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Identification of Cashmere Dermal Papilla Cells Based on Single- Cell RNA Sequencing Technology ZHANG WeiDong,ZHENG YuJie,GE Wei,ZHANG YueLang,LI Fang,WANG Xin Scientia Agricultura Sinica    2022, 55 (12): 2436-2446.   DOI: 10.3864/j.issn.0578-1752.2022.12.014 Abstract （368）   HTML （39）    PDF （3368KB）（105）       Knowledge map    Save 【Objective】 Based on single-cell RNA sequencing, this article aims to explore the marker genes of cashmere dermal papilla cells, and to optimize the methods to identify dermal papilla cells in vitro, thereby laying a cell model for future pertinent research in cashmere hair follicle development. 【Methods】 The single-cell transcriptional data from the skin tissues of Shanbei white cashmere embryonic stage (E60, E90 and E120) were analyzed with Seurat package. After quality control, filter and normalization of raw data, the dimension reduction analysis and cell cluster identification were performed by uniform manifold approximation and projection (UMAP). Moreover, depending on cluster-specific expressed gene expression, the principal cell lineage information was identified. The type-specific marker genes of the dermal papilla were obtained after gene expression analysis. The immunofluorescence staining was used to validate the expression position of marker protein to identify the dermal papilla specific protein in goat skin. Whole hair follicles were isolated mechanically under stereoscope, and combined with enzyme detach, cashmere dermal papilla region was isolated and cultured in vitro until cell separation. The dermal papilla cells were purified by different-speed adherence methods. When the cells were highly pure, the expression of candidate marker protein was verified by immunofluorescence assay. 【Result】 In current study, the key transcription information of goat hair follicle cells was analyzed at single cell level. Information of 17 subsets of cells in cashmere goat skin structure was obtained successfully including dermal cell lineage, epidermal cell lineage, dermal papilla cell, hair stem cell and inner root sheath cell, as well as other functional cell groups such as pericyte cell, macrophage and muscle cell. 427 specific markers of dermal papilla cells including SOX2, FGF7, APOD, BMP3, HHIP, HEY2 and SPON1 were screened. By comparison, the expression of these marker genes in cashmere dermal papilla cells was much higher than that in other cell types, which could be confirmed as the specific genes of hair papilla cells. Immunofluorescence result further proved that SOX2, FGF7 and APOD were specifically expressed in the dermal papilla region, and could be used to trace the dermal papilla cells in vivo. In addition, in current study, the single cashmere goat secondary hair follicle was separated successfully, and the adherent culture of dermal papilla was realized. A large number of cells were observed migrating from the hair papilla area. Immunofluorescence assay showed that SOX2, FGF7 and APOD were all expressed in goat dermal papilla cells, and about 76% of cells were SOX2 positive, while more than 98% of cells were FGF7 and APOD positive. Combined with the immunofluorescence results, SOX2, FGF7 and APOD genes factually could be used to identify the cultured goat dermal papilla cells in vitro. 【Conclusion】 In this study, single cell RNA sequencing technology was used to describe the main transcriptome information of cells in cashmere goat skin, and the specific marker genes of dermal papilla cells were sifted out successfully. And it proved that single-cell sequencing based method was simple and efficient to identify marker genes further identified by immunofluorescence. The discovering of SOX2, FGF7 and APOD not only provided the markers for the localization of hair papilla cells in vivo, but also provided the possibility for the identification of dermal papilla cells with multiple markers, which laid the foundation for further study of the gene functions in regulating hair follicle development. Table and Figures | Reference | Related Articles | Metrics

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DUS Traits Variation Analysis and Application of Standard Varieties of Lolium multiflorum Introduced from Japan FENG JunJie,ZHAO WenDa,ZHANG XinQuan,LIU YingJie,YUAN Shuai,DONG ZhiXiao,XIONG Yi,XIONG YanLi,LING Yao,MA Xiao Scientia Agricultura Sinica    2022, 55 (12): 2447-2460.   DOI: 10.3864/j.issn.0578-1752.2022.12.015 Abstract （320）   HTML （24）    PDF （2382KB）（87）       Knowledge map    Save 【Objective】This study aimed to determine the consistency and specificity of DUS test traits of Lolium multiflorum (annual ryegrass) standard varieties introduced from Japan, and to optimize the DUS test system of L. multiflorum in China, thus identify new varieties (lines) more quickly and accurately. 【Method】In this study, seven standard varieties of annual ryegrass introduced from Japan were planted in Chengdu Plain, China. Fifteen individuals with the similar growth status were selected from each standard variety. Cluster analysis of the standard varieties was conducted based on their DUS test data to identify the DUS trait expression of the standard varieties in Chengdu Plain. In order to screen out the characters with the high intra-population consistency and obvious inter-population specificity, the coefficient of variation and variance values of 18 tested DUS characters were calculated, and the nested variance analysis of the studied characters was also conducted. Furthermore, the probability classification was conducted based on the selected characters, which was used to clustering analysis and identification of the tested national approved varieties and new lines, and to evaluate the correlation coefficients of test traits. 【Result】According to the values of 18 DUS test traits of standard varieties, the standard varieties could be clearly clustered into 7 groups. The DUS traits were fully expressed and could be clearly distinguished from each other, which were suitable for subsequent correlation analysis. The results of intraspecific consistency analysis showed that, six traits, including leaf color degree, growth habit after vernalization, plant width at booting stage, flag leaf width at booting stage, flag leaf length-width ratio at booting stage, and spikelet length, possessed the poor intra-population consistency for multiple varieties. When conducting the inter-population specificity analysis of varieties, however, the poor specificity among the tested varieties was found in plant height at booting stage, ratio of flag leaf length to width at booting stage and spikelet density among cultivars, among which the variance component between populations of flag leaf length-width ratio at booting stage was 54.83%, and the variance component between individuals within populations was 45.17%. The selected ten quantitative traits were conducted the K-S test and χ2 test analysis, however, only the basal spikelet length conformed to χ2 test. Afterwards, the ten quantitative traits were divided into five grades. On the basis of these traits grading criterion, the identification of national approved varieties and new lines of L. multiflorum. The results showed that the tested varieties and new lines of annual ryegrass could be clustered into eight groups according to its classification results and corresponding to varieties origin, which revealed the reliability of traits grading criterion built in this study on variety identification. The heat map drawn based on the trait values of the tested varieties showed that the leaf length of ‘Tetragold’ was significantly different from that of other tested varieties in the vegetative growth period, which was manifested as shorter leaf length; the variety ‘Chuannong No. 2’ showed higher plant height after vernalization; the internode length under spike of ‘Diamond T×changjiang No. 2’ was significantly shorter than that of other varieties; the poor intra-population consistency of ‘Double Barrel’, ‘Chuannong No. 1’ and ‘Chuannong No. 2’ showed good intra-population consistency. The low correction and strong independence were existed within the selected ten traits, indicating that these traits were very well suited for DUS test. 【Conclusion】The character classification system for DUS test based on Japanese standard varieties could be applied to the identification and distinguish of L. multiflorum varieties released in China. This study could provide the important technical and theoretical reference for the optimization of DUS test method system of annual ryegrass varieties. Table and Figures | Reference | Related Articles | Metrics

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Molecular Cloning and Expression Pattern Analysis of NPC2 Gene Family of Apis cerana cerana ZHANG Li,ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang Scientia Agricultura Sinica    2022, 55 (12): 2461-2471.   DOI: 10.3864/j.issn.0578-1752.2022.12.016 Abstract （459）   HTML （35）    PDF （1275KB）（151）       Knowledge map    Save 【Background】As an important native resource insect, the Chinese honeybee (Apis cerana cerana) plays important ecological roles in pollinating the plants that bloom at low temperatures in early winter in China, and the pollination behavior of A. c. cerana is closely related to its olfactory system. According to the analysis of antenna transcriptome data collected from foragers treated at high and low temperatures, it was found that the Niemann-Pick type C2 protein (NPC2) gene family related to insect olfaction was up-regulated expression at low temperatures.【Objective】Therefore, this study aims on the A. c. cerana NPC2 family genes, including cloning and analysis of their structural characteristics and expression profiles. Moreover, the NPC2 family gene expression under high and low temperatures was also studied. It will provide an evidence of the AcNPC2 gene family in the low-temperature adaptation involved in the chemosensory and olfactory function of A. c. cerana.【Method】Based on the results of high and low temperatures transcriptome sequencing of A. c. cerana, the ORF sequence of AcNPC2 genes was cloned by RT-PCR, and phylogenetic tree analysis and three-dimensional structure prediction were performed. Then, the spatio-temporal expression profile of AcNPC2 genes in different developmental stages and different tissues, as well as the amount of expression at high and low temperatures of A. c. cerana were analyzed by qRT-PCR.【Result】The full length ORFs of four NPC2 genes of A. c. cerana (AcNPC2a, AcNPC2b, AcNPC2c, and AcNPC2d) were obtained as 447, 480, 459, and 465 bp, respectively, encoding 148, 159, 152, and 154 amino acids. The predicted protein molecular weight is 16.12-18.53 kD, and the isoelectric points are 7.98, 7.57, 6.56, and 6.34, respectively. Phylogenetic tree analysis showed that AcNPC2 sequences were most close to the NPC2 homologous sequence of Apis mellifera ligustica. qRT-PCR results showed that the expression level of AcNPC2a was the highest in the abdomen of the newborns, followed by the abdomen of the nurses and the larval stage. The expression of AcNPC2b was the highest in the thorax of the newborn bees, followed by the head, thorax and metapodium of the foragers. AcNPC2c was notably expressed in high abundance in the antennae of the nurses and the foragers. AcNPC2d had the highest expression in the head of the foragers. After low temperature treatment, the expression levels of all AcNPC2 genes in the forager antennae increased, but there was no significant difference.【Conclusion】AcNPC2 has the conserved structure of NPC2 protein family, and its members show diversity in the spatio-temporal expression profile of A. c. cerana. Among them, AcNPC2c is highly expressed in the antennae, indicating that it is closely related to the olfactory function of A. c. cerana. The expression of the whole AcNPC2 family genes increased in the antennae of the foragers at lower temperature, indicating that these genes might be involved in the low-temperature adaptability of A. c. cerana and pollination behavior in early winter. Table and Figures | Reference | Related Articles | Metrics

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Association Analysis of the ADIPOQ Variation with Sheep Growth Traits LIANG Peng,ZHANG TianWen,MENG Ke,SHAO ShunCheng,ZOU ShiFan,RONG Xuan,QIANG Hao,FENG DengZhen Scientia Agricultura Sinica    2022, 55 (11): 2239-2256.   DOI: 10.3864/j.issn.0578-1752.2022.11.013 Abstract （336）   HTML （40）    PDF （2205KB）（83）       Knowledge map    Save 【Objective】 The aim of the study was to explore the effects of genetic variation of ADIPOQ on growth traits of sheep, and to find the molecular genetic markers related to growth traits in Ningxia high-quality mutton sheep breeding, so as to achieve the purpose of molecular assisted breeding. 【Method】 The mutation sites of ADIPOQ in Dupo sheep, Tan sheep and Small-Tailed Han sheep were obtained by Allegro Targeted Genotyping. At the same time, the ear tissues of 383 different hybrid progenies of three breeds were collected. The SNPs were genotyped by Sequenom Mass ARRAY®SNP. Haploview was used to analyze linkage disequilibrium and construct haplotypes of the polymorphic loci, as well as association analysis between the SNPs in ADIPOQ with growth traits of newborn and 3-month-old sheep.【Result】 A total of 7 SNPs were screened, and 7 SNPs showed polymorphism in the hybrid population. The dominant genotypes of SNP1-SNP7 were CC, GG, GG, CT, AG, GG and AA, and the dominant alleles were C, G, G, C, G, G and A. X2 test showed that all loci were in Hardy Weinberg equilibrium (except SNP7 site deviated from the equilibrium in all individuals). SNP1 (except F2), SNP4, SNP5 and SNP6 were moderately polymorphic (0.25≤PIC<0.50) in all hybrids and all individuals, while SNP2 and SNP3 were moderately polymorphic (0.25≤PIC<0.50) in F2 and SNP7 were moderately polymorphic (0.25≤PIC<0.50) in H1, and were low polymorphic (PIC<0.25) in other populations. The results of linkage imbalance analysis showed that SNP2-SNP3 and SNP5-SNP6 formed two strong linkage, each of which constructed three haplotypes, and formed 4 and 6 genotypes after combination, respectively, among which the dominant genotype was H1H1 and H4H6. 13 genotypes were produced after the haplotypes formed by SNP2-SNP3 and SNP5-SNP6 were recombined, and the dominant genotype was H1H1H4H6. Single SNP correlation analysis revealed that: SNP1 site in F1 population, the primary chest of GG genotype was significantly higher than that of CG genotype (P<0.05); in H2 population, the primary body length of CC genotype was significantly higher than that of GG genotype (P<0.05). SNP2 site in H1 population, the primary body height of CC genotype was significantly lower than that of CG and GG genotypes (P<0.05). SNP3 site in F1 population, the three months body weight of AG genotype was significantly higher than that of GG genotype; in H1 population, the primary body height of AA genotype was significantly lower than AG and GG genotypes (P<0.05). SNP4 site in F1 population, the three months weight of CC genotype was significantly higher than that of CT and TT genotypes (P<0.05); in F2 population, the primary weight and primary chest of CT genotype were significantly higher than that of TT genotype (P<0.05), which of TT genotype was significantly lower than that of CC and CT genotype (P<0.05) in H1 population; in H2 population, the primary body height and primary body length of TT genotype were significantly higher than that of CT genotype (P<0.05). SNP5 site in F2 population, AA genotype had significantly higher primary body height and primary body length than GG genotype (P<0.05). There were significant differences in primary weight, body height, body length, three months body height and chest among different genotypes of SNP6 (P<0.05). SNP7 site in H2 population, AA genotype had significantly higher primary body length than GA genotype (P<0.05). When combined with all groups, it was found that: the primary weight and body length of CG genotype at SNP2 were significantly higher than that of GG genotype (P<0.05); the primary weight of TT genotype at SNP4 was significantly lower than CC and CT genotypes (P<0.05); the primary weight of AA genotype at SNP6 was significantly higher than GG and GA genotype (P<0.05); and the primary chest was significantly higher than GG genotypes (P<0.05). There was no significant difference in growth traits among the other five genotypes (P>0.05). The results of haplotype association analysis showed that the primary weight and primary body length of H2H3 genotype were significantly higher than those of other genotypes (P<0.05), while the primary weight of H5H5 genotype was significantly higher than that of H5H6 and H6H6 (P<0.05), the primary body height was significantly higher than that of H5H6 (P<0.05), and the primary chest was significantly higher than that of H6H6 (P<0.05); the three months weight of H4H4 genotype was significantly higher than that of H5H5 genotype (P<0.05), the body weight and body length at three months were significantly higher than those of H4H6 genotype (P<0.05), and the chest of three months was significantly higher than that of H4H5, H5H6 and H5H5 genotype (P<0.05). After SNP2-SNP3 and SNP5-SNP6 haplotypes were recombined, the primary weight, body height, body length and chest of H2H3H4H4 genotype were the highest, which were different from other genotypes, and the body length at three months of H1H1H4H6 genotype were significantly lower than that of H1H2H4H4 and H2H2H4H4 genotypes (P<0.05).【Conclusion】 The results showed that the different SNPs and combined genotypes in ADIPOQ had effect on sheep different growth traits, the seven SNPs in this study could be used as potential molecular markers for growth traits in Ningxia high quality mutton sheep breeding. Table and Figures | Reference | Related Articles | Metrics

