mg·kg-1BW of LPS, the rats in group Ⅰ were injected with equivalent amount of sterile saline. Blood and tissue samples were obtained at the time of 6 h after injecting LPS. The following indicators were determinated: The contents of NE,5-HT of Hippocampus; the contents of β-EP, CRF of Hypothalamus; the contents or activities of β-EP, ACTH, COR, IL-1β, IL-6, TNF-α, NO, and iNOS of serum.【Result】Compared with groupⅠ, the contents of NE and 5-HT in hippocampi of groupⅡ were significantly increased (P<0.01), the levels of β-EP in hypothalamus were significantly increased (P<0.01), the contents of β-EP, IL-1β, IL-6, TNF-α, NO, ACTH, and COR in serum were significantly increased (P<0.01), and the activity of iNOS in serum was significantly enhanced (P<0.01). Compared with groupⅡ, the contents of NE and 5-HT in hippocampi of group Ⅲ were significantly decreased (P<0.01), the levels of β-EP in hypothalamus were significantly decreased (P<0.01), the contents of β-EP, IL-1β, IL-6, TNF-α, NO, ACTH, and COR in serum were significantly decreased (P<0.01), and the activity of iNOS in serum was significantly reduced (P<0.01).【Conclusion】The results indicate that CZKJKL can play a role of anti-immunological stress by efficiently alleviating the disorder of stress-related neurotransmitters, cytokines and hormones in HPA axis of nerve-endocrine-immune system of early weanling rats after LPS immunological stress.
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    Development of an ELISA Method for Multi-Residue Detecting of Fluoroquinolones
    LI Xin-peng, JIANG Jin-qing, QIAN Ai-dong, WANG Zi-liang, FAN Guo-ying, SHAN Xiao-feng, KANG Yuan-huan, LI Yi
    Scientia Agricultura Sinica    2014, 47 (23): 4726-2735.   DOI: 10.3864/j.issn.0578-1752.2014.23.017
    Abstract483)   HTML1)    PDF (891KB)(516)       Save
    【Objective】Fluoroquinolones(FQs) are widely used in veterinary medicine for the treatment and prevention of bacterial infection. With the increasing use, FQs residues in animal edible tissues have caused serious public health problems and attracted serious attention by research scholars all over the world. The objective of this study wasto produce class-specific monoclonal antibodies (mAbs) against fluoroquinolones (FQs), establish competitive indirect enzyme linked immunesorbent assay (icELISA), and in order to lay a foundation for detection of multi-residue FQs in animal foods.【Method】 The aminobutyric acid was introduced to carboxyl of ciprofloxacin as hapten (CPFX-A) and was proved by (+) ESI-MS spectrum, which was conjugated to bovine serum albumin (BSA) as immunogen (CPFX-A-BSA) by the N,N'-Dicyclohexylcarbodiimide (DDC) method, and to ovalbumin (OVA) as coating antigen(CPFX-A-OVA) by mixed anhydride method, respectively, which were then identified by infrared ray (IR) and ultraviolet (UV). Balb/c mice immunized by CPFX-A-BSA were selected for cell fusion, which was identified by ELISA and icELISA. Under the effect of PEG-1500, NS0 cells and spleen cells were fused at the ratio of 1﹕5. Hybridoma lines that secrete mAb against FQs were selected and their immunological traits were characterized by titer, subtype, sensitivity and cross reaction rate, which ascites were carried out by injecting 108 hybridoma cells in vivo, and icELISA standard curve was established and optimized. High titer, class-specific monoclonal antibody was used to detect 10 FQs in chicken for calculating recovery rate and variation coefficient. The data were also compared with that of HPLC, and SPSS 17.0 software was used to conduct the significant difference analysis.【Result】 The hapten and artificial antigen were synthesized successfully and antiserum titers of three mice were higher than 1﹕1.28×104, in which the titer and IC50 of No.2 mouse were1﹕2.56×104 and 12.92 ng·mL-1. Three hybridoma cell lines named 2H5, 3D11, and 4F4 were screened after 4 times subclone, which titers were 1﹕1 600, 1﹕1 600, and 1﹕800 in supernatants and 1﹕1.6×106, 1﹕8.2×105, and 1﹕8.2×105 in ascites, respectively. The icELISA procedure was optimized at a concentration of CPFX- A-OVA for 1 μg·mL-1 at 4℃ package overnight by 5% negative serum of pig, monoclonal antibody and GaMIgG-HRP were diluted 1﹕40 000 and 1﹕8 000, respectively. Under the reaction temperature of 37℃, reaction time of standard substance and monoclonal antibody was 15 min, and 25 min after adding GaMIgG-HRP, also 10 min for termination reaction. Cell line named 2H5 showed a good sensitivity and class-specific toward 10 FQs, the linear regression equation was y= -28.022x+56.219,R2=0.9 782,with an IC50 value of 1.67 ng·mL-1 for ciprofloxacin, 1.82 ng·mL-1 for norfloxacin, 1.97 ng·mL-1 for pefloxacin, 1.54 ng·mL-1 for enrofloxacin, 2.79 ng·mL-1 for danofloxacin, 3.38 ng·mL-1 for lomefloxacin, 5.50 ng·mL-1 for ofloxacin, 4.40 ng·mL-1 for marbofloxacin, 11.76 ng·mL-1 for sarafloxacin, 13.60 ng·mL-1 for difloxacin, and the lowest detectable limits (LODs) of 0.09 ng·mL-1-0.64 ng·mL-1 and cross- reactivity (CR) of 12.3%-108.4%, no cross-reactivity to other compounds was found. The recovery ranges of 10 FQs spiked in chicken using icELISA were 80.5%-91.8 %, 85.1%-95.7% for HPLC, both of the coefficient variations (CVs) were below 10.0%, and no significant difference (P>0.05) was observed.【Conclusion】The high-sensitivity and class-specific mAb against FQs was prepared, which laid a solid foundation for FQs multi-residue detection.
