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Effect of Expanded Feather Powder on Growth Performance, Slaughter Performance and Serum Biochemical Index of Broiler CHEN ZhiMin,CHANG WenHuan,ZHENG AiJuan,CAI HuiYi,LIU GuoHua Scientia Agricultura Sinica    2022, 55 (13): 2643-2653.   DOI: 10.3864/j.issn.0578-1752.2022.13.013 Abstract （511）   HTML （39）    PDF （2170KB）（161）       Knowledge map    Save 【Objective】This experiment was conducted to investigate the nutritional indexes, pepsin digestibility, ultra micro morphological changes of expanded feather meal (EFM) and the effects of expanded feather meal (EFM) on broiler performance and related blood indexes, so as to determine whether EFM can be used in broiler feed and the appropriate level.【Method】240 1-day-old healthy AA male broilers were randomly divided into 4 groups with 6 replicates in each group and 10 chickens in each replicate. Four treatment groups were fed with EFM 0, 0.5%, 1%, 2% diet (digestible amino acid balance formula) for 42 days. The average daily gain (ADG), average daily feed intake (ADFI) and feed to weight ratio (F/G) of 1-21, 22-42 and 1-42 days old were calculated. At the age of 21 and 42 days, Blood was collected from heart and serum was separated for determination of AST, ALT, ALP, TP, urea, CREA and ammonia. At the age of 42 days, one chicken was randomly selected for slaughter in each replicate, and the slaughter rate, full evisceration rate, half evisceration rate, leg muscle rate, chest muscle rate and abdominal fat rate were determined.【Result】The results showed puffing treatment could make the feather break, the structure became loose, and the contact area with the environment was larger. EFM had no significant effect on the average daily gain and average daily intake (P>0.05) in the whole period (1-42 days), and the addition of 2% EFM significantly reduced the ratio of feed consumption to weight gain (P<0.05). At the age of 42 days, EFM had no significant effect on the rate of half clean bore, full clean bore, chest muscle, leg muscle and abdominal fat (P>0.05); 2% and 1.5% EFM significantly increased the slaughter rate (P<0.05). At the age of 21 days, EFM had no significant effect on ALT, AST, UREA, CREA and ammonia (P>0.05). Adding 1.5% expanded feather powder significantly increased the TP content (P<0.05). At the age of 42 days, EFM had no significant effect on ALP, AST, ALP, TP and CREA (P>0.05), and significantly reduced urea and blood ammonia (P<0.05). 【Conclusion】 The results showed that expanded feather powder could be added to broiler feed properly, and 2% expanded feather powder could reduce the ratio of feed consumption and weight gain, increase the slaughter rate, and reduce the levels of serum urea nitrogen and blood ammonia. Table and Figures | Reference | Related Articles | Metrics

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Effects of lncFAM200B on the Lipid Deposition in Intramuscular Preadipocytes of Yak RAN HongBiao,ZHAO LiLing,WANG Hui,CHAI ZhiXin,WANG JiKun,WANG JiaBo,WU ZhiJuan,ZHONG JinCheng Scientia Agricultura Sinica    2022, 55 (13): 2654-2666.   DOI: 10.3864/j.issn.0578-1752.2022.13.014 Abstract （327）   HTML （28）    PDF （5919KB）（99）       Knowledge map    Save 【Objective】The aim of this study was to analyze the effects of lncFAM200B in the lipid deposition in intramuscular preadipocytes of yak, which laid a foundation for further mechanism research. 【Method】 The longissimus dorsi muscle tissue of yak was collected and used to separate intramuscular preadipocytes. The adenovirus mediated overexpression technology was used to realize the overexpression of lncFAM200B, and the siRNA interference technology was used to analyze the function of lncFAM200B. The mRNA expression level of fat differentiation marker (PPARγ, C/EBPα and AP2) and the potential target (SIRT1 and PTEN) genes of lncFAM200B were detected via real-time fluorescent quantitative PCR (RT-qPCR). Oil red O staining, triacylglycerol (TAG) determination and CCK-8 determination methods were used to detect intracellular lipid droplet deposition and preadipocyte proliferation.【Result】The overexpression of lncFAM200B not only significantly increased the fat differentiation genes (C/EBPα and AP2, P<0.05) expression level, but also increased the lipid droplets deposition with large lipid droplets in the cells during induced differentiation. Conversely, lncFAM200B interference reduced the expression of PPARγ, C/EBPα and AP2 (P<0.05), and lipid droplet deposition. Furthermore, the triacylglycerol content after lncFAM200B overexpression 4 days was significantly higher than that of the control group (P<0.05), but lower after siRNA interference 6 days. Moreover, the SIRT1 levels increased with time was first decreased and then increased trend, and the PTEN had the opposite trend during lncFAM200B overexpression, and the opposite results of the two genes obtained during lncFAM200B interference. In addition, the results of CCK-8 experiment showed that there was a significant difference in cell proliferation activity both in overexpression or interference group after 72 hours. 【Conclusion】lncFAM200B could regulate yak adipose differentiation by influencing the expression of fat differentiation marker (C/EBPα and AP2), and further affect the triacylglycerol content and the lipid droplet deposition, but the detail mechanism need to be further research efforts. Table and Figures | Reference | Related Articles | Metrics

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Proteome Analysis of the Salivary Gland of Nurse Bee from High Royal Jelly Producing Bees and Italian Bees WANG RongHua,MENG LiFeng,FENG Mao,FANG Yu,WEI QiaoHong,MA BeiBei,ZHONG WeiLai,LI JianKe Scientia Agricultura Sinica    2022, 55 (13): 2667-2684.   DOI: 10.3864/j.issn.0578-1752.2022.13.015 Abstract （413）   HTML （28）    PDF （7038KB）（155）       Knowledge map    Save 【Objective】The objective of this study is to investigate the proteome profile of the postcereberal gland (PGld) and thoracic gland (ThGld) between high royal jelly producing bees (Apis mellifera liguatica, RJBs) and Italian bees (Apis mellifera liguatica, ITBs) with the aim of revealing the molecular basis of salivary gland regulating royal jelly production, and to provide a basis for analyzing the high-yield mechanism of royal jelly.【Method】PGld and ThGld were dissected from nurse bees of RJBs and ITBs. After protein extraction and enzyme digestion, the peptide samples were analyzed by liquid chromatography coupled with tandem mass spectrometry. Furthermore, the mass spectral data were qualified and quantified by MaxQuant software, and the following bioinformatic analysis was conducted using Perseus software, prediction of secretory protein was achieved by SignalP database, biological process and KEGG pathway were enriched by Cluego software.【Result】Totally 2 335 proteins were identified in salivary glands of RJBs and ITBs nurse bees, including 1 823 proteins in PGld and 1 922 proteins in ThGld. The expression profiles of the core proteins in the PGld and ThGld of RJBs and ITBs were similar, mainly involved in RNA metabolism, nucleic acid metabolism, ATP metabolism, protein translation, translation regulation and catabolism. The principal component analysis (PCA) showed that the molecular basis of salivary gland of RJBs and ITBs had exerted varying extent of differentiation during the selective breeding. Quantitatively, the PGld of ITBs and RJBs expressed 254 and 333 up-regulated proteins, respectively, corresponding to the small molecule and carbohydrate metabolism pathway in ITBs, and the organic nitrogen compound synthesis, cell redox homeostasis, amino acid metabolism in RJBs. Those proved that the protein synthesis, amino acid metabolism and energy supply of salivary gland cells in RJBs were more active than ITBs. In the same way, the up-regulated expressions of 412 and 162 proteins were detected in the ThGld of ITBs and RJBs, respectively, which involved in the pathways of oxidative phosphorylation, translation regulation in ITBs, and oxidative phosphorylation, response to toxic substances in RJBs, indicating that the level of resistance of RJBs ThGld cells was increased. A total of 43 secretory proteins were identified in the salivary glands of RJBs and ITBs, in which 15 were detected in royal jelly. Major royal jelly proteins 1, 2, 3, 4, 5 and 7 were detected in PGld and ThGld, indicating that both of the glands were involved in the synthesis of royal jelly main protein. The identification of α-glucosidase related to nectar transformation and odorant binding proteins 3, 13, 17 and 21 involved in the synthesis and release of chemical pheromones in both PGld and ThGld indicated their basic function of nectar transformation and pheromone synthesis. The enhanced expression of major royal jelly proteins 1, 2, 3 and 7, hexamerin 70a and 110, odorant binding proteins 3, 13, 17 and 21, transferrin and apolipophorin-III-like protein in the salivary gland of RJBs demonstrated that the synthesis of pheromone and royal jelly protein was stronger than ITBs.【Conclusion】The core proteome with similar pattern in salivary glands of RJBs and ITBs ensures the synthesis and secretion of royal jelly protein, pheromone and invertase. Molecular variation between the salivary glands of RJBs and ITBs was developed after long-term selective breeding. Relative to ITBs, the salivary glands of RJBs enhance the ability of protein synthesis, amino acid metabolism, cell energy supply and stress resistance, and upregulate the expression of most secretory proteins, which benefits to a better protein synthesis system with promising efficiency and lasting, and contributes to the high producing of royal jelly. Table and Figures | Reference | Related Articles | Metrics

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Evolutionary Relationship Between Transposable Elements and Tandem Repeats in Bovinae Species ZHANG Rui,ZHANG TianLiu,FAN TingTing,ZHU Bo,ZHANG LuPei,XU LingYang,GAO HuiJiang,LI JunYa,CHEN Yan,GAO Xue Scientia Agricultura Sinica    2022, 55 (9): 1859-1867.   DOI: 10.3864/j.issn.0578-1752.2022.09.014 Abstract （593）   HTML （27）    PDF （1506KB）（115）       Knowledge map    Save 【Objective】The repetitive sequence is an important part of eukaryotic genomes and plays an important role in species evolution, gene genetic variation, and transcriptional regulation. The purpose of this study was to reveal the characteristics of tandem repeats in bovinae by investigating the evolutionary relationship between transposons and tandem repeats, so as to provide the theoretical support for the study of tandem repeats in bovinae. 【Method】 In this paper, the six genomes were selected as research object, including Bos taurus, Bos indicus, Bos mutus, Bubalus bubalis, Bison bison and Bos frontalis. The transposable elements and tandem repeats in six genomes was identified through TRF and RepeatMasker software. Meanwhile, the sequence similarity between the two types of tandem repeats was analyzed by BLAST, and single-locus tandem repeats (single-locus TRs, mlTRs), multiple-locus tandem repeats (multiple-locus TRs, mlTRs) and the characteristics of tandem repeat for the transposable elements were investigated too. 【Result】 (1) In the six bovinae genomes, the percent of tandem repeats in Bos taurus was the highest (49.13%), followed by Bos frontalis (46.82%), Bubalus bubalis (46.23%), Bos indicus (42.70%), Bos mutus (42.53%), and Bison bison (42.36%), in which the content of transposable elements in the genome ranged from 40.57%-45.71%, and was higher than that of tandem repeats (1.50%-3.42%). (2) In the tandem repeats, the proportion of mlTRs (76%-99%) was significantly higher than that of slTRs(1%-24%), indicating that the mlTRs was the main component of tandem repeats in six bovinae species. (3) The proportion of TE-derived tandem repeats was 43% to 84%, among them mutiple-locus tandem repeats could reach up to 94%. (4) The analysis of TRs-related transposable elements and their activity showed that these transposable elements were mainly from non-Long Terminal Repeats (non-LTR, including SINE and LINE) and long interspersed nuclear element (LINE), among which SINE/core-RTE (mainly BOV-A2) had the highest number (14 423-24 193) and relative number (4.06%-6.77%), which was considered to be the youngest and the most dynamic transposable elements. (5) The study on transposable elements of tandem repeats’ characteristics indicated that BovB and L1_BT contained a large number of tandem repeats in 0-600 bp and 1 500 bp-2 700 bp, respectively, which were more than 93% and 87% consistent with the consensus sequence, respectively, and the sequences were located in the non-coding region. 【Conclusion】 The repetitive sequence had similar distribution characteristics, non-LTR was an important source of TRs-related TEs, and SINE/Core-RTE(mainly BOV-A2) was the youngest and most dynamic transposable elements. At the same time, the tandem repeats could be used as internal structure component of transposable elements, indicating that tandem repeats and transposable elements interacted with each other in the process of genome evolution. Table and Figures | Reference | Related Articles | Metrics

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miR-221-3p Regulates Ovarian Granulosa Cells Apoptosis by Targeting BCL2L11 in Small-Tail Han Sheep LIU YuFang,CHEN YuLin,ZHOU ZuYang,CHU MingXing Scientia Agricultura Sinica    2022, 55 (9): 1868-1876.   DOI: 10.3864/j.issn.0578-1752.2022.09.015 Abstract （403）   HTML （40）    PDF （1151KB）（149）       Knowledge map    Save 【Objective】BCL2L11 could promote apoptosis in mammals and is involved in the development of tissues and organs related to reproductive traits and in disease treatment. The aim of this study was to explore the effect of miR-221-3p on the regulation of granulosa cell apoptosis in Small-Tail Han sheep by targeting BCL2L11, so as to provide evidence for further study of the regulation of BCL2L11 in granulosa cell apoptosis and atresia of follicles.【Method】Based on the whole transcriptome sequencing analysis of the ovarian tissue of the previous study in our group, the differentially expressed gene BCL2L11 and its regulatory element miR-221-3p were obtained in this study. Analysis of the expression of BCL2L11 in different tissues of the Small-Tail Han sheep by using semi-quantitative and tissue fluorescence quantification (RT-qPCR). The expression of BCL2L11 and miRNA-221-3p was identified in the ovarian tissues of the Small-Tail Han sheep in the follicular and luteal phase by RT-qPCR. The miR-221-3p mimic, BCL2L11 wild-type and BCL2L11 mutant-type were co-transfected in HEK293T cells with negative control, the dual luciferase reporter gene detection system was used to determine the targeting relationship between miR-221-3p and BCL2L11. The miR-221-3p mimic and negative control were transfected into ovarian granulosa cells to achieve the overexpression of miR-221-3p. RT-qPCR was used to detect the effect of miR-221-3p on the expression levels of BCL2L11 and the marker genes of the apoptosis of ovarian granulosa cell gene XIAP and Fas at the mRNA level. At the same time, the changes in proliferation of granulocytes in the miR-221-3p overexpression and negative control groups were also analyzed using the EdU assay. 【Result】The results showed that the expression of BCL2L11 in ovarian tissue was the highest, followed by spleen and lung tissues. RT-qPCR results showed that the expression of miR-221-3p and BCL2L11 was significantly different in the ovarian tissues of Small-Tail Han sheep between follicular and luteal phases. The expression of miR-221-3p was higher in follicular than that in luteal phase ovaries, whereas BCL2L11 was less expressed in follicular than that in luteal phase ovaries, which showed the phenomenon of a negative regulation. Dual luciferase reporter analysis showed that overexpression of miR-221-3p significantly inhibited the activity of BCL2L11 3’UTR vector (P<0.05). The overexpression of miR-221-3p significantly reduced the mRNA level expression of target gene BCL2L11, while follicular granulosa cell apoptosis expression of marker genes XIAP and Fas were also significantly reduced (P<0.05). Analysis of the EdU assay showed that the proliferation rate of granulosa cells overexpressing miR-221-3p was 18.9%, which was significantly higher than that of the negative control group at 10.43% (P<0.01). 【Conclusion】BCL2L11 and miR-221-3p were important genes and regulatory elements that regulate ovarian development in Small-Tail Han sheep. BCL2L11 was one of the target genes of miR-221-3p, and overexpression of miR-221-3p could inhibit granulosa cell apoptosis by target BCL2L11. Table and Figures | Reference | Related Articles | Metrics