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The Response of Leymus chinensis Cloned Offspring to Mowing GUO FengHui,DING Yong,JI Lei,LI XianSong,LI XiLiang,HOU XiangYang Scientia Agricultura Sinica    2022, 55 (11): 2257-2264.   DOI: 10.3864/j.issn.0578-1752.2022.11.014 Abstract （308）   HTML （28）    PDF （473KB）（62）       Knowledge map    Save 【Background】 Grazing could alter the morphological and photosynthetic physiological characters of the Leymus chinensis cloned offspring, but whether grazing influence the adaptability of that to grazing is unclear. 【Objective】 The aim of this study was to investigate whether grazing history enhanced the adaptability of Leymus chinensis cloned offspring to livestock grazing.【Method】 The Leymus chinensis cloned offspring with different grazing histories (enclosed in 1983 VS long-term free grazing) were used to conduct pot experiment in the greenhouse, and their adaptability to simulated livestock grazing (clipping) was compared in terms of individual traits, ramet number, biomass, and biomass allocation. 【Result】 (1) There was a significant interact effect between maternal grazing history and clipping treatment. The individual height and biomass of grazing (GZ) were more resistant to clipping treatment than that of nograzing (NG), while the response of ramet number to clip was not influenced by maternal grazing history. (2) Grazing history altered the responses of aboveground, root, rhizome and total biomass to the clipping treatment. There was significant interaction between grazing history and mowing treatment in terms of the rhizome biomass, while the interaction on other three indicators were not significant. However, the other three indicators of NG had the larger plasticity index and absolute reduction to clipping treatment. Therefore, the maternal grazing experience enhanced the adaptability of L. chinensis cloned offspring to clipping treatment. (3) The cutting biomass of NG was significantly lower than that of GZ, but the cutting degree of NG was significantly higher than that of GZ.(4) The responses of GZ biomass allocation to clipping treatment were not significant, while rhizome biomass allocation of NG significantly decreased under clipping treatment. 【Conclusion】 The grazing disturbance could enhance the adaption of Leymus chinensis cloned offspring to grazing. The maternal grazing history did not enhance the adaptability of L. chinensis cloned offspring to livestock grazing by altering biomass allocation. The response of leaf photosynthetic physiology and grazing avoidance might be the reasons for the enhancement of grazing fitness. In this study, the environmental disturbance factors, such as soil factors, were excluded through control experiments, and the response of L. chinensis cloned offspring traits to grazing was studied from the perspective of the plant itself. Thus, this study provided a new perspective for fully understanding the process of grazing degradation in grassland ecosystem. Table and Figures | Reference | Related Articles | Metrics

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The miR-221 Inhibits the Viability and Proliferation of Ovine Mammary Epithelial Cells by Targeting IRS1 KE Na,HAO ZhiYun,WANG JianQing,ZHEN HuiMin,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin,ZHAO ZhiDong,HUANG ZhaoChun,LIANG WeiWei,WANG JiQing Scientia Agricultura Sinica    2022, 55 (10): 2047-2056.   DOI: 10.3864/j.issn.0578-1752.2022.10.014 Abstract （317）   HTML （29）    PDF （2098KB）（85）       Knowledge map    Save 【Background】MicroRNAs (miRNA) are a type of small RNAs (18-23 nt) that are widely involved in the regulation of mammogenesis and milk traits in livestock animals. In our previous research, the expression level of miR-221 in non-lactating mammary gland was found to be 3.6-time higher than in mammary gland at lactation period in Small-Tailed Han sheep by using RNA-Seq. However, the regulatory mechanism of miR-221 on ovine mammary gland development is still unclear. 【Objective】The aim of this study was to investigate the inhibition of miR-221 on the viability and proliferation of ovine mammary epithelial cells by targeting insulin receptor substrate 1 (IRS1) gene, so as to provide a theoretical reference for revealing the molecular regulation mechanism of miR-221 on ovine lactation performance.【Method】In the study, mammary gland, heart, liver, kidney, spleen, lung, Longissimus dorsi muscle and ovary tissues were collected in Small-Tailed Han sheep, and the expression profiles of miR-221 were constructed in ovine eight tissues by using reverse transcription-quantitative PCR (RT-qPCR). The effects of miR-221 on the viability and proliferation of ovine mammary epithelial cells (OMECs) were investigated by using cell transfection, CCK-8 and Edu assays. The miRDB and miRanda were used to predict the target genes of miR-221. Based on functional enrichment analysis, an investigated target gene was screened. The target relationship between miR-221 and the predicted target gene was investigated by constructing wild-type and mutant-type report vectors for the target gene by using dual luciferase reporter assay. Finally, the effects of over-expressed and silenced miR-221 on expression levels of the target gene and other functional genes in downstream signaling pathways were detected.【Result】The miR-221 was expressed in ovine eight tissues including mammary glands, with the highest expression levels in lung and spleen, and the lowest expression levels in Longissimus dorsi muscle and kidney. The CCK-8 assay result revealed that miR-221 mimic inhibited the viability of OMECs, whereas miR-221 inhibitor promoted the viability of OMECs. The Edu result found that miR-221 mimic reduced the number of Edu-labeled positive OMECs. On the contrary, miR-221 inhibitor increased the number of Edu-labeled positive OMECs. The result from dual luciferase reporter assays showed that the miR-221 mimics reduced the luciferase activity of the 3′UTR region of IRS1, while miR-221 inhibitor increased the luciferase activity. This suggested that IRS1 was a target gene of miR-221. The results from RT-qPCR further found that over-expressed miR-221 reduced expression levels of IRS1 and PIK3R1 in OMECs (P<0.05), while silenced miR-221 enhanced the levels of the two genes in expression (P<0.05). No effect on IGF1R was found for over-expressed and silenced miR-221 in OMECs (P>0.05).【Conclusion】The miR-221 inhibited the viability and proliferation of OMECs by reducing IRS1 expression. Table and Figures | Reference | Related Articles | Metrics

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LKB1 Regulates Steroids Synthesis Related Genes Expression in Bovine Granulosa Cells ZHANG Jing,ZHANG JiYue,YUE YongQi,ZHAO Dan,FAN YiLing,MA Yan,XIONG Yan,XIONG XianRong,ZI XiangDong,LI Jian,YANG LiXue Scientia Agricultura Sinica    2022, 55 (10): 2057-2066.   DOI: 10.3864/j.issn.0578-1752.2022.10.015 Abstract （466）   HTML （38）    PDF （1980KB）（161）       Knowledge map    Save 【Background】 The Steroids synthesis capacity of ovarian granulosa cells plays the important roles in the development and maturation of follicles, however, the key regulators were involved in this process remains largely unknown. Our previously research reported that Liver kinase B1 (LKB1) influenced the cellular lipid metabolism, which is close associated with steroids synthesis. Further, another study showed that knockout of LKB1 caused premature ovarian failure in mice. 【Objective】 The aim of this study was to study the expression pattern of LKB1 in bovine follicle and its regulation on steroid synthesis related genes expression in granulosa cells (GCs),and provided a theoretical basis for the research of the reproductive physiological regulation in the cow.【Method】The expression pattern of LKB1 in follicle was detected by immunohistochemically assay. Then the primary follicular granulosa cells were isolated and identified by immunofluorescence staining incubated by follicle stimulating hormone receptor (FSHR) antibody. Next, these verified granulosa cells were used as the cell model. On one hand, LKB1 loss-of-function was mediated by siRNAs. qRT-PCR was performed to measure LKB1 regulation of steroid hormone synthesis related genes expression. On the other hand, LKB1 gain-of-function was mediated by adenovirus. qRT-PCR and ELISA analysis were carried out to confirm the changes of above detected genes influenced by LKB1 and estradiol (E2) secretion, respectively. 【Result】 The data showed that: 1) LKB1 protein expressed in all cell types of follicles and the positive signal in granulosa cells is significantly higher than that of theca cells, which is verified by quantitative analysis. 2) The morphology of isolated bovine follicular granulosa cells was shape of round, which were specifically labeled by follicle stimulating hormone receptor (FSHR) using immunofluorescence staining, with 95% of positive cells. 3) The interference efficiency of LKB1 treated by siRNA1 and siRNA2 was respectively 48% (P<0.05) and 52% (P<0.05) to that of control. Knockdown of LKB1 significantly down-regulated mRNA levels of STAR (P<0.01), CYP11A1 (P<0.01) and CYP19A1 (P<0.05), with the 60%, 80% and 50% decrease to those of the control. 4) The highly infected efficiency was observed infected by LKB1-OE and control adenovirus. In contrast, overexpression of LKB1 dramatically increased mRNA levels of STAR (P<0.01), CYP11A1 (P<0.01) and CYP19A1 (P<0.05), which was associated with elevation of E2 secretion. 【Conclusion】In summary, LKB1 was highly expressed in follicular granulosa cells, which promoted the expression of steroids synthesis related genes and E2 secretion. This result provides directly theoretical evidence for the LKB1 regulation of steroids hormone synthesis in bovine. Table and Figures | Reference | Related Articles | Metrics

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Mechanism of NMRAL1 Regulating Influenza Virus Replication YAN Ya,WANG GuangWen,KONG FanDi,WANG XuYuan,WANG YiHan,LI JunPing,ZHAO YuHui,LI ChengJun,CHEN HuaLan,JIANG Li Scientia Agricultura Sinica    2022, 55 (10): 2067-2076.   DOI: 10.3864/j.issn.0578-1752.2022.10.016 Abstract （564）   HTML （38）    PDF （2881KB）（111）       Knowledge map    Save 【Objective】Influenza virus is a zoonotic pathogen that often causes a pandemic and poses a great threat to human health, and the influenza viruses are prone to variants and can constantly escape the host cell immune response and develop resistance to existing anti-influenza drugs, so the search for new ways to fight influenza is imminent. This study aimed to explore the effect of NMRAL1 (NmrA-like family domain-containing protein 1) on influenza virus replication, and to reveal the molecular mechanism by which it functioned, so as to provide a potential target for anti-influenza drugs development. 【Method】In this study, siRNA interference technology was used to down regulate the expression of NMRAL1 in A549 cells, and the expression levels of NMRAL1 were detected by Western Blot. Virus titers in cell supernatants at 24 h and 48 h after infection with two different subtypes influenza viruses, including a/Anhui/ 2/2005(AH05)(H5N1) and a/WSN/33(H1N1), were detected using the plaque assay. To determine the specific stage at which NMRAL1 affected influenza virus replication, NMRAL1 was overexpressed by transiently transfecting NMRAL1-Myc-pCAGGS plasmid in HEK293T cells, and the effect of overexpressing NMRAL1 on influenza virus polymerase activity was examined by luciferase reporter system. The influenza virus NP protein was stained by using immunofluorescence, and the down-regulated expression of NMRAL1 on the localization of NP protein at 3, 4, 5, 6 and 8 h post infection was assessed respectively by confocal assay to determine whether down-regulated expression of NMRAL1 affected the process of influenza virus vRNP import and export. Western Blot was used to detect the effect of NMRAL1 knockdown on the expression of viral proteins and on the expression of IFN stimulated genes (ISGs) downstream of type I interferon pathway activated by influenza virus. Indirect immunofluorescence assay was utilized to further verify the effect of NMRAL1 on influenza virus replication. 【Result】Western Blot assay showed that NMRAL1 siRNA could significantly down regulate NMRAL1 expression in A549 cells. With the down-regulated expression of NMRAL1, A549 cells were infected with H5N1 and H1N1 viruses, respectively. Then the virus titers in the cell supernatant were measured by plaque assay, which showed that the virus titers in the supernatant of cells at 24 and 48 h after infection with H5N1 or H1N1 were significantly decreased, meaning that NMRAL1 could promote the replication of different subtypes influenza viruses. To further explore the specific mechanism by which NMRAL1 regulated influenza virus replication, a luciferase reporter system was used to detect influenza virus polymerase activity, and it was found that the overexpression of NMRAL1 had no effect on influenza virus polymerase activity. The results of confocal assay showed that the down-regulated expression of NMRAL1 did not affect the process of NP nuclear import and export, meanwhile Western Blot assay indicated that down-regulated expression of NMRAL1 did not affect the expression of each viral protein. However, the results of the fluorescence quantitative PCR assay showed that down-regulated expression of NMRAL1 was able to promote the up-regulation of IFN-β mRNA levels induced by influenza virus infection, and Western Blot assay found that down expression of NMRAL1 promoted the expression of MxA and IFITM3 antiviral proteins downstream of type I interferon pathway. Meanwhile, the indirect immunofluorescence assay showed that the down expression of NMRAL1 could significantly inhibit influenza virus replication. 【Conclusion】 Those results demonstrated that, during influenza virus infection, NMRAL1 did not affect the process of influenza virus invasion as well as transcription translation, but rather inhibited the expression of antiviral factors, such as MxA and IFITM3, by inhibiting type I interferon pathway activation, which ultimately promoted influenza virus replication. This study confirmed that the host factor NMRAL1 positively regulated influenza virus replication and enriched the network of host factors involved in influenza virus replication. Table and Figures | Reference | Related Articles | Metrics