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    Cited: Baidu(2)
    The Strategy of Parameter Optimization of Bayesian Methods for Genomic Selection in Livestock
    ZHU Bo, WANG Yan-hui, NIU Hong, CHEN Yan, ZHANG Lu-pei, GAO Hui-jiang, GAO Xue, LI Jun-ya, SUN Shao-hua
    Scientia Agricultura Sinica    2014, 47 (22): 4495-4505.   DOI: 10.3864/j.issn.0578-1752.2014.22.015
    Abstract460)   HTML8)    PDF (584KB)(779)       Save
    Variety selection in livestock breeding occupies an important position. Genomic selection, as a novel technology in livestock breeding, has raised considerable concern. It can shorten the generation interval, speed up the genetic progress, and it can select the candidate individuals as breeding stock without phenotypic data. In 2001, Meuviwisen proposed the concept of genomic selection, which was first applied in dairy cattle. Until August 2014, there were 34 member countries of Interbull organization that had applicated genomic selection in their national dairy cattle breeding group. With the popularization and continuous promotion of genomic selection, some problems of the accuracy of genomic estimated breeding value need to be solved. Various methods of genomic selection have been proposed and more efficient models are being developed. So it has great practical significance to exploit better models and algorithm to improve the accuracy of genomic estimated breeding value. So far, there were 17 Bayesian methods that have been successively proposed. This thesis briefly introduced the classical BayesA and BayesB methods for genomic selection. BayesA assumed that all loca have effect, while BayesB supposed that a small part of locus have effect, and the percentage was extremely small. Therefore, BayesA and BayesB had different models and algorithms. After Meuviwisen proposed classic Bayesian methods, other methods were like mushrooms springing up. New Bayesian methods were based on the classical Bayesian methods, which was optimized by improving the hypothetical model and algorithm. For example, BayesC method, which was based on BayesB, optimized the π value in the model. BayesCπ and BayesDπ were the improvement of BayesC, and these two approaches assumed that marker effect variance of each locus had the same value, whereas BayesC assumed that its marker effect variance of each locus was different. BayesDπ, which was based on BayesCπ, optimized the scale parameter of inverse chi-square distribution. Bayes Lasso had the same idea with BayesA. However, its marker effects were assumed to be another distribution for Laplace, so its posterior distributions of marker effects were also changed. BayesRS method assumed that the variances of marker effect were allocated in different percentage of total genetic variance. In order to find proper hypothesis model and parameters, other Bayesian methods were also based on predecessors' research through changing the prior assumption and improving parameters of the model. At present, commonly used methods for genomic selection are classic Bayesian methods and BayesCπ, which have stabile calculation results and high accuracy of genomic estimated breeding value. In the three Bayesian algorithms, the accuracy is generally arranged into BayesB > BayesCπ > BayesA, but accuracy of genomic estimated breeding value of some traits is not the case. Compared with classical Bayesian method, parameter optimization can improve the accuracy of genome estimated breeding value to some extent. In a word, on the basis of the classical Bayesian method,for the purpose of improving the accuracy of genomic estimated breeding value, the extension of bayesian methods and its parameters optimization strategy seeked for the optimal model and parameters optimization through biological genetic algorithm combined with actual population situation. They enriched and expanded the genomic selection algorithm, and can make the genomic breeding value more reference significance. As the animal breeding process is far from the foreign breeding process in China, genomic selection can cultivate new breed, enrich the genetic resources of China and accelerate the pace of livestock and poultry breeding process. Meanwhile, the algorithm study of genomic slection and its application in China was introduced. In face of the advantages of genomic selection, whole genomic selection breeding technology is imperative. Furthermore, the main problems in current researches and the key points in future studies were also proposed, in hope of providing reference for obtaining more reliable and faster algorithm of genomic selection.