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circ-13267 Regulates Egg Duck Granulosa Cells Apoptosis Through Let-7-19/ERBB4 Pathway WU Yan,ZHANG Hao,LIANG ZhenHua,PAN AiLuan,SHEN Jie,PU YueJin,HUANG Tao,PI JinSong,DU JinPing Scientia Agricultura Sinica    2022, 55 (8): 1657-1666.   DOI: 10.3864/j.issn.0578-1752.2022.08.015 Abstract （380）   HTML （28）    PDF （1999KB）（129）       Knowledge map    Save 【Background】 Follicle development is a key factor for laying performance of egg ducks. Previous studies have shown that follicular development is a very complex biological process in poultry. At present, the pattern of follicular development in poultry has been understood. However, as an important factor determining egg production, the specific regulation mechanism of follicular development still needs further study. Granulosa cells are the main functional cells in follicles. They can regulate the growth, differentiation and maturation of theca cells and oocytes. They also regulate the growth and development of follicles, maintain normal ovarian function, such as induce ovulation, maintain maturation division, and provide substrates for oocytes. Circular RNAs (circRNAs) are a new type of endogenous specific non-coding RNA, which plays an important role in follicular development. 【Objective】The objective of this study was to explore the effects and regulatory mechanism of circ-13267 on apoptosis in egg duck granulosa cells, through regulating the expression of circ-13267 by constructing the overexpression vector, so as to provide the evidence for analysis the regulatory mechanism of egg duck follicular development. 【Method】Firstly, the expression levels of circ-13267 in cytoplasm and nucleus of granulosa cells was detected by Q-PCR. The overexpression vector circ-13267-pLCDH was constructed. After transfection of circ-13267 in egg duck granulosa cells, the expression levels of circ-13267, let-7-19, ERBB4, FAS and BCL2 were detected by Q-PCR. The proliferation of egg duck granulosa cells was detected by EdU method after transfection circ-13267-pLCDH and pLCDH-ciR. The linear sequence of circ-13267 or the 3'UTR of ErbB4 was cloned into pmirGLo vector. At the same time, let-7-19 binding site in the wild-type sequence was mutated to obtain the vector expressing the mutant sequence. The targeting relationships between circ-13267 and let-7-19, let-7-19 and ERBB4 were verified by dual luciferase reporter assay, respectively. Then, after transfection of circ-13267-pLCDH and pLCDH-ciR into egg duck follicular granulosa cells, the flow cytometry and Annexin V-FITC were utilized to explore the effects of circ-13267 on duck granulosa cells. 【Result】 In duck granulosa cells, circ-13267 was expressed in both cytoplasm and nucleus. The dual luciferase reporter gene assay confirmed that let-7-19 could bind to ERBB4 and down regulate the activity of luciferase; when the binding site of let-7-19 in ErbB4 sequence was mutated, let-7-19 could not inhibit the expression of luciferase, indicating that ERBB4 was a target gene of let-7-19. The results of Q-PCR showed that, after overexpression of circ-13267, the expression of BCL2 gene decreased significantly (P<0.05), while the expression of FAS and ERBB4 gene increased significantly (P<0.05); after overexpression of let-7-19, the expression of ERBB4 gene increased significantly (P<0.05), while after inhibition of let-7-19, the expression of ERBB4 gene decreased significantly (P<0.05). EdU test results showed that the number of follicular granulosa cells in egg ducks decreased significantly after overexpression of circ-13267, it was shown that circ-13267 promoted the apoptosis of follicular granulosa cells in egg ducks. However, after co-transfection of circ-13267 and let-7-19 into egg duck follicular granulosa cells, compared with the control group, there was no significant change in the expression of BCL2 and FAS (P>0.05); however, compared with overexpression of circ-13267, the expression of BCL2 gene decreased significantly (P<0.01) and FAS increased significantly (P<0.01). It was shown that circ-13267 could inhibit the apoptosis of egg duck follicular granulosa cells. In addition, flow cytometry was used to detect the transfected egg duck follicular granulosa cells. Compared with the co-transfection groups of circ-13267 and let-7-19, the number of late apoptotic cells and total apoptotic cells increased significantly (P<0.05), while the number of living cells decreased significantly (P<0.05). 【Conclusion】 circ-13267 was expressed in the cytoplasm and nucleus of egg duck follicular granulosa cells. circ-13267 could sponge let-7-19 and target ERBB4 gene, which promoted the apoptosis of egg duck follicular granulosa cells. This results provided a theoretical basis for analysis of the regulatory mechanism of egg duck follicular development. Table and Figures | Reference | Related Articles | Metrics

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Effects of Soy Isoflavones on the Proliferation and Apoptosis of Yak Ovarian Granulosa Cells WANG JiaMin,SHI JiaChen,MA FangFang,CAI Yong,QIAO ZiLin Scientia Agricultura Sinica    2022, 55 (8): 1667-1675.   DOI: 10.3864/j.issn.0578-1752.2022.08.016 Abstract （386）   HTML （28）    PDF （1155KB）（102）       Knowledge map    Save 【Background】Soybean isoflavones, mainly including genistein and daidzein, could exert estrogen-like effects, scavenge free radicals and promote cell proliferation. Granulosa cells are an important cell population in follicles and its physiological state is directly related to ovarian function, and the apoptosis of granulosa cells causes oocyte atresia. Yak (Bos grunniens) is one of the endemic species in Qinghai-Tibet plateau, but its reproductive rate is so low, however, the mechanism is still unclear. Ovary granulosa cell is an ideal model to study the regulating mechanism of female animal reproduction. 【Objective】The aim this study was to investigate the effects of soybean isoflavones on the proliferation and apoptosis of granulosa cells of yak ovary, and to provide evidence for the mechanism of soybean isoflavones.【Method】The yak ovarian granulosa cells were isolated and cultured. Immunohistochemistry staining was used to check FSHR for ovarian granulosa cell authenticating. MTT assay was used to detect cell proliferation, then the growth curve was drawn. Ovarian granulosa cells treated with different concentration of genistein and daidzein (0, 1 000, 2 000, 3 000, 4 000 and 5 000 pg·mL-1), the optimal concentration of genistein or daidzein for was selected to treat the second-generation granulosa cells for 48 h, then, the cell culture medium was collected and used to detect the concentration of estradiol and progesterone secreted by granulosa cells by ELISA. At the same time, the cells were collected to extract total RNA and to research the expression of proliferation-related gene AKT1 and apoptosis-related genes Bcl-2, Bax, Bad, Fas, FasL, Caspase-3 and p53 by qRT-PCR.【Result】The yak ovarian granulosa cells were typical epithelial-like cells. After inoculated for 2 h, the granulosa cells began to adhere to the wall. After 12 h, the cells appeared aggregation and growth. After 24 h, the cells were larger and showed long fusiform, star-shaped or polygonal. After cultured for 48 h, the cells looked like paving appearance. Immunohistochemistry staining showed FSHR positive indicated that the cultured cells were ovarian granulosa cells. MTT assay showed that the growth curve of the yak ovarian granulosa cells was S-shape: the growth was slow in 24 h and the cells were in the latent growth stage, and then, which was rapidly proliferated at 24-48 h and entered the exponential growth phase. The granulosa cells, in the plateau phase, steadily proliferated during 48-120 h. After 120 h, the density of living cells began to decline, and the cells entered the phase of degeneration. The second-generation of granulosa cells grew faster and the exponential proliferation phase at 24 h, so the second-generation of granulosa cells was selected for this experiment. MTT assay showed that treated with 3 000 pg·mL-1 genistein or daidzein for 48h, the cell viability were significantly promoted (P<0.01), the secretion of estradiol were induced treated with daidzein, but the progesterone secretion were markedly inhibited treated with genistein. The results of qRT-PCR showed that 3 000 pg·mL-1 genistein or daidzein significantly up-regulated the expression of AKT1, Bcl-2, Bad, p53, Fas and Bcl-2/Bax (P<0.01), down-regulated the expression of Bad and FasL. In addition, genistein significantly down-regulated the expression of Caspase-3 and daidzein significantly up-regulated it (P<0.01). 【Conclusion】The yak ovarian granulosa cells were isolated and cultured to provide an effective cell model for further study on the yak female reproduction regulating mechanism. The results indicated that genistein and daidzein inhibited the progesterone secretion of yak ovarian granulosa cells, promoted cell proliferation, and protected cells from apoptosis by up-regulating Bcl-2, p53, Fas and Bcl-2/Bax and down-regulating Bad and FasL. Table and Figures | Reference | Related Articles | Metrics

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Method Improvement and Its Application of Micro Complement Fixation Test for Brucellosis JIANG Hui,FENG Yu,QIN YuMing,ZHU LiangQuan,FAN XueZheng,DING JiaBo Scientia Agricultura Sinica    2022, 55 (8): 1676-1684.   DOI: 10.3864/j.issn.0578-1752.2022.08.017 Abstract （699）   HTML （21）    PDF （445KB）（95）       Knowledge map    Save 【Objective】 The complement fixation test is used as a confirmatory test for the serodiagnosis of brucellosis. The complement fixation tests are divided into constant complement fixation test (CFT) and micro complement fixation test (mCFT) in diagnostic techniques for animal brucellosis (GB/T 18646-2018). However, there are differences in the related technical parameters and judgment of the result between them, which lead to the inconsistency of CFT and mCFT results. In this study, By the improvement and optimization of the parameters of mCFT, this study made the detection results of mCFT equivalent to classical CFT, and achieved the high-throughput and accurate diagnosis of CFT for brucellosis. 【Method】 According to classical CFT reaction conditions and diagnostic criteria, the reaction time, diluent, titer determination, result judgment and other related parameters for the current national standard of mCFT were optimized and improved. Compared with the original method, the dilution of the tested serum was changed from 1:2-1:64 to 1:10. The dilution method was the same as classical CFT so that the detection critical value was consistent with the constant method. The OD600 value was read by the microplate reader to determine the hemolysis intensity, which reduced the error caused by visual judgment and improved the efficiency and accuracy of result judgment. The reaction time was shortened from 30 min to 20 min in water bath at 37℃, which improved the detection efficiency without affecting the results. The diluent was changed from barbital buffer to normal saline, which used the same diluent of classical CFT and achieved satisfactory results. The 409 clinical bovine and sheep serum samples (163 bovine serum samples and 246 sheep serum samples) from Inner Mongolia, Shandong, Beijing and Jiangsu were detected by improved mCFT, the national standard mCFT, and classical CFT, respectively. The coincidence rate was analyzed and compared. 【Result】By detecting the 409 clinical samples, the result showed that 53 samples were positive, 11 samples were suspicious and 345 were negative by improved mCFT. The 53 positive samples, 9 suspicious samples and 347 negative samples were detected by classical CFT. Compared with the results of the two methods, only two bovine serum samples were suspicious by the improved mCFT, which were negative by classical CFT. The results of the other 407 samples were consistent. The coincidence rate of bovine serum samples was 98.77%, which of sheep serum samples was 100%, and the total coincidence rate was 99.51% between improved mCFT and classical CFT. However, the positive criteria of national standard mCFT were lower than that of classical CFT, and no suspicious interval was set. The results showed that 97 samples were positive and 312 samples were negative. Compared with the results of classical CFT, the coincidence rate of bovine serum samples was 88.34%, which of sheep serum samples was 89.84%; the total coincidence rate was 89.24%, which was far lower than 99.51% of improved mCFT and classical CFT. 【Conclusion】 By optimizing the parameters of mCFT, the mCFT method was established, which was highly consistent with the criterion and the results of the classical CFT. Table and Figures | Reference | Related Articles | Metrics

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Effects of Sublethal Doses of Imidacloprid on the Expression of Neurometabolic Genes in Apis cerana cerana QIU YiLei,WU Fan,ZHANG Li,LI HongLiang Scientia Agricultura Sinica    2022, 55 (8): 1685-1694.   DOI: 10.3864/j.issn.0578-1752.2022.08.018 Abstract （408）   HTML （32）    PDF （1830KB）（132）       Knowledge map    Save 【Background】The target of neonicotinoid insecticides is the acetylcholine receptor in the nervous system of insects. Due to its good systemicity and low toxicity to humans and animals, it has been widely used in agricultural production. Thus there is still a low residue in plants, and this sublethal dose residue can still cause adverse effects on the behavior and nervous system of flower-visiting insects like bees.【Objective】The objective of this study is to clarify the effect of sublethal dose imidacloprid (a kind of neonicotinoid insecticides) on the nervous physiological and metabolism system of Apis cerana cerana.【Method】In this study, the worker bees were treated with two sublethal concentration gradient doses of 5 and 10 μg·L-1 imidacloprid for 10 days (three biological replicates). After total RNA was extracted, the resulting library was analyzed by RNA-seq. Throughput sequencing, and the bioinformatics technology was used to assemble and annotate the sequence de novo, and the differentially expressed genes (DEGs) after sublethal doses imidacloprid treatment were analyzed by clustering and enrichment. Finally, real-time fluorescence quantitative PCR (RT-qPCR) technology was used to verify the DEGs related to the nervous and metabolic systems.【Result】A total of 9 sequencing libraries were obtained, the ratio of effective sequencing data exceeded 94.45%. From the obtained 37 364 unigenes, 571 DEGs were identified. The enrichment analysis of GO and KEGG found that the DEGs were mainly related to multiple pathways such as protein translation, redox, oxidative phosphorylation, and ribosome, indicating that sublethal doses of imidacloprid had an impact on multiple physiological processes and metabolic pathways of A. c. cerana. Nine DEGs (e.g. neuropeptide F (NPF), neuropeptide SIFamide receptor (SIFaR), phosphoinositide 3-dependent kinases (PDK1), A-kinase anchoring protein 1 (AKAP1), carbonic anhydrase 3 (CA3), superoxide dismutase 2 (SOD2), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10 (ND42), cuticle protein 12 (CP12), and odorant-binding protein (OBP17)) related to nerve signal transmission and metabolic function were selected for RT-qPCR verification. Their expression patterns were completely consistent with the transcriptome results.【Conclusion】The sublethal dose of imidacloprid can affect lots of aspects of A. c. cerana such as nerve signal transduction, cellular respiration, immune response, maintenance of homeostasis, and olfactory perception. Table and Figures | Reference | Related Articles | Metrics

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Expression and Localization of LCN5 in Ram Reproductive Organs and Spermatozoa DONG FuCheng,MA ShuLi,SHI JuanJuan,ZHANG JunMei,CUI Yan,REN YouShe,ZHANG ChunXiang Scientia Agricultura Sinica    2022, 55 (7): 1445-1457.   DOI: 10.3864/j.issn.0578-1752.2022.07.015 Abstract （366）   HTML （40）    PDF （5981KB）（115）       Knowledge map    Save 【Objective】The aim of this experiment was to investigate the expression pattern and localization of LCN5 in the testis of ram, the different segments of the epididymis and spermatozoa among them, providing clues to further explore the function of LCN5 in ram reproductive organs. 【Method】All the samples were from 6 healthy 9-month-old rams with similar weight (65.23±1.95 kg). Three rams were used to collect ejaculated sperm via artificial vagina while the others for collecting the testes, epididymis, and vas deferens as well as the regional spermatozoa within these male reproductive ducts using aseptic castration. The right testis, efferent ducts (ED), caput (E1-E2), corpus (E3-E5), cauda (E6-E7) and vas deferens (VS) samples were collected and placed in liquid nitrogen and 4% paraformaldehyde, respectively; the spermatozoa from ejaculated semen, the right testis, various segments of the epididymis and the vas deferens were separated and determined by the CASA system. Western Blot and immunohistochemistry were performed to detect the localization of LCN5 in the testis, the various segments of the epididymis, and vas deferens, respectively. Immunofluorescence was carried out to examine the dynamic localization of LCN5 in the spermatozoa from the testis, different segment of epididymis and vas deferens. 【Result】 (1) The CASA results showed that the proportion of grade A and C spermatozoa and the sperm kinematic parameters were highest, including VAP, VSL, VCL, ALH, BCF, WOB, LIN and STR from the ejaculated sperm and spermatozoa in caudal epididymis, compared with those from the other segments of male reproductive ducts. Except for that, there were the more grade A spermatozoa in VS, which showed better motility than others. However, the spermatozoa from testis were completely immotile. (2)The Western Blot results discovered that the highest expression of LCN5 was found in the E1 segment, next in segment E2 and E7, which were more than in corpus, testis and efferent ducts (P<0.05). The quantitative analysis showed the LCN5 expression levels in these tissues was ordered as E1>E2>E6>E3≈E5≈E4≈E7>VS>ED>T. (3) Immunohistochemistry found that there were slight positive signals of LCN5 in the testicular elongated spermatids, the principal and basal cells of the efferent ducts, while strong positive signals could be detected in the principal and basal cells, stereocilia of epididymal epithelium in the caput, corpus, and cauda. Granular signals of LCN5 were scattered around the sperm in the epididymal lumen. Besides, the weak LCN5 positive signals were found in the smooth muscle cells of the vas deferens. (4) Immunofluorescence results of LCN5 protein on the spermatozoa from different regions showed dynamic changes. Firstly, the local positive signals on the surface of sperm acrosome and midpiece of spermatozoa from testis and ED could be only found. With the spermatozoa transition from the testis to the epididymis, these signals began to expand and spread on the whole surface of sperm acrosome and the intensity of the signals on the sperm acrosome and midpiece reached the peak in the E1 segment. Secondly, the positive signals also existed in cytoplasmic droplets of the sperm flagellum. These signals also showed gradually loss with the removal of cytoplasmic droplets along the epidydimal ducts until E6 or E7. The expression pattern of LCN5 on the cauda and corpus on the sperm surface was similar to that of the caput. 【Conclusion】This research firstly revealed that LCN5 protein was regionally expressed in the ram reproductive system, highly expressed in the epididymal caput and gathered in the E6 segment. In the epididymal cauda, LCN5 was mainly expressed in the surface of acrosome on the spermatozoa from the caput, corpus, caudal of epididymis, as well as the cytoplasmic droplets of the sperm flagellum, which presented a dynamic distribution both on the surface of sperm and midpiece corresponding to the sperm quality and motility along the ram reproductive ducts, highlighting its potential function in sperm maturation. Taken together, LCN5 could play an essential role in spermatogenesis, maturation, storage, which provided the theoretical basis for its functional research and exploitation. Table and Figures | Reference | Related Articles | Metrics