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Effects of Cross-Ventilation System on Physiology and Production Performance of Beef Cattle in Summer FANG HaoYuan, YANG Liang, WANG HongZhuang, CAO JinCheng, REN WanPing, WEI ShengJuan, YAN PeiShi Scientia Agricultura Sinica    2022, 55 (5): 1025-1036.   DOI: 10.3864/j.issn.0578-1752.2022.05.014 Abstract （355）   HTML （45）    PDF （853KB）（150）       Knowledge map    Save 【Objective】 This study was conducted to explore the effects of cross-ventilation system on cowshed thermal environment, physiological and biochemical indexes, and production performance of beef cattle under the high temperature and humidity climate in summer of southern China, to evaluate the technical and economic effects of the environment control system for beef cattle heatstroke prevention. 【Method】 One-factor completely randomized design was introduced in this study. Thirty healthy 8-month-old Simmental bulls with similar body weight ((290.05±7.60)kg) were randomly assigned into two adjacent sheds with the same structure. The experimental group was equipped with the cross-ventilation system, and natural ventilation was used in the control group. The experimental period was from June 30 to July 16, 2019, a total of 17 days, in which the pre-test period was the first 3 days, and the formal period was the last 14 days. The wind speed, dry-bulb temperature and wet-bulb temperature were measured at 5:00, 10:00, 14:00, 18:00 and 22:00 every day in the first 7 days of the formal test period. The temperature-humidity index and sensible temperature were calculated. Meanwhile, the rectal temperature and respiratory rate of beef cattle were measured. During the whole formal period, the feeding amount was recorded every day, and the remaining materials were cleaned and weighed at 6 a.m of the next day to calculate the feed intake. From 7:00 to 8:00 in the morning on the first day and the fourteenth day of the formal test period, all cattle were weighed before feeding to calculate the average daily gain, feed weight ratio and other production performance indicators, and the economic benefit was evaluated. Simultaneously, the blood and fecal samples were collected for determination of inorganic ions, biochemical indexes and hormone levels in serum and cortisol levels in feces. 【Result】 The results showed that: (1) in the experimental group, the cross-ventilation system could significantly increase the wind speed in the shed (P < 0.01), thus significantly reduced the sensible temperature, the rectal temperature at 10:00, 14:00, 18:00, 22:00, and the respiratory rate of beef cattle at 10:00, 14:00, 18:00 (P < 0.01). Compared with the control group, with the increase of ambient temperature, the increase of rectal temperature and respiratory rate in the experimental group decreased by 45% and 42%, respectively. There was no significant difference in dry-bulb temperature, relative humidity and temperature-humidity index between the experimental group and the control group (P > 0.05). (2) At the end of the experiment, the serum calcium content in the experimental group was significantly lower than that in the control group (P < 0.05), while no difference was found concerning the contents of potassium ion, sodium ion, magnesium ion and chloride ion (P > 0.05). The results of serum biochemical indexes showed that the contents of heat stress protein 70, total protein, triglyceride and glucose in bovine of the experimental group were significantly higher than those in the control group (P < 0.05), and no significant difference was observed for the contents of serum albumin, globulin and total cholesterol (P > 0.05). The results of hormone levels showed that the levels of cortisol in feces and serum of the cattle in experimental group were significantly lower than those in control group (P < 0.05), and triiodothyronine and thyroxine had no significant difference in cattle between the experimental group and the control group (P > 0.05). (3) The production performance test showed that there was no significant difference in initial body weight and end body weight between the experimental group and the control group (P > 0.05), while the average daily gain (P < 0.01) and average dry matter intake (P < 0.05) of the experimental group were significantly higher than those of the control group, the feed-to-weight ratio of the experimental group was significantly lower than that of the control group (P < 0.05), and the profit in the experimental group was increased by 10.68%. 【Conclusion】 The cross-ventilation system could significantly increase the air velocity of the shed, reduce the sensible temperature, improve the metabolism of Simmental cattle, promote the production performance, and increase the economic benefits for beef cattle production in high temperature and humidity environment. Table and Figures | Reference | Related Articles | Metrics

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Effects of Additives on the Fermentation Quality of Agricultural By-Products and Wheat Straw Mixed Silage ZONG Cheng, WU JinXin, ZHU JiuGang, DONG ZhiHao, LI JunFeng, SHAO Tao, LIU QinHua Scientia Agricultura Sinica    2022, 55 (5): 1037-1046.   DOI: 10.3864/j.issn.0578-1752.2022.05.015 Abstract （376）   HTML （39）    PDF （493KB）（153）       Knowledge map    Save 【Objective】With the continuous development of food industry and processing industry, a large number of agricultural by-products were produced, including watermelon rind, broad bean pod and beer lees, which caused serious environmental pollution. In order to improve the feeding degree of agricultural by-products and wheat straw and to reduce environmental pressure, in this study, the effects of additives on the fermentation quality of agricultural by-products (watermelon rind, broad bean pod and beer lees) mixed with wheat straw were investigated.【Method】The mixture of agricultural by-products (watermelon rind﹕broad bean pod﹕beer lees = 1﹕3﹕4) and wheat straw, as raw materials, were mixed in ratios at fresh weight of 100%﹕0 (MW), 25%﹕75% (X25), 50%﹕50% (X50) and 25%﹕75% (X75), and the mixed silages were treated with additives: 1×106 cfu•g-1Lactobacillus buchneri (B), 0.2% FW cellulase (C), and 1×106 cfu•g-1Lactobacillus rhamnosus (R), and without additives (CON) as the control. The 200 g of raw materials treated with different additives were put into 250 mL polyethylene plastic bottles. After ensiling for 60 days at room temperature, the silos were opened to analyze the fermentation quality and nutrient composition. The treated silages and raw materials were filtered by two layers of gauze and qualitative filter paper, and the extract was used for measuring the pH value. The raw materials and silage were dried to a constant weight and the dry matter (DM) was measured. The crude protein (CP) was determined by Kjeldahl nitrogen determination method. The contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined by the method of Van’s fiber determination. The water-soluble carbohydrates (WSC) content was determined via the modified phenol-sulfuric acid method. The microorganisms were cultured and counted in the culture medium respectively. The organic acids were analyzed using high performance liquid chromatography. The ammonia nitrogen (NH3-N) content was determined by the phenol-hypochlorite reaction method. 【Result】 Compared with MW-treated silage, NH3-N content was notably decreased in X25-treated silage (P<0.05); pH and NH3-N content were decreased in X50-treated silage, significantly (P<0.05); pH and NH3-N content were notably decreased and the ratio of lactic acid to acetic acid (LA/AA) was notably increased in X75-treated silage, respectively (P<0.05). In addition, compared with the MW-treated silage, the butyric acid content was reduced in X25-, X50- and X75-treated silage, notably (P<0.05). In MW-treated silage, compared with the control, adding C significantly decreased the pH and NH3-N content (P<0.05), and increased the lactic acid (LA) content and LA/AA (P<0.05). In X25-treated silage, compared with the control, adding C decreased the pH and increased the LA content, significantly (P<0.05); adding B decreased the NH3-N content, notably (P<0.05). In X75-treated silage, compared with the control, adding C decreased the pH and increased the lactic acid content, significantly (P<0.05). As the increase of wheat straw mixing ratio, the contents of DM, NDF and ADF was increased, significantly (P<0.05). In contrast, the CP content was decreased, significantly (P<0.05). Compared with MW-treated silage, X25-, X50- and X75-treated silage increased WSC content, notably (P<0.05). In X25-treated silage, compared with the control, the addition of C significantly reduced the contents of NDF and ADF (P<0.05). In X50-treated silage, the addition of B and C significantly reduced the NDF content (P<0.05). In X75-treated silage, compared with the control, the addition of B, C and R reduced the contents of NDF and ADF, notably (P<0.05). 【Conclusion】The fermentation quality of mixed agricultural by-products (watermelon skin﹕broad bean pod﹕beer bad = 1﹕3﹕4) was poor. The mixed silage of agricultural by-products and wheat straw could improve the fermentation quality, and the best mixture ratios of wheat straw and agricultural by-products (watermelon rind﹕broad bean pod﹕beer lees = 1﹕3﹕4) were 50%﹕50% and 75%﹕25%. In terms of nutrition quality, X25 and X50 were the best mixture ratios of wheat straw and agricultural by-products (watermelon rind﹕broad bean pod﹕beer lees = 1﹕3﹕4) were 25%﹕75% and 50%﹕50%. Comprehensively considered the fermentation quality and nutritive value, the optimum mixing ratio of agricultural by-products (watermelon rind﹕broad bean pod﹕beer lees = 1﹕3﹕4) and wheat straw was 50%﹕50%, and the addition of cellulose could further enhance the fermentation quality and improve the nutritional composition. Table and Figures | Reference | Related Articles | Metrics

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Effects of 2-Hydroxy-4-(Methylthio)-Butanoic Acid on Rumen Fermentation and Microbiota in Holstein Female Calves KONG FanLin,LI Yuan,FU Tong,DIAO QiYu,TU Yan Scientia Agricultura Sinica    2022, 55 (4): 796-806.   DOI: 10.3864/j.issn.0578-1752.2022.04.014 Abstract （644）   HTML （36）    PDF （584KB）（134）       Knowledge map    Save 【Objective】2-Hydroxy-4-(Methylthio)-Butanoic Acid (HMBi) is widely used to satisfy the absent of methionine (Met) in ruminal diet. Although the characteristic of HMBi is a rumen protected product, there is still an amount of HMBi degraded in rumen, which should be taken seriously. Hence, this study was conducted to evaluate the effects of HMBi on rumen fermentation and microbiota. 【Method】The experiment was conducted for 97 days with 36 Holstein female calves aged about 84 day-old with (101±10) kg body weight, and those calves were allocated to 2 groups, including PC group (0.40% Met) and PCMet group (0.28% Met). The treatment was achieved by deducting HMBi in diet of PCMet group and made Met level 30% lower than that of PC group. The first 7 d were an adaptation to the diets and the next 90 d for sampling. The body weight was measured at 0 d and 90 d, respectivley. The dry matter intake was recorded daily throughout the whole trial period. The serum and rumen fluid samples from five calves in each group were sampled on day 90 to determine rumen fermentation parameters and microbial communities. 【Result】(1) Compared with PC group, the growth performance of PCMet group was not changed (P>0.05). The Met in serum of PCMet group had trend to be significantly decreased when compared with PC group (0.05<P<0.1); (2) The molar proportion of acetate and microprotein concentration in PCMet group was significantly decreased by Met deduction (P<0.05). There were no significant differences on concentrations of total volatile fatty acid and ammonia nitrogen between two groups (P>0.05). (3) The Shannon index of microbiota in PCMet group was lower than that in PC group (P<0.05). The PCoA and PREANOVA analysis showed the significant distinction between microbiota in two groups (P<0.05). Furthermore, the relative abundance of Firmicutes in PCMet group was decreased and the relative abundance of Bacteroidetes was increased when compared with PC group (P<0.05). At genus level, the relative abundance of Olsenella, [Ruminococcus] gauvreauii group, Acetitomaculum, [Eubacterium] nodatum group, and Coprococcus 1 were decreased in PCMet group (P<0.05). The correlation analysis showed that [Eubacterium] nodatum group and Acetitomaculum were significantly correlated with acetate and [Ruminococcus] gauvreauii group was significantly correlated with MCP (P<0.05, r>0.7). 【Conclusion】 The ruminal microbiota was inhibited by HMBi deduction, which led to the decrease of MCP and Shannon index. Among them, the acetogen was sensitive with HMBi. In conclusion, although HMBi was a rumen protected product, the part of HMBi degraded in rumen still had the ability to regulate rumen fermentation. Table and Figures | Reference | Related Articles | Metrics