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    Effect of Lactobacillus reuteri I5007 on Intestinal Morphology, Disaccharidase Activity and Tight Junction Protein Expression in Newborn Piglets
    YANG Feng-juan, ZENG Xiang-fang, QIAO Shi-yan
    Scientia Agricultura Sinica    2014, 47 (22): 4506-4515.   DOI: 10.3864/j.issn.0578-1752.2014.22.016
    Abstract392)   HTML5)    PDF (782KB)(741)       Save
    【Objective】 The purpose of this experiment was to investigate the effects of Lactobacillus reuteri I5007 on intestinal villi maturation and intestinal barrier function in newborn piglets. 【Method】 Nine litters of 1-day-old Duroc×Landrace×Yorkshire newborn piglets and eight healthy piglets with similar body weight in each litter were selected. Piglets were randomly divided into three groups with three litters per group. The three groups were control group (oral administrated with 0.1% sterile peptone solution), the first 4 days dosed group (1-4 d, daily orally administrated with 1.2×1010 CFU of L. reuteri I5007 per piglet), and the every 4-day dosed group (1, 5, 9, 13, 17, and 21 d, daily orally administrated with 1.2×1010 CFU of L. reuteri I5007 per piglet). The trial was conducted for 21 d. During the trial, each litter of piglets were fed by a sow, respectively. Four healthy piglets closest to the average body weight from each group were fasting weighted, anesthetized and then slaughtered on d 7, 14 and 21. The small intestine was carefully dissected from the mesentery and segments (the same location in all piglets), including duodenum, jejunum and ileum, were collected. Growth performance, intestinal morphology, disaccharidase activity and expression of tight junction (TJ) proteins were measured. 【Result】 Oral administration with L. reuteri I5007 had no significant effect (P>0.05) on growth performance, but had impacts on piglet intestinal morphology at different time points. On d 7, duodenal villus height, villus height/crypt depth and goblet cells density and ileal goblet cells density of piglets in every 4-day dosed group were increased significantly (P<0.05) compared with those of control piglets. Piglets in first 4 days dosed group had distinct higher (P<0.05) duodenal villus height/crypt depth and goblet cells density and ileal goblet cells density than control piglets, but not villus height/crypt depth (P>0.05). On d 14, administration with L. reuteri I5007 in every 4-day dosed group robustly increased (P<0.05) duodenal villus height/crypt depth and ileal goblet cells density. Ileal goblet cells density of piglets in first 4 days dosed group was significant higher (P<0.05) than that of control piglets, but lower (P<0.05) than that of piglets in every 4-day dosed group. On d 21, piglets of every 4-day dosed group had an increased goblet cells density (P<0.05) in both duodenum and ileum. Compared with control group, administration of L. reuteri I5007 in first 4 days dosed group also augmented (P<0.05) ileal goblet cells density, but reduced (P<0.05) duodenal villus height/crypt depth. In addition, piglets in first 4 days dosed group had lower (P<0.05) duodenal villus height/crypt depth and goblet cells density than those of piglets in every 4-day dosed group. For disaccharidase activity, piglets in every 4-day dosed group had higher (P<0.05) both sucrase and maltase activities in jejunum than those of control piglets. Only jejunal maltase activity of first 4 days dosed piglets was enhanced (P<0.05) compared with control piglets, while jejunal sucrase and maltase activities were significantly lower (P<0.05) than those of every 4-day dosed. Furthermore, piglets in every 4-day dosed group had significant higher expression of Claudin-1, Occludin and ZO-1 in both jejunal and ileal epithelial cells (P<0.05). Administration of L. reuteri I5007 in first 4 days dosed group also promoted (P<0.05) expression of jejunum epithelial Claudin-1 and ZO-1 and ileum epithelial Claudin-1. However, the level of jejunum epithelial Occludin of first 4 days dosed piglets was obviously lower (P<0.05) than that of every 4-day dosed piglets. Besides, the levels of Claudin-1 (P = 0.1) and ZO-1 (P = 0.1) in ileum epithelium of first 4 days dosed piglets had a trend of decrease compared with every 4-day dosed piglets. 【Conclusion】 Every 4-day administration of L. reuteri I5007 in newborn piglets can promote the maturation of intestinal villi, improve jejunal disaccharidase activity, and increase expression of TJ protein of intestinal epithelial cells. Thus, L. reuteri I5007 may play a role in protecting or augmenting intestinal barrier function in newborn piglets.