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Establishment and Application of PCR Assay for Mycoplasma Contamination in Cell Culture and Live Virus Vaccine GENG RenHao,LIU Bo,WANG Fang,LUO YuFeng,QU HongFei,FAN XueZheng,QIN YuMing,DING JiaBo,XU GuanLong,SHEN QingChun,QIN AiJian Scientia Agricultura Sinica    2022, 55 (7): 1458-1468.   DOI: 10.3864/j.issn.0578-1752.2022.07.016 Abstract （739）   HTML （29）    PDF （2791KB）（212）       Knowledge map    Save 【Objective】Mycoplasma contamination is prone to occur in biological research and productions. Aimed at the shortage of time-effectiveness and sensitivity of current Pharmacopoeia detection methods, a simple, rapid, specific and sensitive PCR method for mycoplasma was established. 【Method】All the sequences of mycoplasmas were extracted from the datasets file SILVA_123_ SSURef from SILVA database, which contained aligned small (16S/18S, SSU) ribosomal RNA (rRNA) sequences for all three domains of life (Bacteria, Archaea and Eukarya). 181 16S rRNA unique sequences of Mycoplasma (including 139 classified species and 42 unclassified Mycoplasma) were obtained by deduplication of the sequences. After alignment, the hypervariable regions V6 to V9 were selected as the detection target regions, and the detection primers were designed to establish a universal PCR method for mycoplasma detection. 12 kinds of mycoplasmas or 2 kinds of acholeplasmas were selected to verify the detection range of the PCR method; 6 kinds of passage cells from different animal and 3 kinds of common bacteria were selected for specific verification; 5 kinds of mycoplasmas and a kind of acholeplasma were selected for sensitivity test. In order to evaluate the performance of the assay in clinical application, 17 batches of animal live virus vaccines ( for 6 kinds of animals) and 24 samples of 8 kinds of cell cultures were subjected to test, which were compared with the classical culture method for mycoplasmas. 【Result】A general PCR method for mycoplasma was established. The detection primers were composed of 2 upstream primers (5'-GCAAARCTATRGARAYA TAGYVGAG-3' and 5'- GCAAAGGCTTAGAAATAAGTTCGGAG-3') and a downstream primer (5'- CCARCTCYCATRGTKTGA CGG - 3'), and the mixing ratio of the primers was 3:1:4. The optimal annealing temperature was 56℃. The PCR method was used to detect 14 mycoplasma species, and the results showed that 396 bp-413 bp specific bands had been amplified, which indicated that the detection range of the PCR method satisfied the detection requirements; No specific bands were amplified from 6 kinds of animal cells and 3 kinds of common bacteria with the PCR assay. The results of sensitivity test showed that the detection limit of the PCR method was 20-200 CCU, and the corresponding nucleic acid was 1.5-15.0 pg. The detection results of 17 batches of live virus vaccines and 24 cell samples were almost consistent with those of culture method, which indicated that the PCR method established in this study was consistent with culture method well, and was more sensitive. 【Conclusion】The PCR assay established in this study was specific, sensitive, easy and fast, suitable for rapid detection of mycoplasma contamination in cells and live virus vaccine. Table and Figures | Reference | Related Articles | Metrics

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Distribution Characteristics of Prophage in Multidrug Resistant Escherichia coli as well as Its Induction and Isolation LIU Jiao,LIU Chang,CHEN Jin,WANG MianZhi,XIONG WenGuang,ZENG ZhenLing Scientia Agricultura Sinica    2022, 55 (7): 1469-1478.   DOI: 10.3864/j.issn.0578-1752.2022.07.017 Abstract （909）   HTML （32）    PDF （4374KB）（135）       Knowledge map    Save 【Objective】 This study investigated the distribution characteristics of prophage in multi-drug resistant Escherichia coli, induction and isolation, as well as the prevalence of drug resistance and virulence genes in prophage, so as to provide a scientific basis for the study of prophage-mediated resistance genes in the spread of bacteria. 【Method】 131 multi-drug resistant E. coli isolating from poultry origin in Guangdong Province from 2018 to 2019 were selected in the laboratory for nucleic acid extraction and whole-genome sequencing. The results of second-generation sequencing were assembled and spliced into a whole-genome sequence and uploaded to the phage. The PHASTER network database was compared and analyzed with the existing phage genome sequences in the database. Drug resistance genes and virulence genes were compared on the CGE database, and then the distribution of drug resistance genes and virulence genes on the prophage were obtained. The mild phage was induced by mitomycin C, separated and purified by using the double-layer plate method. 【Result】 The results of the drug sensitivity test of 131 strains of Escherichia coli showed that the drug resistance rates of ampicillin, tetracycline, florfenicol and compound trimethoprim were all more than 90%, followed by cephalosporin antibiotics, gentamicin, ciprofloxacin, meropenem and colistin with all around 50%, and the resistance rate of tigecycline reached 0.2%. All strains showed multi-drug resistance, and they were all multi-drug resistant Escherichia coli. A total of 736 prophage fragments were detected in 131 strains of multi-drug resistant E. coli, including 329 complete prophage, 66 suspicious phages and 341 incomplete phage, which matched with 40, 20 and 52 known database phage species in different percentages, respectively. The gene sequence of the complete prophage showed that it matched the known phage species better, and the sequence similarity was the highest, with an average of 58.53%. The average number of prophages in 131 strains of E. coli was 5.6, and the average total content was 152.4 kb. Prophage genome accounted for 0.58% to 5.87% of its host genome, with 3.0% being the dominant. The length of the prophage genome ranged from 2.8 to 107.9 kb, and the 13.0 kb prophage had the highest frequency, accounting for 9.1% of all prophages. CGE comparison results showed that the genomes of 131 strains of multi-drug-resistant E. coli detected resistance genes mdf (A), lnu (G) and mcr-1 in 18 prophage sequences. The detected numbers of mdf (A), lnu (G) and mcr-1 were 16, 1, and 1, respectively. 71 strains of multi-drug resistant E. coli prophage carried 6 different virulence genes, and some strains carried 2 or 3 virulence genes. There were 62 prophages carrying the telomerase RNA gene terC, 16 prophages carrying the serum survival increasing gene iss, and the outer membrane protease ompT, among which the adhesin gene iha, the cvaC gene and the ABC transporter gene mchF were at 2, 2, 1, and 1, respectively. Mcr-a gene were detected in prophage of 1 strain multi-drug resistant Escherichia coli. The mdf (A) gene and terC gene were the most common resistance genes and virulence genes in prophage, respectively. The results of mild phage induction experiments showed that the success rate of prophage induction was 84.0%, but the probability of plaque appearance was still relatively low. 【Conclusion】Prophages were widely distributed in multi-drug resistant E. coli and carried a variety of resistance genes and virulence genes. Mild phages had a high induction rate, and have the risk of horizontal transmission of resistance genes and virulence genes, and need to be strengthened and sustained monitor. Table and Figures | Reference | Related Articles | Metrics

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Definition and Genetic Parameters Estimation for Health Traits by Using on-Farm Management Data in Dairy Cattle WANG Kai,ZHANG HaiLiang,DONG YiXin,CHEN ShaoKan,GUO Gang,LIU Lin,WANG YaChun Scientia Agricultura Sinica    2022, 55 (6): 1227-1240.   DOI: 10.3864/j.issn.0578-1752.2022.06.014 Abstract （459）   HTML （41）    PDF （1798KB）（274）       Knowledge map    Save 【Objective】This study was conducted to define health traits and to estimate their genetic parameters by using on-farm management data in dairy cattle.【Method】In this study, the health event records were collected by extracting from farm management software from 46 large-scale dairy farms in Northern China. Totally, 1 326 kinds of health events were grouped into five categories by standardizing the acronyms of on-farm records, 18 kinds of most frequent health events were selected from five categories. According to whether a health event occurred at least once within a lactation, 18 binary individual health traits corresponding to the 18 selected health events were defined (observations being 0 or 1). Furthermore, in order to define cow’s general resistance to certain type of health problem, five binary general health traits were defined according to whether health problems within a health category occurs at least once in a lactation (observations being 0 or 1). The single trait and two traits linear animal models were used to estimate the genetic parameters for 23 newly defined health traits. 【Result】The estimated heritabilities for 23 health traits ranged from 0 (rumen acidosis) to 0.03 (milk fever). Udder health, reproductive disorders and metabolic disorders had the highest heritability (approximately 0.02) among five general health traits, whereas digestive disorders and hoof health had relatively low heritability (less than 0.01). Clustering health events into categories resulted in higher heritability for reproductive disorders and digestive disorders, while heritabilities of udder health, metabolic disorders and hoof health were lower than that of single health traits with the highest heritability in their respective category. The low genetic correlations between different health category traits were found; however, the moderate genetic correlations among some health traits from same category were observed. The health traits within the hoof health had the high genetic correlations with each other, ranged from 0.63 (Laminitis and Footrot) to 0.99 (Laminitis and Sole ulcer). For reproductive disorders, retained placenta had medium genetic correlations with metritis (0.47) and endometritis (0.46), respectively. For digestive disorders, relatively high genetic correlations were found between diarrhea and enteritis (0.94) as well as dyspepsia and antony of forestomachs (0.80). The ketosis and abomasum displacement had a genetic correlation of 0.42.【Conclusion】Based on the current data quality of health records in Chinese dairy farm and the results from the current study, it was suggested that five individual health traits (clinical mastitis, metritis, ketosis, abomasum displacement and milk fever) and two general health traits (digestive disorders and hoof health) could be considered as target traits, and they should be intensively considered in research and breeding practice for improving health traits genetically in Chinese Holstein population. This study proved the feasibility of defining health traits using on-farm management data in Chinese dairy cattle. The results from the current study provided a reference for research and genetic selection of disease resistance in dairy cattle, and could help to promote balanced breeding in Chinese dairy cattle population. Table and Figures | Reference | Related Articles | Metrics

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Effects of FB1 on Apoptosis and Autophagy of Porcine Oocytes in vitro Maturation LI WenHui,HE YiJing,JIANG Yao,ZHAO HongYu,PENG Lei,LI Jia,RUI Rong,JU ShiQiang Scientia Agricultura Sinica    2022, 55 (6): 1241-1252.   DOI: 10.3864/j.issn.0578-1752.2022.06.015 Abstract （443）   HTML （36）    PDF （4024KB）（139）       Knowledge map    Save 【Objective】The purpose of this study was to investigate the effects of fumonisin B1 (FB1) on porcine oocyte in vitro maturation and its potential mechanism, so as to provide the theoretical reference for effective prevention and treatment of reproductive toxicity injury caused by FB1 in the clinic.【Method】Porcine cumulus oocyte complexes (COCs) were collected and randomly divided into groups, and treated with different concentrations of FB1 (0, 10, 20 and 30 μg·mL-1) for 44 h during in vitro maturation, respectively. Then, the first polar body (PB1) extrusion and embryo development after activation of oocytes were analyzed, and the effects of FB1 on meiotic progression and cytoskeleton structure of oocytes were further detected by immunofluorescence staining and combined with confocal microscopy. In order to further explore the mechanism of FB1 on toxic injury of porcine oocytes, the JC-1, Annexin V-FITC and LC3A/B fluorescence staining were used to detect mitochondrial function, early apoptosis and autophagy levels in oocytes, respectively. On this basis, the expression of apoptosis/autophagy related to proteins were further analyzed by Western blotting.【Result】FB1 treatment had significant inhibitory effects on oocyte maturation, while PB1 extrusion rate decreased in a concentration-dependent manner. When the concentration of FB1 reached more than 20 μg·mL -1, the PB1 extrusion was significantly decreased (P<0.01), and the cleavage rate and blastocyst rate of the embryos after oocytes parthenogenetic activation were significantly decreased (P<0.01), damaging the development potential of oocytes to a certain extent. And the cell cycle analysis showed that FB1 treatment also disordered the meiotic cycle progression, resulting in a significant increase in the proportion of oocytes that arrested at germinal vesicle breakdown (GVBD) (P<0.01), and a significant decrease in the proportion of oocytes that successfully developed to the Metaphase II (MII) (P<0.01), with an increase in spindle abnormal rate (P<0.01) and a decrease in actin distribution at the plasma membrane (P<0.05). Further studies showed that, compared with control group, the mitochondrial membrane potential was significantly decreased (P<0.05), impairing mitochondrial function. At the same time, the rate of early apoptosis was obviously increased (P<0.01), and the level of autophagy was also significantly increased (P<0.01) in FB1-treated oocytes. Western blotting analysis showed that the expressions of pro-apoptotic protein BAX and autophagy protein LC3A/B II in FB1-treated oocytes were significantly up-regulated (P<0.05), and the expression of anti-apoptotic protein BCL2 was significantly down-regulated (P<0.05), indicating the occurrence of early apoptosis and autophagy.【Conclusion】FB1 had obvious toxic effects on porcine oocyte maturation in vitro and embryo development after activation, resulting in meiotic cycle arrest, spindle structure disorder, actin distribution reduction and mitochondrial injury, and its toxic mechanism might be related to the induction of apoptosis and autophagy in oocytes. Table and Figures | Reference | Related Articles | Metrics

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Screening and Identification of HSP90 Interacting Proteins in Silkworm (Bombyx mori) LONG YanBi,WU YunFei,ZHANG Qian,CHEN Peng,PAN MinHui Scientia Agricultura Sinica    2022, 55 (6): 1253-1262.   DOI: 10.3864/j.issn.0578-1752.2022.06.016 Abstract （460）   HTML （36）    PDF （2531KB）（146）       Knowledge map    Save 【Objective】HSP90 is a member of the heat shock protein family and plays an important role in insect resistance and metamorphosis. Studies have shown that HSP90 can promote the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV), but the mechanism of action is unclear. The objective of this study is to identify the interacting proteins of BmHSP90, and to provide a reference for the analysis of its mechanism of promoting BmNPV proliferation.【Method】The BmHSP90 HA eukaryotic overexpression vector linked to pIZ/V5-His was constructed. After transfection in BmN-SWU1 cells for 48 h, BmNPV was infected and cultured for 48 h to collect the proteins. After the total protein was retained, the proteins were divided into two tubes and co-immunoprecipitation was performed. The interacting protein was caught with anti-HA antibody and IgG antibody, respectively, after staining the protein gel with silver nitrate, the different bands were obtained and mass spectrometry analysis was performed. The mass spectrometry results were combined with the information analysis to screen candidate interacting proteins, and then the interacting proteins were cloned and identified. The co-localization of HSP90 and the interacting protein was verified by immunofluorescence, and the co-immunoprecipitation experiment was further used to determine whether they had an interaction relationship.【Result】The results of silver nitrate staining showed that the experimental group and the control group had different bands near 90, 70 and 60 kD, and verified that the different bands at 90 kD were bait proteins. The other two different bands were analyzed by mass spectrometry, and a total of 7 candidate interacting proteins were identified. Two of the candidate proteins were selected for follow-up study through analysis, namely Tubulin-specific chaperone E (Tbce) and Golgin subfamily A member 5 (Golga5). The maximum open reading frame length of the BmTbce is 1 728 bp, which encodes 576 amino acids, and the maximum open reading frame length of the BmGolga5 is 1 854 bp, which encodes 618 amino acids. The homology alignment and phylogenetic tree showed that the microtubule binding domain of BmTbce (cytoskeleton-associated protein-glycine-rich, CAP-Gly) was located at the N-terminus and was highly conserved among different species. The transmembrane domain (TMD) of BmGolga5 was located at the C-terminus and was also conservative. The fluorescence co-localization showed that BmHSP90 co-localized with BmTbce and BmGolga5 in the cytoplasm, and it was further proved by co-immunoprecipitation that BmHSP90 HA and BmTbceFlag, BmHSP90HA and BmGolga5Flag had an interaction relationship.【Conclusion】After screening and identification, in the process of BmNPV infection of B. mori cells, the proteins that interact with the B. mori heat shock protein HSP90 are BmTbce and BmGolga5. Table and Figures | Reference | Related Articles | Metrics