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Effects of CaSR and CCK-1R Mediated Soybean Protein Hydrolysate on Appetite Using Mouse WANG LÜYang,CUI LeiHong,FENG JiangYin,HONG QiuXia,YOU MeiJing,BAO HaoYu,HANG SuQin Scientia Agricultura Sinica    2022, 55 (4): 807-815.   DOI: 10.3864/j.issn.0578-1752.2022.04.015 Abstract （354）   HTML （47）    PDF （621KB）（89）       Knowledge map    Save 【Objective】The study aimed to investigate the effects and mechanisms of soy protein hydrolysate (SPH) on the appetite in mice, so as to provide the new frame work guidelines for strategies towards manipulating feed intake in pigs. 【Method】In this study, the pepsin was used to hydrolyze soy protein to produce SPH. Firstly, the effects on short-term feed intake and the expressions of duodenal peptide sensing receptors calcium sensing receptor (CaSR), G protein-coupled receptor 93 (GPR9)3 and oligopeptide transporter 1 (PepT1) were investigated by intragastrically different concentrations of SPH in mice. Based on this, the CaSR inhibitor NPS2143 and the peripheral cholecystokinin-1 receptor (CCK-1R) inhibitor Devazepide were intraperitoneally injected, respectively, to investigate whether SPH inhibited feed intake by the CASR-CCK-CCK-1R-hypothalamus pathway. 【Result】The amount of 1.5g·kg-1 SPH reduced the 0-1 h feed intake (P<0.05), and increased the CaSR expression (P<0.05). Compared with SPH group, the feed intake of SPH+NPS2143 group were increased at 0-1 h, and the plasma CCK levels were decreased, and there were no differences from the control group (0.05<P<0.5). Meanwhile, SPH reduced 0-1 h gastric emptying rate and increased the expression of hypothalamus anorexia nerve factor pro-opiomelanocortin (POMC) (P<0.05), while the effects disappeared in SPH+Devazepide group. However, SPH had no effect on the small intestine transit rate or the expression of the hypothalamic food-promoting factors neuropeptide Y (NPY) and agouti related peptide (AgRP). 【Conclusion】CaSR mediated SPH to promote CCK secretion, delayed gastric emptying rate through the peripheral CCK-1 receptor, and improved the expression of hypothalamic anorexia nerve factor POMC to suppress appetite. Table and Figures | Reference | Related Articles | Metrics

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Amino Acid of 225 in the HA Protein Affects the Pathogenicities of H1N1 Subtype Swine Influenza Viruses YANG ShiMan, XU ChengZhi, XU BangFeng, WU YunPu, JIA YunHui, QIAO ChuanLing, CHEN HuaLan Scientia Agricultura Sinica    2022, 55 (4): 816-824.   DOI: 10.3864/j.issn.0578-1752.2022.04.016 Abstract （369）   HTML （30）    PDF （491KB）（92）       Knowledge map    Save 【Objective】 The pathogenicities of influenza viruses are determined by multiple viral genes. The results of our previous study indicated that hemagglutinin (HA) gene substitutions of the two genetically similar H1N1 swine influenza viruses altered their pathogenicities in mice. This study aimed to further identify the key amino acids affecting viral pathogenicity. 【Method】 After analyzing the amino acid differences of HA protein between the two H1N1 viruses, the reassortant viruses bearing the single amino acid mutations were constructed using the site-directed mutagenesis primers, and their EID50 values were determined. To determine the growth of the parental, reassortant and mutant viruses in vitro, MDCK cells and A549 cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.001 and 0.1, respectively. The BALB/c mice was further intranasally (i.n.) inoculated with 106 EID50 of each virus, and three mice were euthanized at 3 days post-infection (dpi).The organs, including brain, nasal turbinate, lung, kidney and spleen, were collected from the mice and titrated in eggs to evaluate the viral replication abilities in vivo. The MLD50 values of the indicated viruses were determined by inoculating i.n. groups of five mice with 101-106 EID50 of viruses. The body weight was measured daily for 14 dpi, and the mice that lost more than 25% of their original weight were euthanized for humane reasons. 【Result】 The HA proteins of the ZD71 and SY130 viruses differed at four amino acids at positions 4, 138, 144, and 225 (H3 numbering). Four reassortants were rescued, followed by whole-genome sequencing to ensure the absence of unwanted mutations. The viral replication abilities of the reassortant viruses (rZD71-HA/G225E and rSY130-HA/E225G) were significantly affected in MDCK, as well as in A549 cells, when G225E and E225G substitutions were introduced into the rZD71 and rSY130 virus, respectively. In contrast, the mutations of the other three amino acids had little effect on viral replication in vitro. Further mouse infection experiments also demonstrated that amino acid substitutions at site 225 of HA protein significantly affected the viral pathogenicities in mice. In particular, the substitution G225E increased the pathogenicity of rZD71-HA/G225E virus, with the MLD50 value of rZD71-HA/G225E virus decreasing from 4.32 log10EID50 to 3.0 log10EID50, compared with that of rZD71 virus. And the virus replicated well not only in the nasal turbinate and lung, but also in the spleen and kidney. 【Conclusion】 A single amino acid at position 225 in the HA protein significantly affects the viral replication capacity and virulence of these two H1N1 swine influenza viruses. It is suggested that close monitoring for this residue should be paid in the future virological surveillance, so as to provide a scientific basis for better prevention and control of animal influenza, and even human influenza pandemic. Table and Figures | Reference | Related Articles | Metrics

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Impacts of Somatic Cell Count in Early Lactation on Production Performance over the Whole Lactation and Its Genetic Parameters in Holsteins Cattle ZHU Lei,ZHANG HaiLiang,CHEN ShaoKan,AN Tao,LUO HanPeng,LIU Lin,HUANG XiXia,WANG YaChun Scientia Agricultura Sinica    2022, 55 (2): 403-414.   DOI: 10.3864/j.issn.0578-1752.2022.02.014 Abstract （423）   HTML （27）    PDF （563KB）（108）       Knowledge map    Save 【Objective】 The objective of this study was to explore the relationship between somatic cell count in early lactation (6-35 d) (SCCel) and test-day somatic cell count (SCC), test-day milk yield in different lactation stages, and to estimate genetic parameters of SCSel in Holstein, so as to provide a new idea for the breeding of Holstein mastitis resistance. 【Method】Dairy herd improvement (DHI) records for 182 378 Holstein cows were collected from 141 dairy farms from 2008 to 2018. After quality control, a total of 1 869 976 date records were obtained for 150 864 cattle. The pedigree information of three generations were collected (father, mother, grandfather and grandmother from both father side and mother side) to form the pedigree file, comprising a total of 6 451 bulls and 103 452 cows. The GLM process of SAS software analyzed the factors affecting SCSel, such as measurement scale of pasture, measurement season, measurement year, number of lactation days and fetal secondary, etc, and the relationships between SCSel and test-day SCS and test-day milk yield in different lactation stages by REG procedure using SAS software. The genetic parameters for SCSel traits were estimated by single trait repetition model, single trait and two traits animal model using DMU software, and including heritability and genetic correlation. 【Result】The results showed that the SCC of Holstein changed significantly at 2 weeks postpartum. SCCel of Holstein showed a trend of gradual decline with the increase of lactation days. The farm scale, parity, test season and days in milk had significant impacts on SCCel (P<0.05), and the SCSel of cows with an average annual measurement size of more than 1 000 cows in early lactation was significant lower than the average annual measured size of less than 1 000 (P<0.05). SCSel was firstly decreased and then increased with the increase of parity. The SCSel was the highest in summer and the lowest in winter. There were highly significant regression relationships between SCSel and the test-day SCS in different lactation stages (P<0.01), with regression coefficients ranging from 0.06 to 0.19. There was a highly significant regression relationships between SCSel and the test-day milk yield in different lactation stages (P<0.01), and the regression coefficient ranged from -0.46 to -0.16, and early lactation SCSel could affect breast health and performance during lactation. The heritability of SCSel in 1st, 2nd, 3rd parity and over all parity was 0.05 ± 0.005, 0.07 ± 0.01, 0.04 ± 0.01, and 0.03 ± 0.01, respectively. There were moderate to high genetic correlations among SCSel traits under different parity in Holstein, ranging from 0.54 to 0.87. 【Conclusion】The SCC of Holstein cows in early lactation (6-35 days) was affected by the number of conception and season, and the SCC of early lactation had the population characteristics different from those of the SCC of the measurement date of lactation. A high level of SCCel would have impact on the health and production performance over the whole lactation in Holstein cow. This study provided a theoretical basis for differentiated management by SCCel in postpartum cow pasture, laid a foundation for exploring the genetic mechanism of somatic cell number difference in early lactation of Holstein, and the development of new SCS traits in early lactation was helpful to improve mastitis resistance selection of Holstein in China. Table and Figures | Reference | Related Articles | Metrics

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Isolation and Identification of Brucella melitensis from Ticks on Cattle Surface in Hulunbuir Area HUANG TianPeng,GUO Xu,SUN ChangYun,CHEN JingDiao,CHAOMULIGE ,WU Jie,GERILETU Scientia Agricultura Sinica    2022, 55 (2): 415-424.   DOI: 10.3864/j.issn.0578-1752.2022.02.015 Abstract （660）   HTML （27）    PDF （1509KB）（107）       Knowledge map    Save 【Objective】 The aim of this study was to elucidate the relationship between Dermacentor nuttalli and brucellosis in cattle and sheep. 【Method】 The species of ticks collected in Hulunbuir were identified by traditional morphological and molecular biology methods; the ticks collected from the cattle surface were used as materials, and the suspicious bacteria were isolated from their bodies. The isolated bacterial species were determined by methods, such as bacterial isolation and identification, morphological observation, serological detection and molecular biology. 【Result】 The dominant tick species were identified as D. nuttalli in this area, and four strains of suspicious bacteria were successfully isolated in their bodies. The isolated bacteria were gram-negative Brevibacterium by Gram staining, and the bcsp31 and omp22 gene sequences of Brucella were successfully cloned by PCR method. After gene sequencing and BLAST analysis, it was found that the similarity with the Brucella reference sequence was above 99.0%. The phylogenetic tree was successfully constructed by using Mega 7.0 software. The isolated strains were clustered in the same branch with the known sequences of Brucella. Using AMOS-PCR and Brucella typing test, four isolated strains were identified as Brucella melitensis. 【Conclusion】 In this experiment, four strains of B. melitensis were isolated from D. nuttalli collected from cattle in Hulunbuir area, which indicated that as a blood sucking arthropod, D. nuttalli might play an important role in the transmission of brucellosis among different hosts. This paper provided basic data for the investigation and control of Brucella in Inner Mongolia. Table and Figures | Reference | Related Articles | Metrics

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Expression Differences and Functional Analysis of Exosomes microRNA in Porcine Mature and Atretic Follicles CHEN HuiFang,HUANG QiLiang,HU ZhiChao,PAN XiaoTing,WU ZhiSheng,BAI YinShan Scientia Agricultura Sinica    2021, 54 (21): 4664-4676.   DOI: 10.3864/j.issn.0578-1752.2021.21.015 Abstract （460）   HTML （25）    PDF （1674KB）（527）       Knowledge map    Save 【Objective】 To explore the regulatory role of follicular fluid Exosomes (EXs) miRNA in follicular development and atresia, the difference of miRNA expression between mature follicular fluid Exosomes (mffEXs) and atretic follicular fluid Exosomes (affEXs) were analyzed. 【Method】In this study, the follicular fluid of 4-6 mm porcine mature development and atresia follicles was extracted. Then EXs were identified by particle size analysis and Western Blot detection, respectively. the sequencing analysis of the characteristic EXs carried miRNA and functional enrichment analysis were carried out, and then the key signal pathways and differential genes were screened. Finally, mffEXs and affEXs were used as additives for granular cell culture, and Q-PCR detection technology was used to analyze the expression of key genes to verify and analyze the regulatory functions of EXs miRNA in the two types of follicular fluid in follicular development. 【Result】This study successfully separated mffEXs and affEXs. The sequencing results showed that compared with mffEXs, 90 miRNAs in affEXs were up-regulated and 220 miRNAs were down-regulated, indicating that the level of miRNA expression in follicular fluid could directly regulate follicular development. KEGG enrichment analysis showed that the differential signaling pathways of the two types of follicles were mainly concentrated in the signal pathways, such as Ras, cAMP, P53 and MAPK, which involved in the regulation of biological functions, such as oocyte development, meiosis, and granulosa cell cycle. In atretic follicles, the up-regulated expression of ssc-let-7a and ssc-miR-133a-3p potentially targeted and regulated cyclin-dependent kinase (CDK1) and insulin growth factor (IGF1), which inhibited G1 and G2/M Phase operation, and steroid hormone metabolism promoted the obstruction of granular cell cycle and the apoptosis of granular cells, causing follicular atresia; down-regulated ssc-miR-21-5p potentially targeted tumor suppressor gene (P53) and inhibited cell cycle operation to promote the apoptosis of granular cells. mffEXs and affEXs were added to granular cells cultured in vitro, and Q-PCR results showed that CDK1 was significantly up-regulated in mffEXs, while P53 was significantly down-regulated, indicating the reliability of the sequencing analysis results. These results all showed that changes in miRNA expression levels in affEXs promoted granular cell apoptosis and cell cycle arrest, causing follicular atresia. 【Conclusion】 Porcine affEXs carry miRNAs increased the regulation of CDK1, IGF1 and P53 gene expression, and inhibited the cell cycle of granulosa cells and steroid hormone metabolism and other signal pathways, causing granulosa cell apoptosis and follicular atresia. Table and Figures | Reference | Related Articles | Metrics