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    Influence of Ammonia Concentration on Growth Performance and Meat Quality of Broilers
    LI Cong, LU Qing-ping, TANG Xiang-fang, ZHANG Ji-ze, DING Ze-min, ZHANG Hong-fu
    Scientia Agricultura Sinica    2014, 47 (22): 4516-4523.   DOI: 10.3864/j.issn.0578-1752.2014.22.017
    Abstract501)   HTML8)    PDF (338KB)(541)       Save
    【Objective】This study was conducted to investigate the influence of ammonia concentration on growth performance and meat quality of broilers.【Method】The experiment was conducted in four chambers of the State Key Laboratory of Animal Nutrition. Three hundred and twenty 21-day-old Arbor Acres broilers with similar initial body weight were divided randomly into 4 groups for different treatments, with 4 replicates in each group and 20 broilers for each replicate. Each treatment of broilers was placed in a separated, environmentally controlled chamber, and ammonia concentrations in the four chambers were metered continuously to maintain at (3±3), (25±3), (50±3) and (75±3) µL·L-1, respectively. The broilers were net-rearing and provided ad libitum access to water and diets. The experiment lasted for three weeks. Broilers’ feed intake and health status were recorded, broilers were weighed according to replicate to get the growth performance at the age of 32 and 42 days, at the same time, three broilers were selected and slaughtered from each replicate to measure slaughter performance and meat quality.【Result】 During the age from 22 to 32 days, when ammonia concentration was at 50, 75µL·L-1, ADG and ADFI of broilers were significantly decreased compared with the control group(P<0.05); ammonia concentration did not significantly affect F/G of broilers(P>0.05). During this period, ammonia concentration did not significantly affect the dressing percentage, eviscerated carcass percentage and half-eviscerated carcass percentage of broilers (P>0.05). During the age from 33 to 42 days, compared with the control group, ADFI of broilers was significantly decreased when ammonia concentration was 25-75 µL·L-1 (P<0.05); ammonia concentration did not significantly affect ADG and F/G of broilers (P>0.05). During the whole experiment period, compared with the control groups, ADG and ADFI of broilers in all three experiment group had a significantly decrease(P<0.05), F/G of broilers was significant increased(P<0.05); abdominal fat percentage of broilers had a trend of rising with the increase of ammonia concentration. Compared with the control group, when ammonia concentration was at 75µL·L-1, abdominal fat percentage of broilers was significantly improved(P<0.05); ammonia concentration did not significantly affect the breast meat pH(after 24 h),color L* value, color a* value and color b* value (P>0.05). Compared with the control groups, drip loss rate of breast meat in all three experiment group had a significant increase (P<0.05). 【Conclusion】These results indicate that ammonia concentration at 25µL·L-1 in chamber can inhibit the growth performance and meat quality of broilers, and with the increase of ammonia concentration, the influence become worse, abdominal fat percentage and drip loss rate of breast meat of broilers can be significantly improved when they are under the high ammonia concentration conditions, so, by integrating the foregoing study outcomes, the author suggested that ammonia concentration in chamber should be controlled within 25µL·L-1.