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Relationship Between Expression Levels of Guangxi Partridge Chicken m6A Methyltransferase Genes, Myofiber Types and Myogenic Differentiation SHU JingTing,SHAN YanJu,JI GaiGe,ZHANG Ming,TU YunJie,LIU YiFan,JU XiaoJun,SHENG ZhongWei,TANG YanFei,LI Hua,ZOU JianMin Scientia Agricultura Sinica    2022, 55 (3): 589-601.   DOI: 10.3864/j.issn.0578-1752.2022.03.013 Abstract （576）   HTML （29）    PDF （1487KB）（134）       Knowledge map    Save 【Objective】 It is widely accepted that the regulation of myofiber type composition and transition is an important source of variation to improve meat quality. Recently, several studies revealed that N6-methyladenosine (m6A) RNA methylation played a important biological role in the regulation of muscle differentiation. In the present study, the myofiber types of different location of skeletal muscles were identified in the native chicken breed-Guangxi partridge chicken, and the expression levels of the m6A methyltransferase genes (METTL3, METTL14, WTAP and KIAA1429) were first studied in different location of skeletal muscle and myogenic differentiation, which would provide a reference for clarifying the regulation mechanism of chicken myofiber type composition and transition.【Method】 Myosin ATPase staining was used to identify the myofiber types and to measure the myofiber size and density of seven location of Guangxi partridge chicken muscles, including the Pectoralis major (XD), pectoralis minor (XX), Sartorius (FJ), Medial head of sciatic pubis (CN), medial head of gastrocnemius (FN), Lateral iliotibial muscle (QW) and Latissimus dorsi (BK). Colorimetric method was used to detect the endogenous m6A methylation levels at different time points before and after myogenic differentiation. Expression of METTL3, METTL14, WTAP and KIAA1429 was quantified by RT-PCR in these seven muscles, as well as at the myoblasts separated on 0 h, 19 h, and cultured in differentiation medium on D0, D2, and D5, and the correlations between the gene expression levels and myofiber characteristics as well as m6A methylation levels were also analyzed by SPSS20.0 software.【Result】 The XD and XX muscles of the breast muscle group were all composed of white myofibers, CN, FN and FJ muscles of the leg muscle group were mainly composed of red myofibers, and QW muscles were mainly composed of white myofibers, while the BK muscles of the back muscle group were mainly composed of red myofibers. The average fiber densities of muscles mainly composed of red myofibers were significantly higher than those mainly composed of white myofibers. The expression patterns of METTL3, METTL14, WTAP and KIAA1429 showed significant differences in tissue and time-specific fashion. Overall, the expression levels of the four genes in muscles mainly composed of red myofibers were higher than those mainly composed of white myofibers, and in differentiated myoblasts were significantly higher than those in proliferative myoblasts. The muscle METTL3, METTL14 and WTAP gene showed similar expression patterns, the highest expression level was appeared in BK muscles, which were significantly higher than that in all the other studied muscles; the expression levels in CN and FN muscles were also significantly higher than those in FJ, XD, XX and QW muscles. The highest expression level of KIAA1429 was appeared in FN muscles, which were significantly higher than that in all the other studied muscles; the expression levels in BK muscles were also significantly higher than those in CN, FJ, XD, XX and QW muscles; the lowest expression level was appeared in XD muscles, but the differences were not significantly when compared to XX and QW. In chicken myoblasts, both METTL3 and METTL14 gene showed continuous increasing and then decreasing expression pattern, the lowest expression level was appeared at 0h newly separated myoblasts, and the highest level was appeared at D2 induced differentiation myoblasts, which were significantly higher than those in proliferative myoblasts and D0 induced differentiation myoblasts. The consistent expression pattern was also found in WTAP and KIAA1429 gene, showed continuous increasing expression pattern; the lowest expression level was appeared at 0h newly separated myoblasts, significantly lower than that in other studied time points, then significantly increased at 19 h, and then slightly increased at D0, then significantly increased from D0 to D5. The endogenous m6A RNA methylation levels increased along with myogenic differentiation, and the levels in differentiation myoblasts also showed significantly higher than those in proliferative myoblasts, the highest methylation level was appeared at D2 induced differentiation myoblasts, then significantly increased from D2 to D5; however, the methylation level at D5 was higher than that at D0. Significant positive relationships were observed for the expression of studied genes in different muscle tissues as well as in myoblasts. Meanwhile, the skeletal muscle expression of these four genes was all showed significant positive relationships with red myofiber ratio, and significant negative relationships with red myofiber ratio. Positive relationships were also observed between the expression of studied genes in myoblasts and m6A methylation levels.【Conclusion】 There were differences in muscle fiber type composition in different parts of the muscle of Guangxi partridge chicken. Differential expression and coordinated developmental regulation of the selected m6A methyltransferase genes in the chicken skeletal muscles and myoblasts, and these genes might play a role in the regulation of the myofiber type composition, maintain, and myogenic differentiation. Table and Figures | Reference | Related Articles | Metrics

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Relationship Between Biofilm Formation and Molecular Typing of Staphylococcus aureus from Animal Origin TANG ZiYun,HU JianXin,CHEN Jin,LU YiXing,KONG LingLi,DIAO Lu,ZHANG FaFu,XIONG WenGuang,ZENG ZhenLing Scientia Agricultura Sinica    2022, 55 (3): 602-612.   DOI: 10.3864/j.issn.0578-1752.2022.03.014 Abstract （609）   HTML （26）    PDF （5020KB）（128）       Knowledge map    Save 【Objective】 The aim of this study was to investigate the epidemiological characteristics of Staphylococcus aureus (S. aureus) in the biofilm producing strains and to explore the correlation between biofilm forming ability and molecular typing, so as to provide the theoretical basis for the treatment of S. aureus infection. 【Method】 The biofilm producing ability of all strains of S. aureus was determined by crystal violet semi-quantitative method. The minimum inhibitory concentrations of 22 common antibiotics were determined by the membrane producing strains. Molecular typing was conducted by three common typing methods of S. aureus, including spa typing, MLST typing and PFGE typing, and the correlation between membrane production capacity and molecular typing was analyzed. Finally, whole genome sequencing technology was used to analyze the antibiotics resistance gene and virulence genes in the biofilm producing strains. 【Result】 The semi-quantitative results of crystalline violet showed that a total of 23 strains (23.47%) of 98 S. aureus strains were able to produce biofilm, including 22 strains (25.29%, 22/87) from cow milk source, 14 strains (60.87%, 14/22) from Zhejiang dairy farms, 8 strains (39.13%, 8/22) from Fujian dairy farms, and 1 strain from pig source (9.10%, 1/11) from Guangdong slaughterhouse, indicating that the film-producing potential of S. aureus from cow's milk source was higher than that of pig source, 22 strains (95.65%) of which were from cow's milk source and 1 strain (4.35%) was from pig source. The film-producing ability was classified into strong, medium and weak, and among the 23 film-producing strains, 2 strains (8.70%, 2/23) were strong film-producing strains, 9 strains (39.13%, 9/23) were medium, and 12 strains (52.17%, 12/23) were weak. The results of the drug sensitivity test showed that the bovine milk-derived membrane- producing strains were sensitive to all the tested antibacterial drugs, while the pig-derived membrane-producing strains showed resistance to 13 antibacterial drugs, including penicillin, amoxicillin, ceftiofur, cefoxitin, enrofloxacin, ciprofloxacin, clindamycin, doxycycline, erythromycin, flupenthixol, cotrimoxazole, tiamulin, and tilmicosin. The spa typing results showed that 98 strains of S. aureus obtained 8 spa types, and 23 strains of film-producing S. aureus accounted for 3 of them: 1 strain t2922 from porcine origin in Guangdong, 14 strains t2119 from Zhejiang cow milk source, and 8 strains t189 from Fujian cow milk source. MLST typing results showed that 98 strains were classified into 9 ST types, of which 6 ST types did not have the ability to produce biofilm, namely ST398, ST522, ST705, ST1651, ST479 and ST151, and only 3 strains of ST type had biofilm production ability, namely ST9, ST7 and ST188. It was found that the molecular types of strong film-producing strains were mainly ST7-t2119, the medium film-producing strains were mainly ST7-t2119 and ST188-t189, and the weak film-producing strains were ST9-t2922, ST7-t2119 and ST188-t189. The ST type of weak film-producing strains could be well distinguished from the medium and strong film-producing strains, and only the specific ST type of S. aureus had the ability to produce biofilm. 23 film-producing strains PFGE typing all successful PFGE typing results showed that the results show that each strain of film-producing bacteria in Guangdong, Fujian and Zhejiang provinces were divided into 3 PFGE types, and there were geographical distribution characteristics of PFGE types; the strains isolating from the same region had clonal transmission, and the strains in the province were clonal to each other, but there were significant differences in biofilm production ability between clones.; the whole genome sequencing results showed that the drug resistance genes and virulence genes in the film-producing strains were diverse according to the molecular type. 【Conclusion】 S. aureus from different sources had different potential to produce biofilm and all carried different film-producing genes. The film-producing potential of S. aureus from bovine milk source was much higher than that of porcine source, and all carrid different film-producing genes. Whether strains could produce film or not may be strongly correlated with ST type, and the specific ST types, such as ST9, ST7 and ST188, were more likely to produce biofilm; however, at the same time, the strains with the same molecular type had different abilities to produce biofilm. Table and Figures | Reference | Related Articles | Metrics

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Expression and Binding Properties of Odorant Binding Protein AcerOBP7 in Apis cerana cerana ZHAO HuiTing,PENG Zhu,JIANG YuSuo,ZHAO ShuGuo,HUANG Li,DU YaLi,GUO LiNa Scientia Agricultura Sinica    2022, 55 (3): 613-624.   DOI: 10.3864/j.issn.0578-1752.2022.03.015 Abstract （542）   HTML （30）    PDF （732KB）（172）       Knowledge map    Save 【Objective】 Apis cerana cerana is the indigenous bee of China, which is also one of the important pollinator and economic insect. Odorant binding proteins (OBPs) are the key proteins in the olfactory perception of A. c. cerana. Based on the previous analysis about the sequence characteristics of AcerOBP7, the present study intends to further research its expression profiles and binding properties, so as to provide basic data for revealing the role of OBPs in the olfactory system of A. c. cerana.【Method】 In this paper, qRT-PCR was used to detect the spatio-temporal expression characteristics of AcerOBP7 in worker bees. AcerOBP7 protein was obtained by prokaryotic expression system, and the recombinant protein was purified using Ni-NTA column. Competitive fluorescence binding method was adopted, with 1-NPN (N-phenyl-1-naphthylamine) was used as a fluorescent probe, to determine the binding affinity of AcerOBP7 with pheromone and plant volatiles. dsAcerOBP7 was designed and synthesized, and the gene was silenced by feeding method, the silencing efficiency was detected by qRT-PCR. Combined with RNAi method, EAG was conducted to test and compare the difference of response values of AcerOBP7 to the candidate volatiles between the control and RNAi group.【Result】 qRT-PCR results showed that AcerOBP7 expressed significantly higher in the antennae of worker bees than that in other tissues (P<0.01), and the expression reached to the highest level at 20-day-old and the lowest at 1-day-old. The pET28a/AcerOBP7 expression vector was successfully constructed, and the high-purity recombinant protein was obtained using the Escherichia coli prokaryotic expression system. Fluorescence competition binding assay showed that AcerOBP7 had the strongest binding affinities with 9-ODA and 1-nonanol among 37 ligand molecules, with the Ki values was both 1.85 µmol·L-1, followed by (+)-limonene, 1-octene-3-ol, linalool, trans-ethyl-cinnamate, eucalyptol, (+)-3-carene and nonanal, and the Ki values were 1.87, 2.66, 2.72, 3.05, 3.88, 4.14 and 4.40 µmol·L-1, respectively, while AcerOBP7 had no binding ability with all the tested larval pheromone components. AcerOBP7 was successfully silenced by feeding dsRNA, and the highest interference efficiency could reach to 70.63%. The results of EAG assay after RNAi showed that the EAG values response to the tested odorant chemicals were all decreased, and the relative EAG values of 1-octen-3-ol, nonanal, eucalyptol and 9-ODA decreased significantly (P<0.05).【Conclusion】 AcerOBP7 is highly expressed in antennae of the forager bees and the recombinant protein can bind to a variety of odor molecules, suggesting that AcerOBP7 is a binding protein with broad spectrum, which may play an important role in the foraging behavior and feeding the queen of the worker bees. In addition, 1-octen-3-ol, nonanal, eucalyptol and 9-ODA are the ligands with high binding specificity to AcerOBP7. Table and Figures | Reference | Related Articles | Metrics

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Knockdown Goat KLF12 to Promote Subcutaneous Adipocytes Differentiation DU Yu,WANG Yong,MENG QingYong,ZHU JiangJiang,LIN YaQiu Scientia Agricultura Sinica    2022, 55 (1): 184-196.   DOI: 10.3864/j.issn.0578-1752.2022.01.015 Abstract （403）   HTML （33）    PDF （3647KB）（101）       Knowledge map    Save 【Background】Subcutaneous adipose tissue (SAT) under the skin is an important factor affecting the taste of meat. Exploring the molecular regulation mechanisms of SAT deposition is very important for breeding improvement and the development of animal husbandry. Krüppel-like factors 12 (KLF12) is a conserved transcription factor that evolutionarily conserved, and it was found that it could be expressed in a variety of cell types and control a wide range of cellular processes. 【Objective】 This study aimed to obtain the coding sequence (CDS) of goat KLF12 and to explore its molecular characteristics. Moreover, the study also intended to clarify the expression pattern of KLF12 in goat tissues and subcutaneous adipocytes, and to explore the role of KLF12 in goat subcutaneous preadipocytes differentiation via interference KLF12, so as to provide a theoretical basis for further research on the potential role of KLF12 in the process of fat deposition. 【Method】 In this study, the goat KLF12 CDS sequence was cloned by Reverse Transcription PCR ( RT-PCR) method, and the nucleotide sequence and amino acid sequence of goat KLF12 were analyzed on online bioinformatics analysis software. The Quantitative Real-time PCR (qRT-PCR) technology was used to detect the expression levels of KLF12 in goat heart, liver, abdominal fat, subcutaneous fat, triceps brachii, longissimus dorsi and other 14 tissues. Furthermore, the expression level of KLF12 in subcutaneous preadipocytes in different differentiation periods was investigated. Then, the goat KLF12 small interfering RNA (si-KLF12) was chemically synthesized and transfected into goat subcutaneous preadipocyte in vitro by using Lipofectamine RNAiMAX transfection reagent. Subsequently, 100 µmol·L-1 oleic acid induced adipocyte differentiation. Oil red O and Bodipy staining methods and qRT-PCR techniques were used to clarify the effects of interference KLF12 on the accumulation of lipid droplets in subcutaneous preadipocytes and the mRNA expression levels of adipose differentiation marker genes from the perspectives of morphology and molecular biology. 【Result】 The goat KLF12 (1 315 bp) were successfully obtained, which contained an Open Reading Frame (ORF) (1 209 bp) and encoded 402 amino acids. The subcellular localization results showed that KLF12 was mainly located in the nucleus. In addition, KLF12 had no transmembrane domain and signal peptide but 3 typical zinc finger domains (ZnF_C2H2) at amino acids 317-341, 347-371 and 377-399. Tissue expression profile showed that the expression level of KLF12 in goats’ heart and spleen were significantly higher than that in other tissues (P<0.01). Moreover, during subcutaneous preadipocytes differentiation, the expression level of KLF12 was peaked at 60 h. After transfection of si-KLF12 into goat subcutaneous preadipocytes, the results of oil red O and Bodipy saining showed that accumulation of lipid droplets in adipocytes were significantly increased. At the same time, the results of qRT-PCR showed that the expression levels of key adipogenic regulatory genes like lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor γ (PPARγ) were significantly increased (P<0.05), while the expression level of preadipocyte growth factor (Pref-1) was extremely significantly reduced (P<0.01). Combined with the morphological observation results and the changes in the expression levels of key adipogenic regulatory genes, it was speculated that KLF12 played a negative regulatory role in the differentiation of subcutaneous adipocytes. 【Conclusion】 By investigating the basic molecular biological characteristics and its expression pattern between tissues and cells of goat KLF12 and analyzing the potential regulatory effects of KLF12 on differentiation process of goat subcutaneous adipocytes, the results suggested that KLF12 played a negative role in goats subcutaneous preadipocytes differentiation, and this effect achieved by regulating LPL, PPARγ and Pref-1, which laid a foundation for further exploring the molecular mechanism of KLF12 in regulating the differentiation of adipocytes. Table and Figures | Reference | Related Articles | Metrics

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Development and Application of a Real-Time Fluorescent RPA Diagnostic Assay for African Swine Fever ZHANG JingYuan,MIAO FaMing,CHEN Teng,LI Min,HU RongLiang Scientia Agricultura Sinica    2022, 55 (1): 197-207.   DOI: 10.3864/j.issn.0578-1752.2022.01.016 Abstract （826）   HTML （31）    PDF （1623KB）（367）       Knowledge map    Save 【Objective】 After the first outbreak of African Swine Fever (ASF) in Shenyang, China in 2018, it has rapidly spread to the whole country, severely hitting the pig industry. This study aimed to establish an optimized nucleic acid testing technique for African Swine Fever Virus (ASFV), so as to provide a fast and accurate method for early diagnosis and accurate treatment of ASF outbreaks. 【Method】 Appropriate primers and probes were designed and screened for the conserved gene B646L (p72) of ASFV, and a real-time fluorescent RPA assay based on recombinase polymerase amplification (RPA) was established. The reaction system, reaction conditions and sample treatment steps were optimized. Specificity and sensitivity of the optimized detection method were evaluated by using quality controls. In addition, 1 009 clinical samples were tested by the optimized real-time RPA, after which the results were further confirmed by the real time PCR recommended by OIE and through virus isolation. 【Result】 A pair of primers-probe combinations was successfully screened, and a real-time fluorescence RPA for detection of ASFV p72 gene was developed. The total volume of optimized reaction system was 25 μL. The reaction conditions were set as 39℃ 10 s, 39℃ 20 s, 40 cycles on the fluorescence quantitative PCR instrument, and the whole amplification reaction needs about 20 min. The analysis method at room temperature could replace the traditional nucleic acid extraction method, thus the whole process of sample treatment, nucleic acid amplification and result reading could be completed in 30 min. Specific evaluation showed that the real-time RPA was negative for porcine parvovirus (PPV), pseudorabies virus (PRV), circovirus type1/2 (PCV1/2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV); the sensitivity evaluation showed that the assay could detect type I/II/IX ASFV samples, and could detect 10 copies/μL of ASFV positive simulated blood samples and 1﹕103.0 dilution of positive clinical samples, which was as sensitive as the OIE-recommended qPCR method. Seventeen out of 1 009 clinical samples were tested positive using the real-time RPA, with the same results as by qPCR, 17 positive cultures were obtained from virus isolation. 【Conclusion】 A real-time RPA diagnostic method for ASF was developed, which was proved to be simple, less time consuming with high sensitivity and specificity, providing a new, simple, specific and rapid diagnostic method for ASF. Table and Figures | Reference | Related Articles | Metrics