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To Evaluate the “Two-Step” Genomic Selection Strategy in Pig by Simulation TANG ZhenShuang,YIN Dong,YIN LiLin,MA YunLong,XIANG Tao,ZHU MengJin,YU Mei,LIU XiaoLei,LI XinYun,QIU XiaoTian,ZHAO ShuHong Scientia Agricultura Sinica    2021, 54 (21): 4677-4684.   DOI: 10.3864/j.issn.0578-1752.2021.21.016 Abstract （528）   HTML （37）    PDF （1231KB）（416）       Knowledge map    Save 【Background】 Since genomic selection (GS) was proposed by MEUWISSEN et al. in 2001, it has been widely used in the breeding of dairy cows, pigs, and other livestock, and has significantly improved the speed of genetic gain of various economic traits. In 2017, with the organization and coordination of the National Grazing Headquarter Station and within the framework of the National Swine Improvement Program, the genomic selection platform for pig breeding was officially launched. Although genomic selection has made positive achievements in pig breeding, and the developing of advanced genotyping technology reduced the costs dramatically, some issues were still existed, including the insufficient number of genotyped individuals in majority of core breeding farms and the inappropriate implementation processes has restricted its wide application in practice.【Objective】In combination with the actual situation of domestic pig breeding, the “two-step” strategy for genomic selection was proposed in this study, that is, the off-test evaluation and the early-stage prediction. Off-test evaluation referred to the genetic evaluation of replacement pigs by SSGBLUP after off-test, and early-stage prediction was carried out when the number of chips reached a certain scale. 【Method】 In this study, the 50 K chip datasets of three breeds consisting of Duroc, Landrace, and Yorkshire were used as the base group to simulate the large-scale population of different breeds, respectively. The four generations were simulated: the first three generations were treated as the base population, and the fourth generation as the test population, two traits with medium and low heritability was simulated for each individual. The estimated breeding values of SSGBLUP and traditional BLUP model for different traits were calculated by the pig genomic selection platform based on the HIBLUP software. The predictive performance of early-stage was evaluated according to whether the individual’s testing records have influence their genomic estimated breeding values (GEBV) in test population. 【Result】The results showed that the predictive performance of off-test evaluation and early-stage for traits with medium heritability were better than those with low heritability. The selection accuracy of SSGBLUP was better than traditional BLUP. Moreover, with the increase of the number of chips and the expansion of the population size, the prediction accuracy was higher. The early-stage predictive performance of SSGBLUP was better than that of traditional BLUP, the early-stage prediction could be carried out when the number of genotyped pigs reached about 2 000, and castrating the last 30% individuals according to GEBV could ensure that the top 1% excellent individuals would not be mistakenly eliminated. And the prediction accuracy performance was increasing with the increased number of genotyped pigs. 【Conclusion】 The “two-step” strategy pretty was conformed to the state of domestic breeding program, and was easy to implement and promote the pig breeding in China. When the number of genotyped pigs was small, off-test evaluation could be carried out to improve the accuracy of selection, as well as efficiency, to a certain extent; when the number of genotyped pigs was large, early-stage prediction could be performed by castrating the pigs on the lower rank of GEBV, which could increase the amount of testing for more excellent pigs, and could also strength the selection intensity and accelerate the genetic gain. The “two-step” strategy was in line with the actual requirements of genomic selection in pig industry. The implementation of this strategy could further promote the application of genomic selection and speed up the genetic gain in pig breeding. Table and Figures | Reference | Related Articles | Metrics

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Interference in TP53INP2 Gene Inhibits the Differentiation of Bovine Myoblasts DU JiaWei,DU XinZe,YANG XinRan,SONG GuiBing,ZHAO Hui,ZAN LinSen,WANG HongBao Scientia Agricultura Sinica    2021, 54 (21): 4685-4693.   DOI: 10.3864/j.issn.0578-1752.2021.21.017 Abstract （370）   HTML （30）    PDF （2258KB）（370）       Knowledge map    Save 【Background】Muscle can maintain the motor function of mammals and regulate body metabolism. Its quantity and distribution have an important influence on meat quality. The growth and development of skeletal muscle and its genetic characteristics influence and even determine the meat production and meat quality to a large extent. It is of great significance to study the growth and development of skeletal muscle. The regulatory effect of TP53INP2 on autophagy and the regulation mechanism on the differentiation of preadipocytes have been studied, but whether it affects the differentiation of bovine myoblasts has not been reported. 【Objective】 This study aims to explore the effect of TP53INP2 on the differentiation of Qinchuan bovine myoblasts in order to provide a theoretical basis for the molecular breeding of beef cattle meat traits. 【Method】 The real-time fluorescence quantitative PCR (RT-qPCR) technology was used to detect the expression characteristics of TP53INP2 in different tissues of Qinchuan cattle at 24 months of age. At the same time, the expression patterns of bovine skeletal myoblasts cultured in vitro at different stages of differentiation were analyzed. TP53INP2 gene siRNA was synthesized and transfected to Qinchuan bovine myoblasts, cells were induced to differentiation 12 hours after transfection, phenotypic changes of myoblasts were observed at different time pionts, and RT-qPCR and Western Blot technologies were performed respectively to detect the expression of differentiation marker genes and proteins on the fourth day of induced differentiation. 【Result】 1. RT-qPCR results showed that the expression level of TP53INP2 was the highest in adult Qinchuan cattle Longissimus dorsi muscle tissue and the lowest in the small intestine tissue. It is higher in adult bovine heart, liver, kidney, reticulum and rumen, and lower in other tissues (compared with longissimus dorsi). 2. With the differentiation of myoblasts, the expression of this gene increased from 0 to 4 days and reached a peak on the 4th day, and then decreased. 3. After interfering with TP53INP2 siRNA in myoblasts, the number and length of myotubes in the test group were significantly lower than those in the control group. 4. RT-qPCR results showed that the expression of myoblast differentiation marker genes myogenin (MYOG) and myosin heavy chain protein 3 (MYH3) was significantly lower than that of the control group. Western Blot results showed that the protein expressions of MYOG, MYH3 and MYOD in the test group were reduced and the differences were extremely significant compared with the control group.【Conclusion】Interfering of TP53INP2 has an inhibitory effect on the differentiation of bovine myoblasts, suggesting that this gene may have an important regulatory effect on the growth and development of Qinchuan cattle muscle tissue, and it can be used for in-depth functional research for molecular breeding of beef cattle practice. Table and Figures | Reference | Related Articles | Metrics

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Transcription Factor TEAD4 Regulates Early Embryonic Development in Pigs ZHANG DanDan,XU TengTeng,GAO Di,QI Xin,NING Wei,RU ZhenYuan,ZHANG XiangDong,GUO TengLong,SHENTU LuYan,YU Tong,MA YangYang,LI YunSheng,ZHANG YunHai,CAO ZuBing Scientia Agricultura Sinica    2021, 54 (20): 4456-4465.   DOI: 10.3864/j.issn.0578-1752.2021.20.018 Abstract （466）   HTML （33）    PDF （1144KB）（511）       Knowledge map    Save 【Background】 TEA domain transcription factor 4 (TEAD4) is known to be a member of the TEAD family of transcription factors and plays a key role in determining the characteristics of the preimplantation embryo in rodents. In mouse embryos, it was found to be involved in regulating the genealogical differentiation of trophectoderm cells in preimplantation embryos by promoting Cdx2 expression. The absence of the TEAD4 gene in mouse embryos can lead to failure of mouse blastocyst formation. However, the role of TEAD4 in early porcine embryonic development is still unclear. 【Objective】This study aimed to preliminarily elucidate the effect of TEAD4 on early porcine embryonic development, in order to lay the theoretical foundation for further exploring the molecular mechanisms of transcription factors on early porcine embryonic development. 【Method】In this study, the bioinformatics analysis of the porcine TEAD4 gene was performed by using web-based tools, including analysis of the porcine TEAD4 gene sequence, comparison of homology between pigs, human and mice, and comparison of the evolutionary relationship of TEAD4 between different species. The role of TEAD4 in early embryonic development in pigs was then tested. The mRNA expression level of TEAD4 gene in porcine oocytes and the early embryos was firstly detected by fluorescence quantitative PCR. and then, siRNA targeting TEAD4 was designed and injected into mature oocytes by microinjection technique to reduce the level of endogenous TEAD4 gene in the oocyte cytoplasm, and to determine that TEAD4 siRNA acts only on TEAD4 gene, with a view to determining the role of TEAD4 gene in early porcine embryonic development. 【Result】Sequence analysis showed that the porcine TEAD4 gene contained 11 exons and localized on chromosome 5, with spanning 37.188 kb, 1 473 bp in full mRNA length, and 1305 bp in full coding region, which encoded 434 amino acids. Homology analysis with human and mouse revealed that TEAD4 was highly conserved in different species and had the closest affinity on pig and cow. The results of fluorescence quantitative PCR showed that TEAD4 mRNA was expressed in both porcine oocytes and early embryos; compared with GV-stage oocytes, the expression of TEAD4 mRNA was lowest in MII-stage oocytes and remained low until the 4-cell stage, but reached the highest expression in the 8-cell stage, and then gradually decreased in the morula and blastocyst stages. Microinjection of siRNA targeting TEAD4 revealed that TEAD4 siRNA only acted on the endogenous TEAD4 gene in oocytes, but not on TEAD1 and TEAD3, and compared with the control and negative control siRNA groups, the injection of TEAD4 siRNA significantly reduced TEAD4 mRNA expression at the 8-cell and morula embryo periods. When TEAD4 gene expression was knocked down, observation of the developmental efficiency of porcine orphan activation and in vitro fertilization embryos showed that the developmental efficiency of TEAD4 siRNA knockdown group from 8-cell to blastocyst stage was significantly reduced compared to the control and negative control siRNA groups. 【Conclusion】 The results of this study indicated that the TEAD4 gene was highly conserved across species, with the closest affinity on pigs and bovine, and that TEDA4 might be involved in regulating the development of early porcine embryos.. Table and Figures | Reference | Related Articles | Metrics

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Alternative Splicing of TNNT3 and Its Effect on the Differentiation of MuSCs in Goat CHEN Yuan,CAI He,LI Li,WANG LinJie,ZHONG Tao,ZHANG HongPing Scientia Agricultura Sinica    2021, 54 (20): 4466-4477.   DOI: 10.3864/j.issn.0578-1752.2021.20.019 Abstract （482）   HTML （32）    PDF （3340KB）（1196）       Knowledge map    Save 【Objective】As a member of the troponin (Tn) family, TNNT3 (Fast Skeletal Troponin T3) involves skeletal muscle contraction, growth, development, and even meat characteristics of domestic animals. This study initially aimed to identify the alternative splicing of goat (Capra hirus) TNNT3. 【Method】Based on goat TNNT3 (NM_001314210. 1) and cattle TNNT3 (XM_010821200) mRNA sequence from NCBI (National Center for Biotechnology Information), the primers were designed by using the Primer Premier 6.0 software, subsequently, TNNT3 was amplified from skeletal muscles of embryo Jianyang Bigear Goat. The obtained TNNT3 sequences were then bioinformatically analyzed by using ORF Finder, EditSeq, DNAMAN, ClustalW, and MEGA_X_10.1.8. Furthermore, the levels of TNNT3 isoforms were quantified by using real-time fluorescence quantitative PCR (RT-qPCR) and semi-quantitative PCR in longissimus dorsi (LD) muscle, semimembranosus (SM) muscle, heart, liver, spleen, lung, and kidney, at seven stages (E75, E90, E105, B3, B45, B150, and B300), respectively. Additionally, the coding ability of transcript TNNT3_3 in vitro and its effect on the differentiation of goat skeletal muscle satellite cells (MuSCs) were explored. 【Result】① A total of five isoforms (named TNNT3_1-5) of the TNNT3 gene were identified in pooled RNA extracted from goat muscles, and the complete coding sequence (CDS) sequence mainly contained 18 exons. ② Nucleotide sequence and amino acid sequence of the goat TNNT3 gene were highly consistent with sheep, cattle, pig, and other mammals, but low with that of fish and reptiles, indicating the high evolutionary conservation of the TNNT3 gene in mammals. ③ The TNNT3 mRNA was presented in all seven detected tissues but highly enriched in LD and SM muscles (P < 0.01), followed by cardiac muscle and lung. Furthermore, the levels of TNNT3 mRNA in SM muscles were higher than that in LD muscles in prenatal goats (P < 0.05), while the verse results were presented postnatal (P < 0.05). ④ The conserved exon 9-11 (138 bp) of goat TNNT3 was repeated in the transcript TNNT3_3. TNNT3_3 was amplified, and it was found out that which could encode protein, and the protein size was basically the same as expected (37 kDa) in TnT transcriptional translation system in vitro. Moreover, the transfection of TNNT3_3 into goat MuSCs induced mRNA levels of myogenic differentiation marker genes, including Myomaker, MyoG, and MyH4, comparing with the control (P < 0.01). 【Conclusion】Five isoforms with complete CDS region of the TNNT3 gene were obtained in goat muscles. The sequence and expression of TNNT3 were highly conserved in mammals and enriched in muscles, indicating that potentially TNNT3 gene functions critically in muscle growth and development. Table and Figures | Reference | Related Articles | Metrics