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    Cited: Baidu(5)
    Detection and Analysis of Resistance Genes in Quinolone-Resistant Escherichia coli Isolates from Different Livestocks in Xinjiang
    NAN Hai-chen, DI Li-na, XIA Li-ning
    Scientia Agricultura Sinica    2014, 47 (20): 4096-4108.   DOI: 10.3864/j.issn.0578-1752.2014.20.018
    Abstract355)   HTML1)    PDF (2790KB)(459)       Save
    【Objective】The objective of this study is to investigate the prevalence and characteristics of plasmid mediated quinolone resistance (PMQR) determinents in quinolone-resistant E. coli from different animal origins in Xinjiang, and their coexistence with the major β-lactamases and 16s rRNA methylation enzyme genes. 【Method】 Polymerase chain reaction (PCR) was used to detect PMQR (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB, aac(6’)-Ib-cr), b-lactamase gene (blaTEM, blaCTX-M, blaSHV, blaKPC, blaCMY-2, blaLAP-1) and 16S rRNA (armA, rmtB) genes in 79 strains from pigs, 8 strains from cattle and 96 strains from sheep quinolone-resistant (ciprofloxacin, norfloxacin, enrofloxacin) E. coli. The positive strains were performed by using DNA sequencing to determine the purpose of the belt.Minimal inhibitory concentrations (MICs) of the related antibiotics to these isolates carrying the β-lactamase and 16S rRNA methylase genes,were tested and the relationship between the genotype and resistant phenotype was analyzed.【Result】The results showed that the qnrS (6.33%, 5/79), aac(6')-Ib-cr (5.06%, 4/79), oqxA (44.3%, 35/79), oqxB (50.6%, 40/79) were main PMQR determinants, blaTEM (100%, 79/79) was main β-lactamase genes and rmtB (3.80%, 3/79) was main16S rRNA methylase genes in E. coli from pigs; the qnrS (12.50%, 1/8), oqxA (12.5%, 1/8), oqxB (12.5%,1/8), aac(6')-Ib-cr (12.50%, 1/8) and qepA (12.50%, 1/8) were main PMQR determinants, the blaTEM (100%, 8/8) and blaSHV (12.50%, 1/8) were main β-lactamase genes in E. coli from cattle; the qnrS (6.25%, 6/96), oqxA (33.3%, 32/96) oqxB (38.5%, 37/96) and aac(6')-Ib-cr (22.91%, 22/96) were main PMQR determinants, the blaTEM (100%, 96/96) and blaCTX-M (2.08%, 2/96) were main β-lactamase genes and the rmtB (2.08%, 2/96) was main16S rRNA methylase genes in E. coli from sheep. The qnrA, qnrB, qnrC, qnrD, blaKPC, blaCMY-2 and blaLAP-1 genes were not detected in any of the isolates. The co-harboring of resistant genes was common among these E.coli from different animals, and the resistant phenotype of E.coli from different animal sources carrying β-lactam enzyme and 16S rRNA methylase genes has some correlation.【Conclusion】The PMQR determinants were existed in E. coli from different animals in Xinjiang, and they coexist with the main b-lactamase and/or 16s rRNA methylation enzyme genes. In addition, the PMQR determinants and β-lactamase genes were firstly detected in the E. coli from sheep.
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    Effects of HMGCS1 Gene on Skeletal Muscle Growth and Development and Regulated by miR-18a/b of Pig
    SUN Wei, TANG Zhong-Lin, TAN Lin, LEI Chu-Chao, LI Kui
    Scientia Agricultura Sinica    2013, 46 (12): 2543-2549.   DOI: 10.3864/j.issn.0578-1752.2013.12.015
    Abstract487)      PDF (527KB)(869)       Save
    【Objective】The purpose of the present research was to explore whether HMGCS1 impacts the skeletal muscle growth and development of pig and validate its target gene.【Method】In this study,the expression of HMGCS1 in different tissues and the expression change during Longissimus dorsi muscle development (28 stages) was investigated by real-time qPCR.【Result】The result showed that the HMGCS1 gene was strongly expressed in lung of adult pig, and the expression level of HMGCS1 gene in embryonic period 33 d and 65 d were higher than those in other periods in Tongcheng pigs, and lower expression at postnatal 20 d, 30 d, 40 d and 60 d. The luciferase activity was lower in miR-18a/b minics than in the control.【Conclusion】The results of study indicated that HMGCS1 regulates skeletal muscle growth and development by miR-18a/b, and is a potential candidate target gene of meat production traits.