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Identification and Analysis of MicroRNAs in the Larval Gut of Apis cerana cerana FENG RuiRong,FU ZhongMin,DU Yu,ZHANG WenDe,FAN XiaoXue,WANG HaiPeng,WAN JieQi,ZHOU ZiYu,KANG YuXin,CHEN DaFu,GUO Rui,SHI PeiYing Scientia Agricultura Sinica    2022, 55 (1): 208-218.   DOI: 10.3864/j.issn.0578-1752.2022.01.017 Abstract （423）   HTML （26）    PDF （3398KB）（1025）       Knowledge map    Save 【Objective】 In this study, transcriptome-wide identification and analysis of miRNAs in the larval guts of Apis cerana cerana was conducted using a combination of small RNA-seq (sRNA-seq) technology and bioinformatic method, aiming to enrich the information of A. c. cerana miRNAs and offer a basis for further investigation of miRNA-regulated molecular mechanism underlying A. c. cerana larval gut development.【Method】 Gut samples of A. c. cerana 4-, 5-, and 6-day-old larvae (Ac1, Ac2, and Ac3 ) were sequenced using sRNA-seq technology, and clean tags were obtained after quality control. By using Blast tool, clean tags were continuously mapped to Apis cerana genome and miRBase database to identify known miRNAs and novel miRNAs. TPM method was used to perform normalization of miRNAs’ expression. The ratio of sRNAs, length distribution of miRNAs and first base bias were calculated with GraphPad Prism 7 software. Using related software, target mRNAs of above-mentioned miRNAs were predicted followed by GO and KEGG database annotation. Further, regulatory networks between genes associated with development and immune-related pathways and corresponding target miRNAs were constructed and analyzed, followed by visualization of regulatory networks with Cytoscape software. The authenticity of miRNA expression and sequence was verified by using Stem-loop RT-PCR, molecular cloning and Sanger sequencing.【Result】 In total, 371 known miRNAs and 64 novel ones of A. c. cerana were identified; their length was distributed among 18-25 nt, and the first base had an U bias. The aforementioned miRNAs could target 14 750 mRNAs, involving 2 270 GO terms such as ion binding, metal ion binding, membrane, membrane part and single-organism process, as well as 332 KEGG pathways including endocytosis, apoptosis, mTOR signaling pathway, RNA transport and insect hormone biosynthesis. Further investigation suggested that 156 miRNAs could target 67 genes relative to development-associated pathways such as Wnt, Hippo, Notch and mTOR signaling pathways, while 145 miRNAs could target 21 genes relevant to immune-associated pathways such as Toll, Imd/JNK, Jak-STAT signaling pathways and antimicrobial effectors. Stem-loop RT-PCR result indicated that specific fragments with expected sizes could be amplified from miR-8-y, miR-9-z, miR-14-y, miR-281-y, miR-283-x and miR-306-x; Sanger sequencing result demonstrated that sequences of above-mentioned six miRNAs were in accordance with those in deep sequencing result.【Conclusion】 Our findings provide number, structural characteristics and expression profile of A. c. cerana miRNAs, and unraveled that miRNAs in A. c. cerana larval gut potentially regulate a lot of life processes and cellular activities, part of miRNAs can participate in regulation of development-related and immune-related pathways by targeting corresponding mRNAs. Table and Figures | Reference | Related Articles | Metrics

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Genetic Diversity and Origin Characteristics of Chicken Species Based on Mitochondrial DNA D-loop Region TANG XiuJun,FAN YanFeng,JIA XiaoXu,GE QingLian,LU JunXian,TANG MengJun,HAN Wei,GAO YuShi Scientia Agricultura Sinica    2021, 54 (24): 5302-5315.   DOI: 10.3864/j.issn.0578-1752.2021.24.012 Abstract （354）   HTML （24）    PDF （4094KB）（132）       Knowledge map    Save 【Objective】 The purpose of this study was to investigate the genetic diversity and origin characteristics of the whole sequence of mitochondrial DNA D-loop region in chicken breeds (complete set line) with different growth rates, so as to provide a theoretical basis for breeding and traceability of broiler breeds. 【Method】 15 broiler breeds with different growth rates were used as research materials, including eight yellow-feathered broiler lines (5 medium-fast and 3 slow lines), two local chicken breeds (Gushi chicken and Tibetan chicken), two introduced chicken breeds (Recessive White and Anka), one white-feathered broiler (Ross 308), 817 hybrid broiler and one commercial layer line (Dawu Brown Eggshell Hens). Chicken blood was collected, and DNA was PCR amplified. The full sequences of mtDNA D-loop region of 683 individuals from 15 chicken breeds were sequenced, and the genetic diversity and haplotype characteristics of each chicken breed were analyzed using DnaSP 5.10 software. The genetic distance between breeds was calculated using MEGA 4.0 software, and then a phylogenetic tree was constructed between different haplotypes and the red original chicken. 【Result】The full sequence size of the D-loop region of 15 chicken breeds ranged from 1 231 to 1 232 bp, and individuals with sequence length of 1 231 bp had C-base deletion at 859 bp. 45 variant loci were detected in 683 individuals, which were combined into 53 haplotypes and could be divided into four haplotype groups, including A, B, C and E. Among them, the medium-fast broilers, 817 hybrid broilers and high-yielding laying hens were all haplotype E as the dominant haplotype (≥48.89%); the dominant haplotypes were B haplotypes for Hongguang black chickens, A haplotypes for Jinghai yellow chickens, and four haplotypes were relatively balanced for Xueshan chickens (the proportion of E haplotypes ≤38.46% for three chicken breeds); the haplotypes of local chicken breeds were A and C haplotypes for Gushi chickens and A and B haplotypes for Tibetan chickens. The genetic diversity of the 15 chicken breeds ranged from 0.496 to 0.853 for Hd and from 0.00146 to 0.00673 for Pi. The relatively rich genetic diversity was found in the Xinxing Dwarf Yellow Chicken, Xueshan Chicken, Jinghai Yellow Chicken and Ross 308; the relatively low genetic diversity was found in the Tibetan Chicken, the High Laying Chicken, the Anka Chicken, the Xinxing partridge Chicken No. 4 and the Xugang Yellow Chicken No. 1. The range of Kiumura two-parameter distance of 15 chicken breeds was 0.0016-0.0113, among which the intra-breed genetic distance of Ross 308 was the largest while the intra-breed genetic distance of 817 hybrid broiler and high-yielding chicken was the smallest; the inter-breed genetic distance was the largest between high-yielding chicken and Tibetan chicken, and the smallest between high-yielding chicken and 817 hybrid broiler; the genetic distance between medium-fast broiler was relatively small while the genetic distance between medium-fast broiler, slow-fast and local chicken breeds was relatively large; the genetic distance between Jinghai and local chicken breeds was relatively large. Cluster analysis showed that the haplotypes A, B, and gallus gallus spadiceus were clustered in one group. Haplotype E and gallus gallus murghi were clustered in another group. Haplotype C was clustered with four subspecies of jungle fowl, including gallus gallus murghi, gallus gallus spadiceus, gallus gallus gallus and gallus gallus bankiva. 【Conclusion】The genetic diversity of mitochondrial D-loop region varied among different chicken breeds; E haplotypes were strongly correlated with broiler growth rate, and E haplotypes were the dominant haplotypes in all medium and fast populations, while the proportion of E haplotypes in slow populations was less than 40%; the national chicken population had multiple red proto-chicken maternal origins, indicating that it was domesticated under neutral selection. The results of the study provided a theoretical basis for broiler breed selection and tracing as well as resource exploitation. Table and Figures | Reference | Related Articles | Metrics

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Advance in Genome-Wide Scan of Runs of Homozygosity in Domestic Animals ZHANG PengFei,SHI LiangYu,LIU JiaXin,LI Yang,WU ChengBin,WANG LiXian,ZHAO FuPing Scientia Agricultura Sinica    2021, 54 (24): 5316-5326.   DOI: 10.3864/j.issn.0578-1752.2021.24.013 Abstract （956）   HTML （63）    PDF （728KB）（272）       Knowledge map    Save Runs of homozygosity (ROH) is a long tract of homozygous genotypes commonly found in individuals and populations, which generates on the offspring’s genome inherited identical haplotypes from each parent. ROH contains a wealth of genetic information about populations, which makes it a useful tool for providing information to study how populations change over time. Moreover, ROH can estimate the genetic relationships between individuals to minimize the inbreeding mating rates. In addition, ROH can expose harmful mutations in the genome. The frequencies, sizes and distributions of ROHs in the genome are influenced by natural and artificial selection, recombination, linkage disequilibrium, population history, mutation rate and inbreeding level. Recently, with the use of high-throughput genotype technology and the reduction of second-generation sequencing costs, livestock and poultry breeding have entered into the genomic era. The selection intensity of the elites in livestock and poultry significantly increase to improve their performances, but it will increase inbreeding and cause inbreeding depression as well. Based on ROH molecular information, it is more accurately to detect past and nearest in close relative mating. The ROH-based inbreeding coefficient (FROH) can obtain an individual's true inbreeding coefficient, i.e. the realized inbreeding coefficient, and the pedigree-based FPED is the expectation value of inbreeding coefficient. In the absence of genealogical information, FROH can be used to infer information about a group's history and the inbreeding levels. Meanwhile, the selection reshapes ROH patterns in different regions of the genome. In addition, the selection can increase the homozygosities around the target point, and harmful mutations are thought to occur more frequently in the ROH region, which can be detected by ROH to reduce the risk of complex diseases. After long-term selection, one ROH appeared in multiple individuals’ genomes in the same population, resulting in ROH islands. It has confirmed the correlation between ROH and the selected genomic region. The candidate genes related to economic traits can be annotated on the ROH islands by means of biological information. In addition, ROH also provides a new perspective for assessing the genetic diversity in domestic animals. Genome-wide ROH detection on the population can used to investigate the genetic structure of this population, and FROH can evaluate the impact of inbreeding in the current breeding program, which can adjust breeding plans to protect the genetic diversity of varieties. Therefore, ROH has gradually become an important index to explore the historical population structure, the level of inbreeding, candidate gene identification. There are mainly two kinds of methods to identify ROH: observation genotype counting method and model-based analysis. Commonly used softwares include PLINK, GERMLINE, BEAGLE, GARLIC, etc. In practical applications, PLINK is the most common ROH detection tool. Since the SNP chip for cattle was firstly used in domestic animals, the cattle population was firstly conduct genome-wide ROH detection. Now, studies on ROH are becoming more popular in pigs, sheep and other domestic animals. This review mainly described the principle of ROH formation and its detection methods, as well as progress of its application in livestock and poultry, so as to provide reference for the genetic breeding of livestock and poultry. Table and Figures | Reference | Related Articles | Metrics

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Comparison of Genomic Prediction Accuracy for Meat Type Chicken Carcass Traits Based on GBLUP and BayesB Method ZHU Mo,ZHENG MaiQing,CUI HuanXian,ZHAO GuiPing,LIU Yang Scientia Agricultura Sinica    2021, 54 (23): 5125-5131.   DOI: 10.3864/j.issn.0578-1752.2021.23.016 Abstract （610）   HTML （41）    PDF （429KB）（394）       Knowledge map    Save 【Objective】 Predicting the breeding value is the core content of livestock breeding and the accurate predicting of breeding value is an important approach to improve the selection accuracy of breeding. Carcass traits are economic important traits for broilers. However, carcass traits can only be measured postmortem. Genomic selection may be a powerful tool because of its accurate prediction of breeding values of animals without own phenotypic information. At present, there are few reports on genomic selection on carcass traits for broilers. The purpose of this study was to compare the accuracy of genomic prediction of broiler carcass traits by using GBLUP and BayesB method. 【Method】 This study investigated the efficiency of genomic prediction in a white-feathered broiler population, which was collected from 3 362 white-feathered broilers. Five carcass traits, including breast muscle rate (BrR), breast muscle weight (BrW), carcass weight (CW), thigh muscle rate (ThR) and thigh muscle weight (ThW) were taken into account. All the individuals were genotyped with “IASCHICK” chicken 55 K SNP array. PLINK software was used for the quality control of genotype data. GBLUP and BayesB method were implemented with ASReml and hibayes package in R software, respectively. Generation validation were utilized to evaluate the accuracy of genomic prediction for twenty replicates for each trait.【Result】The results showed that the accuracy of genomic selection was almost positively correlated with the heritability of each trait. The validation results indicated that the prediction accuracy of BrR was the highest with GBLUP method, and the accuracies of BrR, BrW, CW, ThR and ThW were 0.3262, 0.2871, 0.2780, 0.2153, and 0.2126, respectively. Meanwhile, the accuracy of BrR was the highest with BayesB method, and the accuracies of BrR, BrW, CW, ThR and ThW were 0.3765, 0.2257, 0.1376, 0.2525, and 0.2844, respectively. The results showed that the prediction accuracy of BayesB method was slightly higher than that of GBLUP method. It took about 1 h and 7 h for GBLUP and BayesB method, respectively, to carry out the calculating procedure for one trait. 【Conclusion】 In conclusion, the prediction accuracy of BrR, ThR and ThW with BayesB method was higher than that with GBLUP method, while the prediction accuracy of BrW and CW with GBLUP method was higher than that with BayesB method. However, the calculating time for BayesB method was longer than that for GBLUP method. In breeding practice, the balance of prediction accuracy and computational efficiency should be comprehensively considered to predict the genomic estimated breeding value. Table and Figures | Reference | Related Articles | Metrics

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Effects of miR-31-5p on the Proliferation and Apoptosis of Hair Follicle Stem Cells in Goat FENG YunKui,WANG Jian,MA JinLiang,ZHANG LiuMing,LI YongJun Scientia Agricultura Sinica    2021, 54 (23): 5132-5143.   DOI: 10.3864/j.issn.0578-1752.2021.23.017 Abstract （380）   HTML （30）    PDF （1939KB）（402）       Knowledge map    Save 【Objective】 The Yangtze River Delta White Goat is the only goat breed that can produce superior-quality brush hair in China and the world. The transcriptome sequencing results of the research team showed that there were significant differences in the expression level of MAP3K1 in the individual skin tissues of superior-quality brush hair and normal-quality brush hair. This study aimed to explore the key miRNAs that interacted with MAP3K1 during the formation of superior-quality brush hair and their effects on the proliferation and apoptosis of goat hair follicle stem cells. 【Method】 The bioinformatics websites (StrBase, miRDB, TargetScan, miRWalk, DAVID, KEGG, and RNAhybrid) were used to predict and select the miRNAs, with targeted relationship with MAP3K1, and use the online website Venny 2.1 to draw a Venn diagram. Through the construction of miR-31-5p overexpression vector, wild-MAP3K1, wild/Mut-RASA1 Luciferase Reporter assay vector, the relationship between miR-31-5p and MAP3K1, RASA1 was verified, and the effects of miR31-5p on MAP3K1, RASA1 mRNA and protein expression were detected by qPCR and Western Blot. In order to explore the effect of overexpression of miR-31-5p on cell proliferation and apoptosis, the mRNA and protein expression levels of proliferation marker gene (PCNA,CDK1,CCND2), anti-apoptotic gene (Bcl-2) and pro-apoptotic gene (Bax) in hair follicle stem cells transfected with miR-31-5p were detected, and the effects of overexpression of miR-31-5p on the viability, cell cycle and apoptosis of hair follicle stem cells were verified by CCK-8, EdU, flow cytometry. 【Result】 Through the database, the final score of the three miRNAs were predicted, which might relatively highly interact with MAP3K1. Then, combined with the known miRNAs studies in skin and hair follicle cells, miR-31-5p with highest score was selected as research object. After transfection of miR-31-5p, the relative expression of miR-31-5p in cells was detected, and it was found that the expression of miR-31-5p was significantly higher than that in the control group and blank vector group (P<0.01). The results of double luciferase reporter genes showed that overexpression of miR-31-5p could increase the activity of MAP3K1. Combined with Target Scan and KEGG database, it was predicted that miR-31-5p could target RASA1, the upstream inhibitor of MAP3K1 in MAPK signal pathway. In order to verify the relationship between miR-31-5p and RASA1, it was found that overexpression of miR31-5p inhibited the activity of RASA1 (P<0.01); qPCR and Western Blot assays showed that overexpression of miR-31-5p significantly inhibited the expression of mRNA and protein of RASA1 and promoted the expression of MAP3K1 (P<0.01). CCK-8 assays showed that overexpression of miR-31-5p increased the ability of cell proliferation. EdU staining showed that the rate of positive cells overexpressing miR-31-5p was significantly higher than that in the blank group (P<0.01), and promoted cell proliferation. Cell cycle data showed that after overexpression of miR-31-5p, the proportion of cells in G1/G0 phase was 52.23%, which was significantly lower than that in Control group (56.81% P<0.01). It slowed down the cell arrest in G1/G0 phase, but there was no significant difference between S phase and G2/M phase, however there was still an upward trend. Through apoptosis experiment, it was found that the survival rate of miR-31-5p group was 93.8%, and the total apoptosis rate was 4.9%; while that of control group was only 90.1%, and the total apoptosis rate was 8.41%, which indicated that the apoptosis rate decreased significantly after overexpression of miR-31-5p. Finally, the effects of miR-31-5p on proliferation and apoptosis-related genes were detected. It was found that overexpression of miR-31-5p significantly increased the mRNA and protein expression levels of proliferation marker genes and anti-apoptosis genes (Bcl-2), and decreased the mRNA and protein expression levels of pro-apoptosis gene (Bax). According to the results of the research, the molecular mechanism of miR-31-5p in hair follicle stem cells was revealed. 【Conclusion】 miR-31-5p targeted RASA1 and up-regulated the expression level of MAP3K1, thereby promoting the proliferation of hair follicle stem cells and inhibiting their apoptosis. It provided a theoretical basis for further investigating the molecular mechanism that regulates the characteristics of superior-quality brush hair of the Yangtze River Delta White Goats. Table and Figures | Reference | Related Articles | Metrics