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Establishment and Preliminary Application of Lawsonia intracellularis IPMA Antigen Detection Method Based on SodC Monoclonal Antibody LI MinXue,LI JianNan,ZHOU Hong,XIAO Ning,LIN HuiXing,MA Zhe,FAN HongJie Scientia Agricultura Sinica    2021, 54 (20): 4478-4486.   DOI: 10.3864/j.issn.0578-1752.2021.20.020 Abstract （800）   HTML （34）    PDF （1989KB）（430）       Knowledge map    Save 【Objective】 Lawsonia intracellularis (L. intracellularis) is an enteric pathogenic bacteria that causes porcine proliferative enteropathy (PPE), which mainly shows the decline of animal welfare and causes serious economic losses to the world swine industries. The objective of this study was to prepare monoclonal antibodies against SodC of L. intracellularis, and to establish an immunoperoxidase monolayer assay (IPMA) method for detecting L. intracellularis base the monoclonal antibody, while test its application in clinical practice, so as to provide a scientific and effective means for the diagnosis of L. intracellularis. 【Method】In this study, the commercial live attenuated L. intracellularis vaccine was selected as target strain. The sodc gene was amplified by PCR and cloned into the prokaryotic expression vector pGex-6p-1. The recombinant plasmid pGex-6p-1-sodc was confirmed to be constructed successfully and induced expression of recombinant SodC protein. The reactivity of the recombinant protein was analyzed by Western Blot. The primary antibody was a mouse anti-GST labeled antibody. BALB/c mice aged 4-6 weeks were immunized with purified SodC protein, and hybridoma cells were screened by conventional cell fusion, limited dilution and indirect ELISA, then ascites were prepared. It was confirmed that two monoclonal antibodies had good specificity through indirect immunofluorescence (IFA). Using the monoclonal antibody as the primary antibody, a method of IPMA for detecting L. intracellularis was developed, and the specificity, sensitivity and repeatability of the method were evaluated. The optimized IPMA method was used to detect ileal tissue samples from pig farms in Jiangsu Province and to evaluate the clinical value of the method. 【Result】After purification, the concentration of SodC protein was higher, and it specifically bound to the antibody against GST tag, indicating that the protein had good regenicity. After three times of subcloning, two strains positive hybrid tumor cells were screened, named 1D6 and 1F7, respectively. The titers of two monoclonal antibodies were both reached 1﹕204 000 by ELISA. The subclass identification results of antibodies showed the subclass of 1D6 was IgA,and subclass of 1F7 was IgG3. The result IFA showed that 1D6 and 1F7 had specific reaction with L. intracellularis, but did not cross-react with S. Cholerasuis, PEDV and TGEV. The ascites of the two monoclonal antibodies were both 1﹕1 024 000 by ELISA; IFA confirmed that the two monoclonal antibodies had good specificity. The optimized IPMA reaction conditions showed that when the dilution ratio of the primary antibody was 1:800 for 45 min, and the dilution ratio of the secondary antibody was 1﹕2 500 for 1h, the established IPMA exhibited the best performance. The specificity and sensitivity tests showed that S. Cholerasuis, PEDV, TGEV, PCV2 and PRV were all negative, and the minimum detection limit was 103L.intracellularis. The optimized IPMA method was used to detect the ileum tissue samples from pig farms in the surrounding areas of Jiangsu Province. A total of 92 positive samples were detected from 146 samples of ileal tissues. The positive rates of 3 different pig farms were 65.6%, 68.1% and 53.7%, respectively, and the overall positive rate was 63.0%. 82 positive samples were detected by PCR method. and the positive coincidence rate of the two methods was 94.6%. These results indicated that this method had clinical value. 【Conclusion】The monoclonal antibodies against the recombinant SodC protein were successfully prepared, and the IPMA method for L. intracellularis was established with good specificity and sensitivity, and the clinical samples were tested. In summary, these results further proved that the IPMA had certain clinical value, and provided an effective technical means for the isolation and identification of L. intracellularis in the laboratory, localization in infected cells, epidemiological investigation and quarantine. Table and Figures | Reference | Related Articles | Metrics

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Evaluation of Feeding Value for Whole Broussonetia papyrifera Silage in Diet of Wuchuan Black Beef Cattle CHEN GuangJi,XIONG XianQin,HE RunXia,TIAN Xiong,SHEN YingLong,ZOU XiaoMin,YANG Hong,SHANG YiShun,ZHAO MingKun,LI XiaoDong,LI ShiGe,ZHANG Rong,SHU JianHong Scientia Agricultura Sinica    2021, 54 (19): 4218-4228.   DOI: 10.3864/j.issn.0578-1752.2021.19.016 Abstract （421）   HTML （34）    PDF （642KB）（546）       Knowledge map    Save 【Objective】 This study aimed to explore the feeding value of whole Broussonetia papyrifera silage (WBPS) on Wuchuan black beef cattle, so as to provide a reference for rational utilization of Broussonetia papyrifera as feed.【Method】 A completely randomized trial design was used, and fifty head of black beef cattle with similar weight and age ((108.06±14.51) kg, 9 months) were randomly assigned to five dietary treatments: (A, B, C, D, and E). Each treatment had 1 replicate, with 10 steers in each replicate. Each treatment group was fed diets with the same concentrate to forage ratio () for 288 days, and the ratio of A, B, C, D, and E was 0, 17%, 41%, 66% and 83% WBPS, respectively. The dry matter intake (DMI), daily gain (ADG) and feed to weight ratio (DMI/ADG) were measured at the beginning, the middle stage (175th day and 220th day ) and the end of the experiment (288th day). The body size, rumen fermentation parameters and carcass quality were measured at the end of the experiment. 【Result】 ADG of C and D were higher than those of other groups at 0-175th day, 175-220th day, 220-288 day and the whole period (P<0.05), while DMI/ADG value was lower (P<0.05). In addition, the dietary factors affected the ADG time gradient of each group: with the prolongation of the experimental period, the ADG of A and B decreased significantly, while that of C, D and E increased firstly and then decreased. The increment of body height and oblique length of D was higher than that of other groups, and the second was group C (P<0.05). The ruminal acetic, propionic and total volatile fatty acids of A and B were higher than those of other groups, but the rumen microbial protein production of D was the highest, which was 5.27 times higher than that of A. The trial factor had no significant effect on slaughter rate and net meat rate of Wuchuan black beef cattle (P>0.05), while increasing the proportion of WBPS in the diet decreased the carcass fat percentage and muscle shear force (P<0.05). There was no significant difference in the composition and content of amino acids in muscle of Wuchuan black beef cattle (P>0.05), but WBPS decreased the content of saturated fatty acids and increased the content of polyunsaturated fatty acids (P<0.05). 【Conclusion】 WBPS replacing whole plant corn silage as a dietary component of Wuchuan beef black cattle could improve daily gain, reduce feed to weight ratio, increase rumen microbial protein production, reduce carcass fat rate, and positively change muscle fatty acid composition. Table and Figures | Reference | Related Articles | Metrics

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Correlation Analysis of Inosine Monophosphate Specific Deposition Related LNC_003828-gga-miR-107-3P-MINPP1 in Jingyuan Chicken Muscle Tissue YU BaoJun,DENG ZhanZhao,XIN GuoSheng,CAI ZhengYun,GU YaLing,ZHANG Juan Scientia Agricultura Sinica    2021, 54 (19): 4229-4242.   DOI: 10.3864/j.issn.0578-1752.2021.19.017 Abstract （442）   HTML （35）    PDF （4456KB）（1851）       Knowledge map    Save 【Objective】The aim of this study was to explore the regulatory role of key regulatory factors in the process of inosine monophosphate deposition in the muscle tissue of Jingyuan chickens, and to use lncRNA-miRNA-mRNA association analysis to identify LNC_003828, gga-miR-107-3p and MINPP1 related to inosine monophosphate specific deposition, so as to provide a theoretical basis for molecular-assisted breeding to improve chicken muscle quality.【Method】 The inosine monophosphate content of the breast and leg muscles of 15 Jingyuan chickens was determined, and three samples of the breast muscles with high inosine monophosphate content and the leg muscles with low inosine monophosphate content were screened to extract total RNA. The cDNA library was constructed after passing the quality test, and PCR amplification test was carried out. Then, the cDNA library quality was evaluated by using Agilent 2100, which was sent the library to the Illumina-Hiseq platform for transcriptome sequencing. Using bioinformatics methods, the differentially expressed MINPP1, gga-miR-107-3p and LNC_003828 in different parts of the muscle tissue of Jingyuan chicken were screened out, and GO annotation and protein interaction network was used to analyze the function of MINPP1. The qRT-PCR method was used to detect the expression of LNC_003828, gga-miR-107-3p and MINPP1 in the breast and leg muscles of Jingyuan chickens, and the correlation between them and the content of inosine monophosphate was analyzed. 【Result】 R2, the correlation of gene expression levels between sequenced samples, was greater than 0.9, that is, gene expression between experimental samples could be used for subsequent differential gene analysis. Three differentially expressed genes, including MINPP1, PKM, and ALDH9A1, were detected in the glycolysis/gluconeogenesis pathway involving in the synthesis and metabolism of inosine monophosphate. Interaction analysis found that there were 17 miRNAs (9 up-regulated, 8 down-regulated), 44 mRNAs (16 up-regulated, 28 down-regulated), and 155 lncRNAs (68 up-regulated, 87 down-regulated) in the lncRNA-miRNA-mRNA network diagram, of which the target gene of the core node gga-miR-107-3p interaction was MINPP1, and the target lncRNA was LNC_003828. GO enrichment analysis found that the MINPP1 gene had functions such as phosphatase activity and bisphosphoglycerate phosphatase activity; the MINPP1 gene in the protein interaction network were all interact with PGAM1 and ENO1, which were involved in glycolysis/gluconeogenesis and amino acid biosynthesis pathways BPGM genes. The results of qRT-PCR showed that the relative expression of LNC_003828 and gga-miR-107-3p in breast muscle of Jingyuan chicken was lower than that of leg muscle, but the difference was not significant; the relative expression of MINPP1 in breast muscle was significantly lower than that of leg muscle (P<0.05). The expression of gga-miR-107-3p in the breast and leg muscle tissues of Jingyuan chicken was positively correlated with the expression of LNC_003828 and negatively correlated with the expression of MINPP1. The expression of LNC_003828 and gga-miR-107-3p in breast and leg muscle tissues were positively correlated with inosine monophosphate content, and the difference was not significant; the expression of breast muscle MINPP1 was negatively correlated with inosine monophosphate content and the expression of leg muscle MINPP1. The amount was significantly negatively correlated with the content of inosine monophosphate (P<0.05). In summary, it was speculated that gga-miR-107-3p in the muscle tissue of Jingyuan chicken was used as a core regulator to adsorb LNC_003828, which affected the MINPP1 gene to regulate the specific deposition of muscle inosine monophosphate, thereby improving meat quality. 【Conclusion】 LNC_003828, gga-miR-107-3p, and MINPP1 were selected as candidate regulatory factors affecting the specific deposition of inosine monophosphate. Table and Figures | Reference | Related Articles | Metrics

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Detection of Interaction Between Porcine Type I Complement Receptor and C3b Active Fragment in Vitro SUN YuChen,JIA RuiPu,FAN KuoHai,SUN Na,SUN YaoGui,SUN PanPan,LI HongQuan,YIN Wei Scientia Agricultura Sinica    2021, 54 (19): 4243-4254.   DOI: 10.3864/j.issn.0578-1752.2021.19.018 Abstract （404）   HTML （35）    PDF （984KB）（717）       Knowledge map    Save 【Objective】In order to provide scientific data for elucidating the molecular mechanism of porcine erythrocyte immune adhesion function, it was investigated whether CR1-like (Complement receptor 1-like, CR1-like) of porcine erythrocyte could bind to the C3b or not.【Method】In this study, the recombinant plasmids of CR1-like(3-6) and CR1-like(8-11) functional domain fragments were constructed first, which were used to establish a yeast two-hybrid detection system. The bait plasmid (recombinant pGBKT7-CR1-like) and capture plasmid (recombinant pGADT7-C3b) were co-transformed into Y2HGold yeast cells. The single deficient SD/-Leu, SD/-Trp and double-deficient SD/-Leu/-Trp (DDO) media were used to strictly screen the co-transformed yeast cells. Then, according to the expression of report factor, the growth of transformants were identified on the double-deficient medium SD/-Leu/-Trp/X-α-Gal (DDO/X) or SD/-Leu/-Trp/X-α-Gal/Aba (DDO/X/A) combined with the color change phenomenon of the colony to comprehensively determine whether CR1-like active fragments and complement C3b bind to each other in yeast cells or not. The CR1-like-C3b binding complex in yeast cells was then separated by immunoprecipitation, and the specificity of the complex was identified by Western blot. 【Result】The co-transformed yeast clones showed normal growth on SD/-Leu, SD/-Trp, DDO and DDO/X, DDO/X/A media with blue color colonies, and this indicated that positive yeast colonies were successfully obtained. The results of PCR reverse identification showed that the co-transformed yeast contained the target genes CR1-like(3-6) and CR1-like(8-11). The C3b gene fragment appeared after the plasmid was digested, indicating that the recombinant plasmid pGBKT7-CR1-like and pGADT7-C3b were successfully co-transformed into yeast cells. In the immunoprecipitation test, the tag antibody c-Myc of the pGBKT7 vector was used to precipitate the fusion protein in yeast cells. Western blot detection with c-Myc as the primary antibody revealed that the fusion protein transformed pGBKT7-CR1-like(3-6) and pGBKT7-CR1-like(8-11) separately showed a specific band at 50 kDa; the yeast fusion protein co-transformed with pGBKT7-CR1-like(3-6) + pGADT7-C3b and pGBKT7-CR1-like(8-11) + pGADT7-C3b showed a specific band at 83 kDa; when the HA monoclonal antibody was used as the primary antibody for Western blot detection, no specific bands appeared in the pGBKT7-CR1-like(3-6) and pGBKT7-CR1-like(8-11) fusion proteins, and only the yeast fusion protein co-transformed in lane 3 and 4 showed a specific band at 83 kD. It showed that there was a complex of CR1-like and C3b in Y2HGold yeast cells. Using CR1-like monoclonal antibody to precipitate the fusion protein in yeast cells, Western blot detection with CR1-like as the primary antibody revealed that the fusion protein transformed with pGBKT7-CR1-like(3-6) and pGBKT7-CR1-like(8-11) separately showed a specific band at 50 kD; the yeast fusion protein co-transformed with pGBKT7-CR1-like(3-6) + pGADT7-C3b and pGBKT7-CR1-like(8-11) + pGADT7-C3b showed a specific band at 83 kD; when the C3 monoclonal antibody was used as the primary antibody for Western blot detection, no specific bands appeared in the pGBKT7-CR1-like(3-6) and pGBKT7-CR1-like(8-11) fusion proteins, lanes 3 and 4 showed that only the co-transformed yeast fusion protein had a specific band at 83 kD. This indicated that there was a biologically active CR1-like and C3b binding complex in Y2HGold yeast cells. The bait plasmid expression products CR1-like(3-6), CR1-like(8-11) fragments and capture plasmid expression products C3b fragment could be combined in yeast cells.【Conclusion】In summary, the recognition ligand for porcine erythrocyte CR1-like to exert immune adhesion function was C3b, which provided an important data basis for the further analysis of the molecular structure of CR1-like functional domain. Table and Figures | Reference | Related Articles | Metrics