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    Cited: Baidu(4)
    Study on Network Database Platform of Hill-Black Swine Breeding Data
    YANG Liang, XIONG Ben-Hai, 吕Jian-Qiang , CHANG Le, SUN Xiu-Kun
    Scientia Agricultura Sinica    2013, 46 (12): 2550-2557.   DOI: 10.3864/j.issn.0578-1752.2013.12.016
    Abstract391)      PDF (765KB)(786)       Save
    【Objective】 The aim of the study is to preserve resource data and to increase efficiency of breeding and genetic improvement of Hill-Black breeding pigs. 【Method】 Based on the North Hill Black pigs production process from progenitor to parents and to its commodity pigs, the pigs in breeding farm were divided into breeding boar, mothball boar, breeding sow, mothball sow, grew boar, grew sow, finishing boar, finishing sow and so on, and the standards and specifications of essential information of breeding pigs were designed. 【Result】 The study constructed pig breeding data management and analysis platform by using. Net and SQL Server 2008 network database technology, and realized remote network database management of essential breeding process information or data, and it could analyze some derived indexes including calving interval, low production sow and high production sow so on on-line, and it also realized intelligent alert of various production events such as mating, parturition, weaning, transfer group and culling.【Conclusion】 Regardless of how complicated in types, quantity and generation reproduction of the Hill-Black breeding pigs, it is possible to do a integrated data management, analysis and intelligent decision-making by developing a network computing system in terms of a series of databases, business logics and corresponding models.
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    Cited: Baidu(4)
    Polymorphism of Chicken Prolactin Receptor Gene and It’s Association with Broodiness and Egg Reproduction Traits
    DU Xiao-Hui, LI Cong-Yan, LIU Jun-Ying, NIU Wei-He, ZHANG Xi-Quan
    Scientia Agricultura Sinica    2013, 46 (12): 2558-2565.   DOI: 10.3864/j.issn.0578-1752.2013.12.017
    Abstract474)      PDF (632KB)(813)       Save
    【Objective】 In order to reveal the relationship between bloodiness and egg reproduction of Chinese indigenous chicken and prolactin receptor (PRLR) gene,which closely related to reproductive traits among amimals.【Method】Using the PCR-SSCP method and 576 Ningdu Yellow chicken, the experiment studied the polymorphism of PRLR gene and its association with nesting and egg production traits.【Result】The results confirmed previous studies that SNP1 (T10862C, exon 3) and SNP2 (T25670C, exon 6) had no significant association with broody traits (P>0.05). The effect of SNP2 on egg production of 300 d (EN-300 d) was significantly (P<0.05). The new SNP3 (G30716A, intron 8) significantly influenced the age at first egg (AFG) (P<0.05). The novel SNP4 (A31900G, exon 10) had an extremely remarkable (P<0.01) impact on the nesting rate, significant (P<0.05) impact on the total broody days and EN-300 d. The differences among different haplotypes constructed by the four SNPs, in AFG reached an extremely significant level (P<0.01), in other traits were no significant differences (P>0.05). 【Conclusion】 PRLR gene polymorphism was associated with broodiness and egg production traits, especially the effect of SNP4.