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Biofilm-Forming Phenotype, Antibacterial Resistance Genes, Integrase Genes and Virulence Genes Detection of Escherichia coli Isolated from Yaks and Tibetan Pigs in Northwest Sichuan Plateau CHEN ChaoXi,LI YuHan,TAN Min,WANG Lu,HUANG ZhiHong Scientia Agricultura Sinica    2021, 54 (23): 5144-5162.   DOI: 10.3864/j.issn.0578-1752.2021.23.018 Abstract （404）   HTML （25）    PDF （1984KB）（392）       Knowledge map    Save 【Objective】To improve the understanding of biological properties of drug resistance status, virulence characteristics and the predominant phylogroups of Escherichia coli (E.coli) isolated from yaks and Tibetan pigs in northwest Sichuan Plateau, biofilm-forming ability, antibacterial resistance genes, virulence genes, integrase genes and phylogenetic analyses were carried out in current study.【Method】Fecal samples and gastrointestinal contents from yaks and Tibetan pigs were collected for E.coli isolation and identification using MacConkey agar and 15e enterobacteriaceae bacterial biochemical coding identification tube. Modified semi-quantitative crystal violet staining method and microdilution broth method were used for biofilm-forming ability and antibacterial sensitivity testing to 24 antibacterial agents, respectively. Meanwhile, the detection of 28 antibacterial resistance genes, 2 integrase genes and 15 virulence genes and phylogenetic analyses were performed by conventional PCR or multiple PCR method. 【Result】The results showed that: (1) 329 strains of E.coli were isolated and identified from 471 feces and gastrointestinal samples collected from yaks and Tibetan pigs, and the isolation rate was 78.9%. (2) Most of the 329 strains of E.coli performed weak or absent biofilm-forming ability, and only 2 strains showed strong biofilm formation phenotype (one isolated from yak and the other isolated from Tibetan pig). (3) Most of the 329 strains of E.coli revealed drug resistance to 24 antibacterial agents and were multi-drug resistant, among which, the drug resistance rates to Sulfamethoxazole, Sulfadimidine, Streptomycin, Chloramphenicol, Ampicillin, Rifampicin, and Oxytetracycline were relatively high, and were sensitive to Aminoglycosides (Kanamycin, Amikacin, Spectinomycin), β-lactams(Ceftiofur, Ceftriaxone, Cefazolin), Quinolones (Nalidixic acid, Sarafloxacin, Enrofloxacin, Ciprofloxacin, Danofloxacin, Levofloxacin) and Colistin B. (4) Twenty-one antibacterial resistance genes (ARGs) were detected positive and the other seven ARGs (cat1, cat2, blaCMY-2, blaSHV, tetC, tetG, and tetX) were detected negative, among which aac(6')-Ib was the most prevalent gene, followed by sul1 and floR, with the detection rates over 30%. There existed correlation between drug resistance to quinolones antibiotics and qnrA in Tibetan pig-derived E.coli, and for yak-derived E.coli, the strains resistant to β-lactam antibiotics existed correlation between blaTEM and blaDHA. (5) The detection rates of integrase genes intⅠ1 and intⅠ2 were 30.09% (99/329) and 4.56% (15/329), respectively. And integrase genes intⅠ1 and intⅠ2 were simultaneously detected in 10 isolates (2 yak-derived and 8 Tibetan pig-derived, respectively). (6) Virulence genes of agn43, sitA, ompT, eaeA, bcsA, fimC, LT, fyuA and irp2 were all positively detected, but stx1, stx2, ehxA, bcsB, hlyA, and hlyE were not detected. There existed 38 different virulence genotypes in 329 strains of E.coli and 285 of which carried at least one of the seven virulence genes except for agn43 and bcsA, some strains carried six virulence genes at most. (7) Among the 21 ARGs, types of ARGs in phylogroup A and B1 were more abundant than those of phylogroup B2 and D; In phylogroup A, sul3, qnrS, tetM were the most widely distributed ARGs, and for phylogroup B1 most widely distributed ARGs were sul1 and aac(6')-Ib, without tetM and qnrA; Seven virulence genes were mainly distributed in phylogroup A and B1, fimC, sitA and ompT genes were mainly distributed in phylogroup A and B1, and eaeA, fyuA and irp2 were the mainly distributed genes in phylogroup B1; LT was mainly distributed in phylogroup A (only one distributed in phylogroup D).【Conclusion】 In summary, the resistance status of 329 strains of E.coli was serious, revealing various drug resistance profiles and virulence genotypes. The current study could provide data support and theoretical basis for yak and Tibetan pig colibacillosis treatment, mechanism of pathogenesis and rational use of antibacterial agents in northwest Sichuan Plateau. Table and Figures | Reference | Related Articles | Metrics

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Effects of Dietary Supplemental Pattern of Trace Eloments on the Growth Performance, Carcass Traits and Meat Quality of Broilers ZHANG Lan,WANG LiangZhi,HUANG YanLing,LIAO XiuDong,ZHANG LiYang,LÜ Lin,LUO XuGang Scientia Agricultura Sinica    2021, 54 (22): 4906-4916.   DOI: 10.3864/j.issn.0578-1752.2021.22.016 Abstract （467）   HTML （35）    PDF （476KB）（447）       Knowledge map    Save 【Objective】 This experiment was conducted to determine the effects of dietary supplemental pattern of trace elements on growth performance, carcass traits and meat quality of broiler chicks, so as to provide the experimental basis for the reasonable addition of trace elements to broiler diets.【Method】 A single-factor completely randomized design was adopted in this experiment. A total of 240 one-day-old Arbor Acres (AA) broiler chicks were randomly allotted by body weight to 1 of 5 treatments with 6 replicate cages of 8 birds per cage. The trace elements were added to the corn-soybean basal diet as follows: the inorganic trace elements according to NRC (1994) recommendation for broiler chicks (T1: the added levels of copper (Cu), Iron (Fe), manganese (Mn), zine (Zn) and selenium (Se) were 8, 80, 60, 40 and 0.15 mg·kg-1 during 1-42 days, respectively), the inorganic trace elements according to the recommendation for broiler chicks in Chinese Feeding Standard of Chicken (NY/T 33-2004) (T2: the added levels of Cu, Fe, Mn, Zn and Se were 8, 100, 120, 100 and 0.3 mg·kg-1 for 1-21 days old, respectively; and 8, 80, 120, 80 and 0.3 mg·kg-1 for 22-42 days old, respectively), the inorganic trace elements according to the previous results of trace elements requirements from our lab (T3: the added levels of Cu, Fe, Mn, Zn and Se were 4, 40, 110, 60 and 0.35 mg·kg-1 during 1-21 days, respectively; and 0, 30, 80, 40 and 0.35 mg·kg-1 during 22-42 days, respectively), the decrement levels of organic trace elements according to the previous results of from our lab (T4: the added levels of Cu, Fe, Mn, Zn and Se were 2, 30, 80, 40 and 0.25 mg·kg-1 during 1-21 days, respectively; and 0, 30, 80, 40 and 0.25 mg·kg-1 during 22-42 days, respectively), and the organic trace elements according to the recommendation for broiler chicks in NY/T 33-2004 (T5: the added levels of Cu, Fe, Mn, Zn and Se during 1-21 and 22-42 days were the same as those in T2), respectively. The inorganic trace element sources (feed grade) were Cu sulfate pentahydrate, Fe sulfate monohydrate, Mn sulfate monohydrate, Zn sulphate monohydrate and sodium selenite, and the organic trace element sources (feed grade) were Cu mothionine, Fe glycine, Mn methionine, Zn glycinate and Se yeast, respectively. The experiment lasted for 42 days.【Result】The results showed that those different supplemental patterns of trace elements had no significant effects (P>0.05) on the average daily feed intake and average daily gain. Broilers from T2 had higher (P<0.05) feed to gain ratio during 22-42 days than those from T1, T4 and T5, and no difference was detected between T2 and T3 (P>0.05). Broilers from T2 had higher (P<0.05) feed to gain ratio during 1-42 days than those from other groups, and there were no differences (P>0.05) among other groups. The different supplemental patterns of trace elements had no significant effects on (P>0.05) the carcass traits, L* and a* values, pH values and drip losses of breast and thigh muscles. The breast muscle b* value of broilers from T5 was higher (P<0.05) than that of broilers from T1 and T3, and no difference was observed (P>0.05) between T5 and T4. The shear force of thigh muscle from T4 was lower (P<0.05) than that from T1 or T5, and the muscle tenderness was relatively well. 【Conclusion】Under this experimental conditions, the group with decrement supplement of organic trace elements based on our previous results (T4: the added levels of Cu, Fe, Mn, Zn and Se were 2, 30, 80, 40 and 0.25 mg·kg-1 during 1-21 days, and 0, 30, 80, 40 and 0.25 mg·kg-1 during 22-42 days, respectively) was better than other groups in improving the growth performance and meat quality of broiler chicks. Table and Figures | Reference | Related Articles | Metrics

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Effects of Glucose Oxidase on Growth Performance, Immune Functions and Intestinal Health of Ducks Challenged by Escherichia coli LIU Jiao,CHEN ZhiMin,ZHENG AiJuan,LIU GuoHua,CAI HuiYi,CHANG WenHuan Scientia Agricultura Sinica    2021, 54 (22): 4917-4930.   DOI: 10.3864/j.issn.0578-1752.2021.22.017 Abstract （483）   HTML （31）    PDF （698KB）（359）       Knowledge map    Save 【Objective】 This experiment was conducted to study the effects of glucose oxidase (GOD) on growth performance, immune functions and intestinal health of ducks challenged by Escherichia coli (E. coli) O88 and its mechanism to find a substitute for antibiotics to prevent duck colibacillosis. 【Method】 A total of 144 one-day-old healthy male lean Peking ducklings were selected and randomly divided into three groups with six replicates and eight ducks per replicate. Ducks were fed the three diets supplemented with nothing (Control group), 30 mg·kg-1virginiamycin (Antibiotic group) and 200 U·kg-1 GOD in basal diet, respectively. On day 7, all ducks were orally taken 0.2 mL E. coli O88 (3×109 CFU/mL) twice, 8 hours apart. The experiment lasted for 28 days. 【Result】The results showed as follows: 1) Compared with the control group, adding antibiotic or GOD in the diet significantly increased the average daily gain and average daily feed intake of 1 to 14-day-old attacking ducks (P<0.01). 2) The number of serum white blood cell of 28-day-old attacking ducks was significantly reduced and the percentage of serum lymphocyte was significantly increased by adding antibiotic and GOD in diet (P<0.01), and the number of serum red blood cell of 28-day- old attacking ducks was significantly reduced by adding GOD in diet (P<0.05). 3) Serum MDA at day 14 (P<0.05) and CAT contents at day 28 (P<0.01) were significantly reduced by adding antibiotic and GOD, serum T-AOC at day 28 was significantly increased (P<0.05), and CAT at day 14 showed a tendency to decrease by adding GOD (P=0.087). 4) Supplementation of antibiotic and GOD significantly decreased endotoxin of 14 and 28-day-old attacking ducks (P<0.01). 5) Adding antibiotic or GOD significantly decreased the concentration of IL-1β and IL-6 of ducks at day 14 and 28 and TNF-α at d 28 (P<0.05), no significant differences between the GOD and the antibiotic groups（P>0.05）. 6) The concentration of serum diamine oxidase and D-lactic of 14 and 28-day-old attacking ducks were significantly reduced by adding antibiotic and GOD (P<0.05), no significant differences between the GOD and the antibiotic groups (P>0.05). 7) The addition of GOD increased the number of unique OTUs in ileum, reduced the contents of E. coli, and increased the relative abundance of probiotics such as Lactobacillus and Bifidobacterium. 【Conclusion】 In conclusion, addition of GOD in the diet can balance the structure of intestinal flora, reduce the production of bacterial endotoxin and decrease oxidative stress, and then maintain the integrity of intestinal mucosa and prevent activation of inflammatory signal pathway by endotoxin and occurrence of inflammatory reaction, so as to improve intestinal health and growth of ducks infected with E. coli. The GOD can be used as antibiotics substitute to prevent or alleviate colibacillosis of ducks. Table and Figures | Reference | Related Articles | Metrics

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Identification of circINHBB During Follicular Atresia and Its Effect on Granulosa Cell Apoptosis MA MengNan,WANG HuiMing,WANG MiaoMiao,YAO Wang,ZHANG JinBi,PAN ZengXiang Scientia Agricultura Sinica    2021, 54 (18): 3998-4007.   DOI: 10.3864/j.issn.0578-1752.2021.18.017 Abstract （394）   HTML （29）    PDF （2129KB）（470）       Knowledge map    Save 【Background】The follicular development is related to the reproductive capacity and production performance of mammals. Follicular development includes the growth and development of oocytes, the initiation and growth of the primordial follicles, and the development of primary follicles. In addition, these biological processes are closely related to the proliferation and apoptosis processes of follicle granulosa cells. The main inducement of follicular atresia is the apoptosis of granulosa cells, which is extremely complex and regulated by various cytokines. Previous studies have shown that non-coding RNAs (ncRNAs) are involved in various biological processes as regulators, including cell proliferation and apoptosis. Circular RNA (circRNA) is a new type of ncRNAs, widely existing in organisms. The circRNA is involved in the regulation of various physiological processes, but there are few studies in the field of livestock reproduction, especially, its expression, distribution and biological function in the pig ovary and follicle are rarely studied. Inhibin (INH) is a gonadal glycoprotein hormone, which is mainly produced by granulosa cells of ovarian follicles in female animals. It is an important factor to control ovulation in mammals. Previous studies have shown that the coding gene of the precursor of INH βsubunit in porcine follicles may form a circular RNA, i.e. circINHBB. 【Objective】In this study, the sequence structure and cell distribution of circINHBB in porcine follicular tissue were verified, and its expression difference in healthy and atresia follicles analyzed to explore the effect of circINHBB on cell apoptosis in porcine granulosa cells cultured in vitro, so as to broaden the research ideas of circRNAs in the field of livestock breeding, and provide reference for improving the reproductive capacity of livestock. 【Method】 Firstly, the porcine follicles were collected, amplified by PCR and sequenced by Sanger sequencing to verify the sequence structure of circINHBB. Secondly, the follicles were divided into two groups, including healthy and atretic, and the expression difference of circINHBB was detected by qRT PCR in each group. Then, the FISH experiment was used to verify the distribution of circINHBB in the granulosa cells. Finally, the porcine ovarian granulosa cells were cultured in vitro, circINHBB was knocked down by siRNA, and the effect of circINHBB on granulosa cell apoptosis was detected by flow cytometry. 【Result】Sanger sequencing confirmed the existence of circINHBB in porcine follicles, and it was a circular RNA formed by reverse splicing of the INHBB mRNA 5’UTR. qRT PCR results confirmed that the expression level of circINHBB was higher in healthy follicles, but significantly decreased in atresia follicles. FISH further verified the distribution of circINHBB in the cytoplasm of porcine granulosa cells. After siRNA knockdown, the expression of circINHBB was significantly decreased, and the apoptosis rate of granulosa cells increased significantly. 【Conclusion】The circular structure of circINHBB was verified in the porcine ovary, and its distribution in cytoplasm suggested that circINHBB might be involved in post-transcriptional regulation. The fact that the expression of circINHBB was lower in atresia follicles, and knockdown of circINHBB could significantly increase the level of apoptosis, indicating that circINHBB had a significant inhibitory effect on the apoptosis of porcine granulosa cells. Thus, it might be an active regulatory factor in the growth and development of follicles. This study explored the role of circRNA in follicular atresia and granulosa cell apoptosis, which was an important supplement to the regulatory mechanism of follicular atresia. Table and Figures | Reference | Related Articles | Metrics