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Index System for Quantitative Evaluation of Pasture Degradation in Meadow Grassland of Inner Mongolia YAN RuiRui, GAO Wa, SHEN BeiBei, ZHANG Yu, WANG Miao, ZHU XiaoYu, XIN XiaoPing Scientia Agricultura Sinica    2021, 54 (15): 3343-3354.   DOI: 10.3864/j.issn.0578-1752.2021.15.017 Abstract （454）   HTML （41）    PDF （1216KB）（721）       Knowledge map    Save 【Background】 Inner Mongolia grassland is an important natural ecological barrier in northern China, among which meadow steppe is located in the transition zone from forest to grassland, and it is a very valuable natural renewable resource in China. The proportion of meadow grassland in Inner Mongolia is the largest in China, most of which are pastures. Grazing is one of the most important ways for human beings to affect grassland ecosystem. Excessive grazing will lead to retrograde succession of grassland community, and grassland production performance will be continuously reduced, thus limiting the stable development of grassland animal husbandry. 【Objective】 Comprehensive, accurate and timely assessment of pasture degradation is of great significance for maintaining and promoting sustainable grassland utilization. 【Method】 In this study, the degradation succession law and driving mechanism of grassland pasture were summarized, and the degradation index system of grassland pasture in Inner Mongolia was established by using analytic hierarchy process (AHP), expert investigation and comparative matrix analysis methods, which included 8 indexes, such as aboveground biomass, coverage, average height, plant species, litter, proportion of degradation indicator plant, soil organic carbon content and soil bulk density. Based on the establishment of the comprehensive evaluation index model, the parameters of the reference index were put forward, and the comprehensive index of quantitative evaluation was used to reflect the overall situation of grassland degradation. At the same time, the quantitative evaluation index system of grassland degradation in Inner Mongolia and its technical method were discussed and studied. This method was evaluated and verified based on the controlled grazing experiment in Xeltala of Hulunbuir. 【Result】 The results showed that the weight of the eight indexes from the largest to the smallest in the evaluation index system of Inner Mongolia meadow steppe were aboveground biomass, coverage, average height, proportion of degraded plants, number of plant species, litter, soil organic carbon content, and proportion of soil bulk density increase. Meadow grassland degradation could be classified into four grades: non-degradation, mild degradation, moderate degradation and severe degradation. When the grazing was equal to zero or very light grazing, the grassland belonged to the scope of non-degraded grassland. When the grazing rate was above 90%, the grassland belonged to the range of severely degraded grassland. 【Conclusion】 It was suggested that a longer period of discussion should be carried out in the future research to further improve and update the benchmark reference value, which was conducive to the improvement and maturity of the evaluation index system of pasture degradation, and could provide a basis for quantitative assessment of pasture degradation. Table and Figures | Reference | Related Articles | Metrics

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Advances of Biosynthesis and Toxicity of Cereulide Produced by Emetic Bacillus cereus CUI YiFang,ZHENG Min,DING ShuangYang,ZHU Kui Scientia Agricultura Sinica    2021, 54 (12): 2666-2674.   DOI: 10.3864/j.issn.0578-1752.2021.12.016 Abstract （796）   HTML （37）    PDF （2807KB）（2148）       Knowledge map    Save Bacillus cereus (B. cereus) is a gram-positive facultative anaerobe that can produce spores to survive adverse environments. And it is widely present in soil, water, air and a variety of foods. Pathogenic B. cereus is one of the most common food-borne pathogens, and the toxins produced by B. cereus are the main cause of food poisoning. Cereulide is a major toxin produced by pathogenic B. cereus, which is a small molecule lipophilic cyclic dodecadepsipeptide with stable structural properties. Cereulide can cause mild food poisoning with emetic symptoms, such as nausea and vomiting, and it may induce severe fatal diseases such as hepatic encephalopathy or acute liver failure. Current researches believed that cereulide caused vomiting by stimulating the vagus nerve, and induced the loss of mitochondrial membrane potential by acting as a potassium ionophore, which ultimately led to cell death. However, the toxic mechanism of hepatic encephalopathy or acute liver failure caused by cereulide remains unclear. Cereulide is encoded by the cereulide synthetase gene cluster (ces) and is synthesized by the non-ribosomal peptide synthetase (NRPS) system. Cereulide is composed by two hydroxy acids and two amino acid residues [-D-HIC-D-Ala-L-HIV-L-Val-], which forms a trimer phenolphthalein after three iterations and shows a structural specificity and representativeness. However, isocereulides may be produced due to the flexibility of the NRPS system. Therefore, the toxicity of cereulide is closely related to its biosynthesis process. Based on previous studies, this review summarized and proposed the biosynthesis mechanism of cereulide. Firstly, the CesA and CesB domained in ces recognize D-α-ketocarboxylic acid, L-alanine, L-α-ketoisovalerate and L-valine, respectively, which formed the main synthetic unit dipeptide of cereulide by covalent bonding. Secondly, a tetrapeptide was synthesized by repeating the above process. Thirdly, the second tetrapeptide was synthesized through repeated reactions, and the two tetrapeptides formed an octapeptide through esterification. Fourthly, the above reaction was repeated to form a ternary complex product peptide. Lastly, because the surface structure of the active center of the thioesterase domain in ces-NRPS prevented external water molecules from entering, it induced an internal nucleophilic attack reaction and finally released a circular cereulide. The risk of food poisoning caused by cereulide producing B. cereus was underestimated. In addition, our previous studies have found that some probiotic Bacillus products were contaminated with cereulide-producing B. cereus strains. This posed a potential risk to food safety and public health. This review briefly summarized the characteristics and toxic mechanisms of cereulide, which would provide a scientific basis for the prevention of cereulide. This review also summarized and proposed the biosynthesis process of cereulide. Functions of two domains in the synthesis process need to be focused. The main function of ketoreductase (KR) domain was that it could catalyze the formation of esters of keto acid at the beginning of the biosynthesis processes. The important role of thioesterase (TE) domain was to form repeating units and the cyclic peptide in the last link of synthesis. These could serve as a model for other cyclic peptides synthesized by the non-ribosomal peptide synthetase system. Table and Figures | Reference | Related Articles | Metrics

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Protective Effects of Chinese Propolis Extract Against Lipopolysaccharide- Induced Acute Mastitis and Mammary Barrier Functions in Mice SONG MeiJie,OU AiQun,XUE XiaoFeng,WU LiMing,SHOU QiYang,WANG Kai Scientia Agricultura Sinica    2021, 54 (12): 2675-2688.   DOI: 10.3864/j.issn.0578-1752.2021.12.017 Abstract （443）   HTML （37）    PDF （2428KB）（684）       Knowledge map    Save 【Background】 Dairy cow mastitis is a common and frequent dairy cow disease, which not only threatens the health of dairy cows, but also causes significant economic losses. The traditional medicine for the treatment of dairy cow mastitis is largely dependent on the usage of antibiotics, which easily caused antibiotic resistance/abuse. Therefore, it is with practical significance to develop an alternative approach of antibiotics for the prevention and treatment of mastitis in dairy cows. Propolis is a natural product with good anti-inflammatory and antibacterial activities. Propolis is collected by western honeybees from plant resin and mixing with bees’ maxillary gland and wax gland secretions. Accordingly, propolis has great potential for the prevention and treatment of mastitis in dairy cows. Despite that there have some previous reports on the prevention and treatment of mastitis using propolis, scant information is available on the effects of propolis on the breast blood barrier function. 【Objective】The aim of this study was to evaluate the protective effect of ethanol extract of Chinese propolis, EECP, against bacterial lipopolysaccharide- induced mouse acute mastitis, and we focused on its effect on the expression of tight junction proteins in mammary epithelial cells. This outcome of this study would lay a foundation for the in-depth study for the usage of propolis for prevention and treatment on dairy cow mastitis. 【Method】 The type and content of the main polyphenolic compounds in EECP were determined by ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). Female ICR mice were divided into four groups, including the normal control group, model group (injecting LPS into the mammary gland, 1mg·kg-1 b.w.), EECP group and positive control group (dexamethasone). The female mice were subjected to continuous gavage by EECP or positive control drugs for seven days. The LPS model group, EECP group and positive control group were injected with 1 mg·kg-1 LPS through the fourth and the fifth pair of nipple tubes, to induce acute mastitis in mice. After 24 h, they were killed for collecting the mammary tissue samples. Hematoxylin-Eosin (HE) staining method and Sirius red staining method were used to evaluate pathological changes in mouse mammary tissue. Enzyme-linked immunosorbent assay (ELISA) was used to determine the release of inflammatory factors and quantitative real-time PCR was applied to determine mouse mammary tissue gene expressions of tight junction proteins (Occludin, Cluadin-1, ZO-1). Finally, the immunohistochemical technology was used to study the expression and distribution of mouse mammary tight junction proteins (Occludin and ZO-1). These above indicators were applied to comprehensively evaluate the anti-mastitis activity and its effect on mammary tight junction proteins by EECP. 【Result】 Based on UHPLC-QqQ-MS/MS, an accurate quantitative method was established for the 17 main polyphenolic compounds in EECP. The results showed that the five compounds with the higher content and their contents were: galangin (12.88 ±0.57 μg·mg-1), 3-O-Acetylpinobanksin (12.93±0.59 μg·mg-1), pinocembrin (8.56±0.27 μg·mg-1) and pinobanksin (8.52±0.25 μg·mg-1). Animal study results showed that after a week of regular oral administration of EECP reduced on the infiltrations of inflammatory cells in mammary gland under LPS stimulation, and the structural damages were alleviated by EECP. Moreover, Chinese propolis administration inhibited the overexpression of inflammatory factors (IL-1β, IL-6, IL-10) in mouse mammary gland tissue, and increased the transcription level of tight junction protein genes (Occludin, Claudin-1, ZO-1) mRNA, and enhanced the expression of tight junction protein Occludin, ZO-1. 【Conclusion】EECP showed good preventive effect against lipopolysaccharide-induced mastitis in mice, with potent anti-inflammatory effects. It also increased the expressions of tight junction proteins in the mammary gland, which maintained the integrity of the tight junction structure in the mammary gland and protected the blood-milk barrier. Nevertheless, the molecular mechanism of Chinese propolis on the regulation of tight junctions needed to be clarified in the future research. Table and Figures | Reference | Related Articles | Metrics