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    Studies on Native Grass Silage of Different Plant Communities in Meadow Steppe
    WANG Hong-Mei, SUN Qi-Zhong, HUA Mei
    Scientia Agricultura Sinica    2013, 46 (12): 2566-2575.   DOI: 10.3864/j.issn.0578-1752.2013.12.018
    Abstract408)      PDF (608KB)(655)       Save
    【Objective】 The silage quality and feasibility of sixteen plant communities in meadow steppe were studied in this paper. 【Method】 Fresh grass from 16 sites in meadow steppe were loaded after cutting into polyethylene bag to be vacuumed and sealed. After 60 days, the silage fermentation quality(pH value, content of ammonia nitrogen(NH3-N), lactic acid(LA), acetic acid(AA), propionic acid(PA), butyric acid(BA)and total acid(TA)) and chemical composition(Buffering capacity(BC), content of dry matter(DM), crude protein(CP), water-soluble carbohydrate(WSC), neutral detergent fiber(NDF)and acid detergent fiber(ADF)) were analyzed. 【Result】 Many of the raw materials were suited to silage, because of their lower buffering capacity and higher content of dry matter and water-soluble carbohydrate. The contents of dry matter, WSC, NDF and ADF in each silage were lower than them in each raw materials, except in sites 5 and 14. But the change of CP content in silage had no trend compared with raw materials. There were significant differences in pH value, NH3-N/TA, contents of four organic acids and total acid and contents of nutrient compositions (DM, CP, WSC, NDF and ADF) among 16 silage materials(P<0.05). The silage pH value was from 4.56 to 5.69, with lower value of NH3-N/TA.【Conclusion】 The rating of silage fermentation quality could be divided into optimal(1, 8, 9, 13, 14, 15), good(2, 10, 12), medium (5, 16) and bad(3, 4, 6, 7, 11).
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    Cited: Baidu(1)
    Immunoregulatory Effects of Compound Ammonium Glycyrrhizin Soluble Powder on Liver Injury Induced by Enrofloxacin and LPS in Chickens
    GUO Fan-Xi, LIU Teng-Fei, GENG Zhi-Xia, JIANG Fan, YU Zu-Gong
    Scientia Agricultura Sinica    2013, 46 (12): 2576-2583.   DOI: 10.3864/j.issn.0578-1752.2013.12.019
    Abstract594)      PDF (821KB)(719)       Save
    【Objective】 The present study was aimed at the effects of compound ammonium glycyrrhizin soluble powder (CAG) on proinflammatory cytokines production and T-cell subsets deviation in peripheral blood in chicken liver injury induced by enrofloxacin and lipopolysaccharide crude extract(LPS).【Method】A total of 104 animals were allocated randomly into 5 groups named I- V: I normal group, II liver injury group(model group), III prevention group, IV high dosage treatment group and V low dosage treatment group. Group II- IV were administered with enrofloxacin(100 mg•kg-1) once a day for three days and on the third day administered with LPS (4 mL•kg-1) at the same time. Group III received prior administration of CAG at the dosage of 40 mg•L-1 ( by ammonium glycyrrhizin, the same as follows) in drinking water for three days before establishment of liver injury model. Groups IV and V received CAG at a dosage of 22.5 and 7.5 mg•kg-1 twice a day for three days after LPS treatment. Every eight chickens were sacrificed at 6, 24 and 48 h following post-treatment with LPS and their blood samples were collected for detection.【Result】Compared with group I, the group II levels of serum ALT, AST, TNF-α, IL-1 and IL-6 were significantly higher at 6, 24, 48 h post-treatment with LPS and CD3(+), CD4(+) and CD8(+) T-lymphocytes were significantly lower at 6 and 24 h higher at 48 h post-treatment with LPS. When compared with group II , there existed a significant fall in the levels of serum ALT ,TNF-α, IL-6 at 6 h, AST at 6 h and 24 h and the number of CD3(+), CD4(+) and CD8(+) T-lymphocytes at 48 h in group III. In group IV, the levels of ALT, AST, TNF-α, IL-6 at 6 h and 24 h , IL-1 and CD3(+) , CD4(+) and CD8(+) T-lymphocytes at 48 h decreased significantly and CD3(+) T-lymphocytes at 6 h and 24 h , CD4(+) T-lymphocytes at 24 h increased obviously. In group V, there was a significant fall in CD8(+) T-lymphocytes at 48 h and no obvious other differences were detected. The liver histopathological changes got a better improvement in group IV than others.【Conclusion】The novel liver injury induced by enrofloxacin and LPS effects might be related to TNF-α, IL-1 and IL-6 production and CD3(+), CD4(+) and CD8(+) T-cell deviation in peripheral blood. CAG prevents the liver injury through inhibiting TNF-α, IL-1 and IL-6 production and regulating the number of T-cell subsets in peripheral blood by bi-directional regulation and the efficacy might be related to dosage and treatment time.
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    Cited: Baidu(4)
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