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Effects of Dietary Zearalenone Adsorbent on the Distribution and Expression of LC3 and PCNA in the Uterus of Gilts HUANG LiBo,WANG JinQuan,GAO WenBo,CHEN HongJu,HOU YanMeng,YUAN XueJun,WANG ChunYang Scientia Agricultura Sinica    2021, 54 (18): 4008-4017.   DOI: 10.3864/j.issn.0578-1752.2021.18.018 Abstract （349）   HTML （28）    PDF （5193KB）（529）       Knowledge map    Save 【Objective】 It has been proved that zearalenone (ZEA) can activate estrogen sensitive gene through estrogen receptor in animals, resulting in reproductive toxicity, and affecting endometrial cell growth, oocyte maturation and follicular granulosa cell proliferation. The purpose of this experiment was to investigate the effects of ZEA adsorbent (zeolite + montmorillonite combinations) on distribution and expression of LC3 and PCNA in the uterus of gilts, and to discuss the detoxification effect of the new ZEA adsorbent from the perspective of histochemistry. 【Method】 A total of 48 healthy gilts with a body weight of 30±2.11 kg were randomly divided into 6 groups (n = 8): control group with basal diet, the ZEA group with basal diet + 1.008 mg∙kg-1ZEA, the 0.1% ZEA adsorbent (ZEA 0.1) group with ZEA diet + 1.0 g∙kg-1 new adsorbent, the 0.25% ZEA adsorbent (ZEA 0.25) group with ZEA diet + 2.5 g∙kg-1 new adsorbent, the 0.5% ZEA adsorbent (ZEA 0.1) group with ZEA diet + 5.0 g∙kg-1 new adsorbent, and the ZEA montmorillonite (ZEA+M) group with ZEA diet + 2.5 g∙kg-1 montmorillonite. The preliminary trail period was 7 d, and the trial period was 21 d. 【Result】 ZEA increased the uterine organ index, and adding 0.25% and 0.5% adsorbents in the diet decreased the index of uterine organs obviously. The LC3 and PCNA positive cells were mainly distributed in the glandular epithelial cells, and the LC3 immunoreactivity of lumen epithelial cells were weaker than that of glandular epithelial cells. The immunoreactivity of LC3 in control group was stronger than that of ZEA group. The immunoreactivity and number of positive cells of LC3 in the ZEA new adsorbent group were significantly higher than those in the ZEA group, and there was a dose-dependent trend, but the effect of the ZEA-M group was increased slightly more than that of ZEA group. The results of PCNA in the luminal and glandular epithelium were contrary to that of LC3. The WB and qRT-PCR results also showed that 0.25% and 0.5% new adsorbents could promote the expression of LC3, increase autophagy, and decrease the PCNA immunoreactive reaction. These results indicated that ZEA inhibited the expression of LC3, inhibited autophagy and destroyed the homeostasis of endometrial cells and glandular epithelial cells. However, the new adsorbent could increase the expression of LC3 protein and decrease the expression of PCNA, which could promote autophagy and resist the abnormal proliferation of endometrial cells caused by ZEA. The new adsorbent had a good protective effect on uterine cells through bidirectional regulation.The experimental results provided theoretical basis for the further application of the new adsorbent. 【Conclusion】In this experiment, ZEA induced the uterus proliferation reaction, the new adsorbent (zeolite + montmorillonite combinations) resisted the negative effect of ZEA on the normal physiological function of uterus within a certain limits. As a result, 0.25% and 0.5% doses of this newadsorbents was suitable. Table and Figures | Reference | Related Articles | Metrics

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The Changes of Eggshell Quality in the Laying Cycle of Hy-Line Brown Layers MA LingLing,FENG Jia,WANG Jing,QI GuangHai,MA YouBiao,WU ShuGeng,ZHANG HaiJun,QIU Kai Scientia Agricultura Sinica    2021, 54 (17): 3766-3779.   DOI: 10.3864/j.issn.0578-1752.2021.17.017 Abstract （915）   HTML （66）    PDF （2546KB）（736）       Knowledge map    Save 【Objective】 In this study, the changes of egg physical and eggshell quality properties from peak to late phase of production (31 to 80-wk-old) of Hy-Line Brown layers were observed to investigate the critical period when eggshell quality deteriorated and the changes of eggshell structure and components, so as to provide reference for improving the eggshell quality of the late phase of production. 【Method】 A total of 84 healthy 30-wk-old Hy-Line Brown layers were randomly divided into 7 replicates with 12 birds each. All hens had free access to corn-soybean meal basal diet and water for 50 weeks. Three eggs per replicate were collected every day for 3 consecutive days at the hen age: 31, 36, 41, 46, 50, 55, 60, 65, 70, 75 and 80-wk-old, and then eggshell physical and mechanical properties were tested. Eggshell from the hens aged: 31, 41, 50, 60, 70 and 80-wk-old was tested the following items, eggshell ultrastructure of cross section and inner surface by scanning electron microscope, the crystal structure by X-ray diffraction analyzer, organic content by burning method, total eggshell protein content by Coomassie brilliant blue method, and eggshell calcium and phosphorus content by inductively coupled plasma atomic emission spectrometry. 【Result】 (1) Egg weight, egg length and eggshell area of 31 to 80-wk-old were increased linearly (P<0.01). Eggshell weight, eggshell percentage, eggshell thickness and eggshell index were increased and then decreased with hen age increasing (P<0.05). After 50-wk-old, eggshell strength and eggshell fracture toughness were decreased significantly (P <0.05), and reached the lowest value from 65 to 80-wk-old (P<0.05). (2) Based on the principal component loading analysis of eggshell quality, in PC1, the load values of eggshell strength, eggshell percentage, eggshell fracture toughness and eggshell index were higher, while in PC2, the loading values of eggshell weight, eggshell thickness and eggshell area were higher. According to properties of eggshell quality, the laying cycle was divided into 2 stages of 31 to 50-wk-old and 55 to 80-wk-old, and the later could further be divided into 55 to 60-wk-old and 65 to 80-wk-old. (3) For eggshell ultrastructure, mammillary thickness and rate of 70 and 80-wk-old were significantly lower than those of 31 to 60-wk-old (P<0.05), and mammillary knob density was significantly lower than that of 31-wk-old (P<0.05). (4) There were no significant changes in the contents of organic matter and total protein, per unit area and per eggshell and no significant change in calcium content per eggshell (P>0.05), but the calcium content per unit area of 70 and 80-wk-old were decreased significantly (P<0.05). The phosphorus content of 60, 70 and 80-wk-old was significantly lower than others (P<0.05), while the phosphorus content per eggshell and per unit area were significantly lower than those of 31-wk-old (P<0.05). (6) The eggshell mechanical properties was significantly positively correlated with the thickness of calcified layer and effective layer, mammillary knob density, effective layer rate, calcium content per unit area and the content of phosphorus, per eggshell and per unit area (P<0.05), was significantly negatively correlated with mammillary layer rate (P<0.05). 【Conclusion】 According to eggshell physical and mechanical properties, the laying cycle of Hy-Line Brown layers could be divided into 2 stages: 31 to 50-wk-old and 55 to 80-wk-old. The eggshell mechanical properties were decreased significantly after 65-wk-old, as the changes of eggshell ultrastructure layer thickness and rate might contribute to the decrease of eggshell mechanical properties in the laying cycle. The decrease of eggshell phosphorus content might be one of the reasons for the decrease of eggshell mechanical properties from 60 to 80-wk-old. From 70 to 80-wk-old, eggshell ultrastructural variation and the decrease of eggshell calcium content per unit area might aggravate the decrease of eggshell mechanical properties Table and Figures | Reference | Related Articles | Metrics

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Analysis of Plasmid-Mediated AmpC β-lactamases Gene and Plasmid in Poultry Proteus mirabilis Strains ZHAO ShiYu,JIAO JiaJie,DONG NingNing,PAN YuanYue,CUI MengMei,PAN YuShan Scientia Agricultura Sinica    2021, 54 (17): 3780-3788.   DOI: 10.3864/j.issn.0578-1752.2021.17.018 Abstract （316）   HTML （33）    PDF （1604KB）（357）       Knowledge map    Save 【Objective】 The aim of this study was to probe the genotype of AmpC β-lactamases gene and the complete nucleotide sequence of the conjugative plasmid carrying blaCMY-2 in poultry P. mirabilis strains, so as to provide a theoretical basis for the prevent spreading of multidrug-resistant poultry P. mirabilis strains. 【Method】 Twenty-one P. mirabilis strains were characterized for the confirmation of AmpC β-lactamases genes by using three-dimensional test, polymerase chain reaction (PCR) amplification and sequencing. The blaCMY-2-carrying P. mirabilis strains were further evaluated using pulse field gel electrophoresis (PFGE) and conjugation experiments. The complete nucleotide sequence of conjugative plasmid pC12 was determined by using high-throughput sequencing platform and compared with closely related plasmids. 【Result】 Six of twenty-one P. mirabilis strains produced AmpC enzymes, all of which carried the blaCMY-2 gene and the detection rate was 28.6%. Antimicrobial susceptibility testing showed that six P. mirabilis strains exhibited high resistance to ampicillin, cefoxitin, doxycycline, florfenicol and colistin, but were susceptible to ceftazidime and amikacin. Conjugation assay revealed the blaCMY-2 gene was successfully transferred from P. mirabilis C12 to E. coli C600 recipient strain, however, conjugation experiments failed to obtain transconjugants for other blaCMY-2-bearing strains, despite repeated attempts. Three PFGE patterns of six P. mirabilis strains were determined. The findings demonstrated the vertical and horizontal dissemination of blaCMY-2 gene in poultry P. mirabilis isolates. Sequence analysis revealed the P. mirabilis C12 harbored a conjugative plasmid, designated as pC12. pC12 was found to be a multi-drug resistant type 1b IncC plasmid with 161 319-bp size and an average GC content of 52.45%, and had at least 161 predicted open reading frames. The complete sequence of pC12 has been submitted to GenBank with the accession number MT320534. The pC12 harbored three antibiotic resistance regions: the first region, antibiotic resistance island ARI-B, carried floR, tet(A), strA, strB, and sul2 genes; the second region, ISEcp1-blaCMY-2-blc-sugE, was a typical structure, and the ISEcp1 was truncated by IS10R; the third region, ARI-A, was a hybrid Tn1696tnp-pDUmer module. The ARI-A contained a sul1-containing class 1 integron with cassette array (aac(6')-Ib-cr|arr3|dfrA27|aadA16), and a mercury resistance cluster merEDBAPTR, and inserted into the plasmid backbone generating 5-bp direct repeats (TTGTA). 【Conclusion】 All the AmpC-producing P. mirabilis strains carried the blaCMY-2 gene, and one of them harbored an epidemic type 1 IncC conjugative plasmid. Three PFGE patterns were identified. The findings demonstrated the vertical and horizontal dissemination of blaCMY-2 gene in poultry Proteus mirabilis isolates. IncC plasmid was one of the predominant vehicles for the dissemination of multiple resistance genes, such as blaCMY-2, tet (A), floR or class 1 integron cassette, which further increased the difficulty for the treatment of the infection caused by P. mirabilis. More attention should be paid on the epidemiology of IncC plasmid in pathogenic bacteria. Table and Figures | Reference | Related Articles | Metrics

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Energy and Protein Requirements of Alpine Merino Growing Sheep WANG Chen,ZHANG HongWei,WANG HuCheng,SUN XiaoPing,LI FaDi,YANG BoHui Scientia Agricultura Sinica    2021, 54 (16): 3537-3548.   DOI: 10.3864/j.issn.0578-1752.2021.16.015 Abstract （425）   HTML （38）    PDF （507KB）（1262）       Knowledge map    Save 【Objective】The objective of this study was to determine the energy and protein requirements of Alpine Merino growing sheep, which could provide basic data and theoretical support for the formulation of feeding standards. 【Method】 It was selected that forty eight 14-month old Alpine Merino growing male sheep (n = 24, an initial body weight of 42.96 ± 3.13 kg) and female sheep (n = 24, an initial body weight of 32.85 ± 3.21 kg). The experiment lasted for 40 days, including 5-day transition period, 10-day pre-trial period and 25-day formal period. In the transition period, oat hay was fed in the morning and evening every day, and a small amount of total mixed pellet feed was fed at noon to realize the transition from forage to pellet feed; in the pre-trial period, the sheep were free to eat the whole mixed pellet feed in a single pen, and the feed intake was recorded; in the formal period, the sheep were grouped according to the feed intake of the pre-trial period feeding, growing male and female sheep were divided into 4 groups, fed with 4 levels of free feeding (AL group), 80% (IR80 group), 60% (IR60 group) and 40% (IR40 group) with 6 replicates in each group and 1 sheep in each replicate. In the last five days of the formal period, the digestion and metabolism trials were carried out continuously with the method of total collection of feces and urine. During this period, the feeds, feces and urine of each sheep were accurately recorded and collected. In the last two days, the respiratory calorimetry was carried out by the indirect calorimetry of respiratory mask to determine the growth performance, energy utilization and nitrogen balance indexes. The energy and protein requirements of Alpine Merino growing sheep were obtained by regression analysis. 【Result】 There was no significant difference in the initial weight between the growing male and female sheep (P>0.05). With the decrease of feed intake level, the final weight, average daily gain, dry matter intake, gross energy intake, fecal energy, digestible energy, metabolic energy, retained energy, nitrogen intake, fecal nitrogen, digestible nitrogen, retained nitrogen, retained nitrogen/nitrogen intake and retained nitrogen/digestible nitrogen decreased significantly, that is, AL>IR80>IR60>IR40 (P<0.05). However, feed intake level had significant effect on gas emission of Alpine Merino growing female sheep (P<0.05). Oxygen consumption and carbon dioxide production of IR40 group were significantly lower than those of the other three groups (P<0.05). With the decrease of feed intake level, the total energy digestibility, total energy metabolic rate and nitrogen apparent digestibility of male sheep increased significantly (P<0.05). The maintenance requirements of NE, ME, the efficiencies of ME utilisation for maintenance, every 1 g·kg -1BW0.75·d-1 body weight gain, DE, ME and NE of Alpine Merino growing male and female sheep were 227, 213 kJ·kg-1BW0.75·d-1, 283, 279 kJ·kg-1BW0.75·d-1, 0.80, 0.76, 760, 830 kJ·kg-1BW0.75·d-1, 570, 750 kJ·kg-1BW0.75·d-1, and 290, 370 kJ·kg-1BW0.75·d-1, respectively. Every 1 g·kg-1BW0.75·d-1 body weight gain, the maintenance requirements of net nitrogen, NPm, CPI and DCP of Alpine Merino growing male and female sheep were 220.8, 190.4 mg·kg-1BW0.75·d-1, 1.38, 1.19 g·kg-1BW0.75·d-1, 7.75, 6.55 g·kg-1BW0.75·d-1, 6.02, and 4.38 g·kg-1BW0.75·d-1, respectively. 【Conclusion】The results of this study showed that the energy and protein requirements of Alpine Merino sheep were different from the established sheep recommended values, which might be related to factors such as breed, physiological condition, age and environment. In addition, the model established in this study could be used to estimate the energy and protein requirements of Alpine Merino sheep. Table and Figures | Reference | Related Articles | Metrics

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Requirement of Vitamin D3 on Fast-Growing Yellow-Feathered Breeder Hens WANG YiBing,CHEN Fang,GOU ZhongYong,LI Long,LIN XiaJing,ZHANG Sheng,JIANG ShouQun Scientia Agricultura Sinica    2021, 54 (16): 3549-3560.   DOI: 10.3864/j.issn.0578-1752.2021.16.016 Abstract （414）   HTML （39）    PDF （576KB）（790）       Knowledge map    Save 【Objective】This experiment was conducted to investigate the effects of vitamin D3 (VD3) supplementation on performance, tibial characteristics and metabolism of calcium and phosphorus of fast-growing yellow-feathered breeder hens and the progeny, so as to establish requirement of vitamin D3 on breeder hens.【Method】Seven hundred and twenty breeder hens at 48 weeks of age were fed up with basal diets supplemented with 0, 800, 1 600, 2 400, 3 200 and 4 000 IU·kg-1 VD3 for eight weeks with six replicates per group and 20 hens per replicate, then the progeny hatched from each of the 6 maternal groups were fed a basal diet supplemented with 1 000 IU·kg-1 VD3 for 63 days. 【Result】Compared with the control group, supplemental 800 IU·kg-1 VD3 enhanced average egg weight (P<0.05); the dietary supplementation with 1 600 and 3 200 IU·kg-1 VD3 increased (P<0.05) egg shell strength and 1 600 IU·kg-1 VD3 increased (P<0.05) egg shell thickness; 4 000 IU·kg-1 VD3 supplementation increased dehydrated and degreased tibial weight/BW and bone density of yellow-feathered breeder hens (P<0.05); 4 000 IU·kg-1 VD3 supplementation also increased content of calcium and phosphorus, and decreased activity of AKP activity in plasma (P<0.05); supplementation of VD3 increased broking strength of tibia of breeder hens (P>0.05). There was no significant effect of maternal VD3 on growth performance of the progeny (P>0.05), However compared with the control group, the broking strength of tibia in the progeny was increased (P<0.05) when 4 000 IU·kg-1 VD3 was added to maternal diet, and tibial density was increased when 800, 1 600, or 4 000 IU·kg-1 VD3 was added; supplementation of 1 600 to 4 000 IU·kg-1VD3 increased dehydrated and degreased tibial weight/BW of the progeny (P>0.05). Addition of VD3 significantly increased content of calcium and phosphorus, and decreased activity of AKP in plasma of hens and their progeny aged 1 day (P<0.05), however, it had no significant effect on those of progeny at 21 or 63 days of age (P>0.05).【Conclusion】Under the condition of this experiment, the dietary supplementation of VD3 improved productive performance of yellow-feathered breeder hens, tibial characteristics and metabolism of calcium and phosphorus of breeder hens and progeny. The VD3 requirement of yellow-feathered breeders was estimated by considering the experimental data and quadratic regressions comprehensively. For yellow-feathered breeder hens, the best productive performance was obtained when supplemented with 800 IU·kg-1 VD3, and the best egg quality was obtained when supplemented with 1 650 to 1 828 IU·kg-1 VD3. The best tibial characteristics of the hens and progeny were obtained when supplemented with high level of VD3 (4 000 IU·kg-1). Table and Figures | Reference | Related Articles | Metrics

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Identification of the Core Promoter of Linc-NORFA and Its Transcriptional Regulation in Erhualian Pig DU Xing,ZENG Qiang,LIU Lu,LI QiQi,YANG Liu,PAN ZengXiang,LI QiFa Scientia Agricultura Sinica    2021, 54 (15): 3331-3342.   DOI: 10.3864/j.issn.0578-1752.2021.15.016 Abstract （365）   HTML （43）    PDF （3721KB）（520）       Knowledge map    Save 【Objective】 In our previous study, linc-NORFA has been proved as a candidate gene for sow fertility and participated in regulating follicular atresia and granulosa cell apoptosis. The aim of this study is to identify the core promoter of linc-NORFA and investigate its transcriptional regulation in Erhualian pig, so as to provide theoretical basis and new ideas for revealing the mechanism of linc-NORFA regulation to ovarian follicular atresia.【Method】 Ear samples of Euhualian pig were collected for genomic DNA extraction. PCR amplification and clone sequencing were used to obtain the 5’-flanking sequence of Erhualian pig linc-NORFA gene. Reporter vectors construction and luciferase activity assays were performed to identify the core promoter of linc-NORFA gene, and bioinformatic methods were conducted to analyze the characterization of linc-NORFA core promoter and the potential binding elements of transcription factors (TFs). In addition, pig FOXO1 gene eukaryotic expression vector was constructed and Western blot, qRT-PCR, and luciferase activity assays were performed to analyze the effects and regulatory mechanism of FOXO1 overexpression on the transcription of linc-NORFA gene. Besides, chromatin immunoprecipitation (ChIP) assay was performed to identify the interaction between FOXO1 and the core promoter of linc-NORFA in porcine granulosa cells (GCs).【Result】 A total of 1 734 bp 5’-flanking sequence of Erhualian pig linc-NORFA was obtained by PCR amplification and clone sequencing technology, which contained two potential CpG islands. Luciferase activity assay was performed and demonstrated that the core promoter of Erhualian pig linc-NORFA was located at -988 — -684 bp (TSS as +1). Multiple potential binding elements of several transcription factors (TFs) were identified within the core promoter of linc-NORFA using bioinformatic analyses, including ESR2, FOXO1, E2F1, BRCA1 and NFIC. In addition, results from ChIP assay proved that FOXO1 directly binds to the core promoter of linc-NORFA by acting as a transcription factor. Furthermore, It was proved that overexpression of FOXO1 could significantly down-regulate the activity of linc-NORFA core promoter (P<0.01), and also notably inhibited the expression level of linc-NORFA in porcine GCs (P<0.01).【Conclusion】 In this study, the core promoter of Erhualian pig linc-NORFA was identified, and FOXO1 acts as a transcription factor was proved, which significantly inhibited linc-NORFA transcription in porcine GCs through binding and further down-regulating the activity of its core promoter. These findings were of great significance for investigating the molecular mechanism of down-regulation of linc-NORFA during follicular atresia. Table and Figures | Reference | Related Articles | Metrics

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Screening, Identification and Functional Analysis of Important LncRNAs for Lactation Traits in Small-Tailed Han Sheep WANG JiQing,HAO ZhiYun,SHEN JiYuan,KE Na,HUANG ZhaoChun,LIANG WeiWei,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin Scientia Agricultura Sinica    2021, 54 (14): 3113-3123.   DOI: 10.3864/j.issn.0578-1752.2021.14.016 Abstract （474）   HTML （43）    PDF （1128KB）（676）       Knowledge map    Save 【Objective】Long non-coding RNAs (lncRNAs) are a type of non-coding RNAs with >200 nt in length, which have been shown to regulate mammary gland development and lactation process in dairy cows and dairy goats. However, little is known about the effect of lncRNAs on milk traits in sheep. The aim of the study was to analyze the effect of lncRNAs on milk performance and then provided a theoretical basis for elucidating molecular mechanism of lactation performance in sheep.【Method】Three high-lactating yield and high-milk-fat-content Small-Tailed Han sheep and three low-lactating yield and low-milk-fat-content Small-Tailed Han sheep were selected to profile the expression of lncRNAs in the mammary gland tissues during lactation using RNA-Seq. The enrichment analysis was performed using GO and KEGG databases for the target genes of differentially expressed lncRNAs between the two groups. The expression levels of 16 differentially expressed lncRNAs were verified using reverse transcription-quantitative PCR (RT-qPCR).【Result】A total of 7 239 expressed lncRNAs were identified in the mammary gland tissues of Small-Tailed Han sheep, including 2 262 known lncRNAs and 4 977 novel lncRNAs. The most of lncRNAs were expressed at low levels. The 120 differentially expressed lncRNAs were found between the two groups of Small-Tailed Han sheep, of which 68 lncRNAs were up-regulated in high-lactating performance Small-Tailed Han sheep, while 52 lncRNAs were down-regulated. The target genes of differentially expressed lncRNAs were significantly enriched in sulfur compound metabolic process, thioester biosynthesis process, acyl-CoA biosynthetic process, Rap1 signal pathway and adhesion junction. The lncRNA- miRNA network showed that some target miRNAs of the six most differentially expressed lncRNAs including MSTRG.125242.6 and MSTRG.59580.8, play important roles in mammary gland development and lactation in domestic animals. The RT-qPCR results showed that the expression tendency of 16 lncRNAs was consistent with the RNA-Seq results, which confirmed the accuracy and authenticity of the RNA-Seq data.【Conclusion】The differentially expressed lncRNAs screened were involved in the regulation of mammary gland development and milk performance in sheep and the results will provide a theoretical basis for analyzing the molecular genetic mechanism of lactation performance in sheep. Table and Figures | Reference | Related Articles | Metrics

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Effect and Mechanism of Tea Tree Oil on LPS Induced Mastitis in Dairy Cows CHEN Zhi,ZHANG Yi,LU QinYue,GUO JiaHe,LIANG Yan,ZHANG MingYiXing,YANG ZhangPing Scientia Agricultura Sinica    2021, 54 (14): 3124-3133.   DOI: 10.3864/j.issn.0578-1752.2021.14.017 Abstract （488）   HTML （34）    PDF （1253KB）（421）       Knowledge map    Save 【Objective】Cow mastitis has been one of the biggest challenges in dairy farming and dairy products industry, which restricts the healthy development of dairy industry. Effective prevention and treatment of cow mastitis can provide a good guarantee for the health of cows and the production of high-quality dairy products. This experiment explored the effects of tea tree oil on LPS-induced mastitis in dairy cows, and explored the feasibility of using tea tree oil instead of antibiotics to treat mastitis in dairy cows. This experiment provides a reference for the treatment of dairy cow mastitis with tea tree oil. 【Methods】The cells in good condition, were selected, which were added 50, 100, 200, 500, and 1 000 μg·mL-1 LPS respectively in the culture of these cells to detect the relevant indicators. The tea tree oil and LPS were added to the model for co-culture. The model of dairy cow mastitis induced LPS was established by the CCK-8 method, flow cytometry, real-time fluorescence quantification and ELISA assay. Antagonistic effect of tea tree oil on LPS in dairy cow mastitis cell model: 0.0002%, 0.0004%, 0.0006%, 0.0008%, 0.001%, 0.002%, 0.004%, 0.006%, 0.008% and 0.01% tea tree oil were added to the dairy cow mastitis cell model induced by 200 μg·mL-1 LPS for 12 hours to detect the related indexes. 【Result】CCK-8 method was used to detect the cell proliferation activity. The results showed that under the condition of 100 μg·mL-1 LPS poisoning, the activity of the cells began to decline in varying degrees. There was not a large number of apoptosis in 100 μg·mL-1 LPS after 12 hours of induction, while about 46% of the cells showed early and late apoptosis in 200 μg·mL-1 LPS. 200 μg·mL-1 LPS induced for 12 hours was the best condition for the establishment of mastitis model. The results also showed that when tea tree oil concentration was 0.0004%, 0.0006% and 0.0008%, the apoptosis rate of the cells decreased. Among them, when tea tree oil concentration was 0.0006%, the protection effect was the most obvious. The proportion of living cells was 71.95%, the proportion of early apoptotic cells was 22.15%, and the proportion of late apoptotic cells was 5.11%; compared with the living cells, the proportion of the mastitis model group of tree oil increased by about 22%. After that, the expression of cytokines and apoptotic factors were detected by qPCR in the three groups with protective effect. With the increase of the concentration of tea tree oil, the expression of TNF-α was down regulated more, the expression of IL-6 was down regulated less (P< 0.01), and the expression of STAT1 was up regulated slightly when 0.0004% tea tree oil was added, while down regulated slightly when 0.0006% and 0.0008% tea tree oil were added, and the expression of tea tree oil with 0.0006% concentration was the lowest (P< 0.05). The expression of NF-κB, MAPK and caspase-3 was significantly reduced in the three groups of tea tree oil adding concentration. Among them, the expression of inflammatory response protein in 0.0006% tea tree oil group was the lowest, about 50% of that in the blank control group. The protein expression was almost the same, about 55% of the blank control group (P< 0.05).【Conclusion】Tea tree oil had a certain antagonistic effect on LPS within the appropriate concentration range, which could reduce the proportion of apoptosis, improve the survival proportion of normal cells, and down regulate the expression of inflammatory factors, apoptosis factors and corresponding proteins. Table and Figures | Reference | Related Articles | Metrics

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Functions of Antibacterial and Inducing Defense Peptide Expression of Medium-Chain Fatty Acid and Its Application in Piglet Feeds YU ZhengWang,ZHOU ZhongXin Scientia Agricultura Sinica    2021, 54 (13): 2895-2905.   DOI: 10.3864/j.issn.0578-1752.2021.13.017 Abstract （387）   HTML （30）    PDF （496KB）（386）       Knowledge map    Save In recent years, more and more studies have shown that medium-chain fatty acid (MCFAs) resistance to pathogenic bacteria is an important component of innate defense system of mammals, and MCFAs can also induce expressions of endogenous host defense peptides (HDPs) in human, pig and chicken. However, these new functions of MCFAs have not attracted much attention. MCFAs also have a synergistic antibacterial synergistic effect with feeding organic acids or feeding plant essential oils, which can reduce the use of these active substances. In addition, compared with long-chain fatty acids, the addition of MCFAs in the diet can significantly increase the oxygen consumption and mitochondrial respiration rate in the body of animals, but it produces less reactive oxygen species, which is in line with the characteristics of rapid energy supply required by intestinal metabolism and liver metabolism in young animals. Adding low concentration of MCFAs (0.1%-0.5%, mass ratio) to the diet can significantly increase the survival rate of newborn or weaned piglets, the digestibility of crude protein and crude fat as well as the feed conversion rate, regulate the intestinal flora, and improve the intestinal epithelial structure, thus promoting the growth of animals. Based on the above advantages of MCFAs, mixing MCFAs with forage organic acid or plant essential oil to prepare coated particles may be a good way to use it as a substitute for antibiotics in piglets. Table and Figures | Reference | Related Articles | Metrics

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The Ability of Acidic Calcium Sulfate to Kill Common Clinical Pathogenic Microorganisms FU XiaLi,ZHENG ZiFang,MA ZhiQian,XU LeLe,LI ZhiWei,LI Yang,XIAO ShuQi,LI Shuang Scientia Agricultura Sinica    2021, 54 (13): 2906-2915.   DOI: 10.3864/j.issn.0578-1752.2021.13.018 Abstract （474）   HTML （34）    PDF （4920KB）（623）       Knowledge map    Save 【Objective】The purpose was to study the effect of disinfectant acid calcium sulfate (ACS) on the killing of microorganisms, and to provide basic data and theoretical basis for disinfectant prevention and control of human and animal diseases.【Method】After incubating ACS with 3% lecithin and 5% Tween-80 as phosphate buffer neutralizer for a period of time, Escherichia coli, Staphylococcus aureus, Salmonellaand porcine reproductive and respiratory syndrome virus (PRRSV) were added respectively for a period of time, We spread the culture mixture with bacteria on nutrient agar plates and cultured at 37℃ biochemical incubator for 18-24 h, the mixture with virus was inoculated on the cell plates and incubate in 37℃ cell incubator for a certain period of time to evaluate the neutralizing effect of the neutralizer. The comparison settings and evaluation criteria were as follows: disinfectant mixed with bacterial suspension or virus suspension (group 1), the mixture of disinfectant and bacterial suspension or virus suspension was mixed with neutralizer (group 2), the mixture of neutralizer and disinfectant was mixed with bacterial suspension or virus suspension (group 3), the mixture of sterile hard water and bacterial suspension or virus suspension was mixed with neutralizer (group 4), the mixture of sterile hard water and bacterial suspension or virus suspension was mixed with PBS (group 5), sterile hard water mixed with PBS (group 6). The evaluation criteria of the neutralizer effect in the bacteria killing test were as follows: group 1 had a very small amount of bacterial growth or aseptic growth; group 2 had bacterial growth, which was significantly less than that of groups 3, 4, and 5, but more than the group 1; the number of bacteria in group 3, 4,5 was close to that of the positive control group, no bacterial growth in group 6. All three repeated tests met the above conditions, and the results were consistent, it was determined that the selected neutralizer and concentration were appropriate. The evaluation criteria of the neutralizer effect in the virus inactivation test were as follows: group 1 had very little virus growth or no virus growth; group 2 had virus growth and was significantly less than that of groups 3, 4, and 5, but more than group 1. The growth of viruses in groups 3, 4, and 5 was similar to the original inoculation; the cells in group 6 grew normally. The results of the three repeated tests were consistent, and it was determined that the selected neutralizer and concentration were appropriate. We used the suspension quantitative sterilization method and the method of determining the virus titer to evaluate the elimination effect of ACS on the above-mentioned bacteria and viruses. We diluted the ACS 200, 300, 400, 500, 600, and 700 times to interact with the above-mentioned bacteria or virus for different time and added neutralizer for neutralization, then spread the bacterial mixture on nutrient agar plates to evaluate the killing effect of ACS by calculating the number of colonies, and determined the virus titer in the virus mixture to evaluate the killing effect of ACS on the virus.【Result】 The selected neutralizer can effectively neutralize the residual effects of ACS 200-fold dilution on bacteria and viruses, and the neutralizer was non-toxic to bacteria, viruses and cells. When ACS interacted with the bacteria for 0 h, it was neutralized immediately. The sterilization rate ofEscherichia coli at the maximum dilution of 300 times was 100%, the sterilization rate of Staphylococcus aureusat the maximum dilution of 600 times was 100%, and when the maximum dilution was 700 times, the sterilization rate of Salmonella was 100%. When ACS was diluted to 700 times, it was neutralized one day or six days after treatment with the above bacteria, and the sterilization rate was 100%. In addition, when ACS was diluted to 200 times, it was neutralized 60 minutes after treatment with PRRSV, and no virus titer was detected.【Conclusion】 When ACS diluted 700 times withEscherichia coli,Staphylococcus aureus and Salmonella for one day or more, it can produce better killing effect; when ACS diluted 200 times with PRRSV for 60 minutes, it can completely kill the virus, which will provide strong data support for the selection of disinfectants in the farms and provide reference for the prevention and control of epidemic diseases. Table and Figures | Reference | Related Articles | Metrics

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