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The Effect of Flumethrin on Metabolism of Worker Larvae of Apis mellifera with LC-MS Technique YU LongTao,YANG HeYan,SU YuChen,YAN WeiYu,WU XiaoBo Scientia Agricultura Sinica    2021, 54 (12): 2689-2698.   DOI: 10.3864/j.issn.0578-1752.2021.12.018 Abstract （473）   HTML （31）    PDF （1581KB）（692）       Knowledge map    Save 【Objective】Flumethrin belongs to the second generation pyrethroid insecticides and acaricides, which is used for the control of honeybee mites. Because of the toxicity of acaricides, it can not only kill mites, but also threat the health of honeybees. The different metabolites of Apis mellifera worker larvae treated with different concentrations of flumethrin were tested using liquid chromatography-mass spectrometry (LC-MS) technology and the metabolic pathways involved of different metabolites were analyzed, so as to explore the toxicological effect of flumethrin on honeybees and provide references for scientific using in beekeeping.【Method】The queen was controlled to lay eggs on an empty worker frame for 12 h, and the spawning area was divided into four groups. From the 5th day, the small larvae of each group were fed with sugar water containing different concentrations of flumethrin (0, 0.5, 5, 50 mg·kg-1), the dose was increased daily from day 5 to day 8 (1.5, 2, 2.5, 3 μL), and lymph fluid from larvae was collected on day 9. The metabolites of A. mellifera larvae were analyzed by LC-MS and the metabolites with significant difference were screened by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), while the metabolic pathways of common differential metabolites in flumethrin treatment groups were analyzed.【Result】Compared with the control group, a total of 190 different metabolites were found and 87 types were identified in 0.5 mg·kg-1 group, and a total of 275 different metabolites were identified and 97 types were identified in 5 mg·kg-1 group, while there were a total of 275 different metabolites and a total of 131 species were identified in 50 mg·kg-1 group. Meanwhile, 29 common differential metabolites in treatment groups were screened, of which 16 metabolites were up-regulated, 12 metabolites were down-regulated, and 1 metabolite was down-regulated in 0.5 and 50 mg·kg-1 groups while it was up-regulated in 5 mg·kg-1 group. These differential metabolites include ribose, purine and its derivatives, fatty acids with their conjugates. After enrichment analysis of metabolic pathways, significant differences (P＜0.05) were found in the metabolic pathways, which include amino sugar and nucleotide sugar metabolism, drug metabolism-other enzymes, α-linolenic acid metabolism and other pathways.【Conclusion】The LC-MS technology can effectively analyze the changes of metabolites in the honeybee larvae treated with flumethrin, and flumethrin can cause the contents of UDP-N-acetylglucosamine, azathioprine, traumatic acid, 9-oxononanoic acid and 13(s)-HPODE abnormal in honeybee larvae. The changes of these different metabolites confirm that flumethrin causes various substance metabolism disorders in honeybees. The analysis of these metabolic processes can further explain the mechanism of honeybees metabolizing toxic compounds, and provide a theoretical basis for the stress of acaricides and other toxic compounds on honeybees. Table and Figures | Reference | Related Articles | Metrics

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Research Progress of Intelligent Sensing Technology for Diagnosis of Livestock and Poultry Diseases LI QiFeng,LI JiaWei,MA WeiHong,GAO RongHua,YU LiGen,DING LuYu,YU QinYang Scientia Agricultura Sinica    2021, 54 (11): 2445-2463.   DOI: 10.3864/j.issn.0578-1752.2021.11.016 Abstract （982）   HTML （90）    PDF （1104KB）（1193）       Knowledge map    Save Animal husbandry is an important part of agriculture. At present, animal husbandry is developing towards large-scale and intensive development, which also increases the difficulty of diagnosis of livestock and poultry diseases. In recent years, in order to improve the level of animal welfare in livestock and poultry breeding, and to reduce the economic losses and public health safety risks caused by animal diseases and health abnormalities in livestock breeding, a number of automated methods for the diagnosis and treatment of livestock and poultry diseases through digital and intelligent means have emerged, such as machine vision analysis, animal audio analysis, infrared temperature perception, deep learning classification, etc. These methods could effectively improve the diagnosis efficiency of diseased or abnormal livestock and poultry, shorten the diagnosis cycle, and reduce the labor force of manual inspection in animal husbandry. The automatic diagnosis and treatment method of livestock and poultry diseases is different from the conventional diagnosis methods based on pathological knowledge, which mainly uses various sensors to automatically obtain various characteristics information of livestock and poultry during the breeding process, such as images, sounds, body temperature, heart rate, and excrement. The collected information is comprehensively analyzed and processed through mathematical models, such as Mel cepstrum coefficient, Logistics regression analysis and intelligent algorithms such as support vector machines and deep learning, and then the animal’s health status is evaluated and predicted. The current research progress of animal disease intelligent diagnosis technology and some basic method principles was summarized from several aspects, such as livestock and poultry morphological diagnosis technology, behavior diagnosis technology, sound diagnosis technology, body temperature diagnosis technology, and other physiological parameter diagnosis technology. Those methods were based on the digital characteristics of animal appearance and body size, behavior and movement, call and sound, body temperature, excrement, respiration and heart rate, the characteristics collected by the sensor, which were analyzed and classified in real time through mathematical models, and the analysis was basically achieved. The current research results on automatic diagnosis and treatment of livestock and poultry diseases were abundant, but most of the related diagnosis methods were carried out in an ideal environment. However, the interference factors in the actual production and breeding environment were very large, and the most of the current diagnostic methods could not eliminate the interference well and accurately extract the required characteristic information. Besides, the current digital livestock disease diagnosis methods were mostly based on the analysis and diagnosis of one kind of livestock feature information, which affected the diagnosis accuracy of the diagnosis system and the diagnosis results were not convincing. At the same time, the most of the current digital diagnosis methods for poultry and livestock diseases still had some problems such as poor diagnosis generalization ability and poor anti-interference ability, which restricted their promotion and application. The focus of future research on automatic diagnosis of livestock and poultry diseases is to improve the accuracy of its sensing algorithms and the applicability and robustness of mathematical models, and to develop an intelligent diagnosis and treatment expert system for livestock and poultry diseases based on multiple feature coupling and data fusion, realize real-time, efficient, intelligent and accurate livestock and poultry health diagnosis. Table and Figures | Reference | Related Articles | Metrics

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The Capping Pheromone Contents and Putative Biosynthetic Pathways in Larvae of Honeybees Apis cernana QIN QiuHong,HE XuJiang,JIANG WuJun,WANG ZiLong,ZENG ZhiJiang Scientia Agricultura Sinica    2021, 54 (11): 2464-2475.   DOI: 10.3864/j.issn.0578-1752.2021.11.017 Abstract （319）   HTML （22）    PDF （1321KB）（423）       Knowledge map    Save 【Objective】 In honeybees, methyl palmitate (MP), methyl oleate (MO), methyl linoleate (ML) and methyl linolenate (MLN) are important capping pheromone components, which trigger the capping behavior of adult workers. The objective of this study is to compare the contents of these four pheromone components in the larvae of workers and drones of Apis cernana at different capping stages, analyze their biosynthetic pathways, and to further explore the mechanism of pheromone communication between larvae and adult workers. 【Method】Using A. c. cernana as the experimental material, the larvae of workers and drones of prior to be capped, in the process of being capped and had been capped were collected for comparing the contents of these four pheromone components by using GC/MS. Simultaneously, RNA-seq was used for gene expression analysis, and the biosynthetic pathways were speculated based on KEGG enrichment of differential expressed genes. 【Result】In worker larvae, the contents of the four capping pheromone components were significantly higher at the capping and capped stage than those of the prior to be capped larvae, and the contents of MP and MO significantly increased with aging of the larvae, while the contents of ML and MLN were not significantly different between the capping and capped stage. Whereas in drone larvae, the contents of the four pheromone components were higher overall and increased with aging, and the content at capped stage was significantly higher than that at prior to be capped and capping. RNA-seq results showed that there were 4 299 and 3 926 differential expressed genes among the larvae groups of three stages of workers and drones, respectively. In addition, 152 and 130 KEGG pathways were obtained from the KEGG annotation analysis of the differential expressed genes, respectively. Furthermore, the possible de novo biosynthetic pathways were proposed for MP, MO, ML and MLN from acetyl-CoA, regulating under 11 related candidate genes, and these biosynthesis pathways were found to be similar to those of Apis mellifera. 【Conclusion】The release contents of MP, MO, ML and MLN were increased during the critical stage of capping in worker and drone larvae of A. cernana, which further verified that these four pheromones were related to capping behavior of honeybees, and it was speculated that they were possibly de novo biosynthesized from acetyl-CoA under the control of related genes. A. cernana larvae and A. mellifera larvae may use the same biosynthesis pathway for pheromone biosynthesis. Table and Figures | Reference | Related Articles | Metrics

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Transient Expression and the Effect on Proliferation and Apoptosis of Granule Cell Stimulating Factor in Ovarian Fibroblasts LI RunTing,CHEN LongXin,ZHANG LiMeng,HE HaiYing,WANG Yong,YANG RuoChen,DUAN ChunHui,LIU YueQin,WANG YuQin,ZHANG YingJie Scientia Agricultura Sinica    2021, 54 (11): 2434-2444.   DOI: 10.3864/j.issn.0578-1752.2021.11.015 Abstract （346）   HTML （24）    PDF （1756KB）（354）       Knowledge map    Save 【Objective】 The purpose of this paper is to study the transient expression of granule cell stimulating factor (GCSF) in ovarian fibroblast cells, and the influence of GCSF on proliferation, cell cycle, and apoptosis, to provide theoretical basis for molecular genetic breeding of sheep pluripotent stem cells induced by GCSF in the future. 【Method】 The sheep GCSF eukaryotic expression plasmid pRTL1-GCSF and the control vector plasmid pRTL1 were transfected into 1×105 cells·mL-1 sheep fibroblasts respectively. After 48 h of culture, the total RNA was extracted by Trizol method and reverse transcribed into cDNA. The transient expression level of sheep GCSF in fibroblasts was detected by real-time quantitative PCR. GCSF dependent cell line NFS-60 was used for the biological activity of GCSF secreted and expressed in the supernatant of sheep fibroblasts 48 hours after transfection, which was determined by cell viability detection reagent alamarBlue. The HEK 293F suspension culture was used to express the secreted GCSF protein. The GCSF protein expressed in the cell culture medium was purified by Ni-NTA resin and detected by SDS-PAGE. After adding the 30 ng·mL-1 purified GCSF protein, the proliferation of sheep fibroblasts was detected by alamarBlue at 24 h and 48 h, and the cell cycle and apoptosis of sheep fibroblasts were detected by flow cytometry. 【Result】 The expression level of GCSF in sheep fibroblasts was significantly increased after transfection for 48 h. In sheep fibroblasts, the expression level of GCSF transfected with pRTL1-GCSF plasmid was 50 615.92 ± 4 738.83 of that of pRTL1 empty control group. The fluorescence intensity of NFS-60 in the experimental group and positive control group was significantly higher than that in the negative control group and blank control group (P<0.01), but there was no significant difference between the experimental group and the positive control group (P>0.05). The results showed that sheep GCSF could significantly stimulate the proliferation of NFS-60 cells, indicating that the GCSF expressed in sheep fibroblasts had biological activity. After eukaryotic expression of secretory GCSF protein in HEK 293F cell line, the sheep GCSF protein was purified. After 30 ng·mL-1 sheep GCSF was added to sheep fibroblasts, the cell viability of GCSF test group was not significantly different from that of culture medium dilution control group for 24 h and 48 h, but the distribution of cell cycle was significantly changed. At 24 h, compared with the control group, the proportion of G1 phase cells increased from (55.29±1.68)% to (69.37±0.24)%, the difference was very significant (P<0.01); the proportion of S phase cells changed from (15.99±0.38)% to (15.39±0.60)%, the difference was not significant (P>0.05); G2/M phase cells increased significantly (P<0.05), and the proportion increased from (22.88±1.00)% to (26.76±0.82)%. The results showed that 24 hours after the addition of sheep GCSF, the number of cells in division and interphase increased significantly. At 48 h, compared with the control group, the proportion of G1 phase cells decreased from (65.96±0.37)% to (45.69±0.26)%, the difference was very significant (P<0.01); the proportion of S phase cells increased from (13.45±1.33)% to (37.87±2.43)%, the difference was very significant (P<0.01); the proportion of G2/M phase cells changed from (16.42±1.29)% to (21.80±1.86)%, the difference was not significant (P>0.05). The results showed that the number of cells in interphase was significantly decreased and the number of cells in DNA replication state increased significantly at 48 h after adding GCSF. Compared with the control group, the apoptosis rates of the control group (Ctr) and the experimental group (GCSF) were (7.51±0.38)% and (9.16±0.46)% respectively at 24 h culture. At 48 h, the apoptosis rates of the control group and the experimental group were (5.73±0.29)% and (5.39±0.27)%, respectively. At 72 h, the apoptosis rates of control group (Ctr) and experimental group (GCSF) were (8.88±0.45)% and (5.41±0.27)%, respectively. There was a significant difference between 24 h and 72 h (P<0.01), but there was no significant difference at 48 h (P>0.05). The results showed that GCSF promoted the apoptosis within 24 hours, and the apoptosis was inhibited with the prolongation of time. 【Conclusion】 In conclusion, sheep fibroblasts can express GCSF instantaneously and have biological activity. GCSF did not affect the proliferation of sheep fibroblasts, but could regulate its cell cycle and affect cell apoptosis. The results laid a foundation for breeding sheep with high immunity and disease resistance by GCSF mediated by sheep fibroblasts. Table and Figures | Reference | Related Articles | Metrics

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Expression Analysis of IGF1-PI3K-Akt-Dependent Pathway Genes in Skeletal Muscle and Liver Tissue of Yellow Feather Broilers SHU JingTing,JI GaiGe,SHAN YanJu,ZHANG Ming,JU XiaoJun,LIU YiFan,TU YunJie,SHENG ZhongWei,TANG YanFei,JIANG HuaLian,ZOU JianMin Scientia Agricultura Sinica    2021, 54 (9): 2027-2038.   DOI: 10.3864/j.issn.0578-1752.2021.09.018 Abstract （763）   HTML （43）    PDF （477KB）（472）       Knowledge map    Save 【Objective】IGF1-PI3K-Akt signal pathway have been implicated in the regulation of growth and development of chicken. In the present study, the expression of IGF, IGF1R and IRS1genes of IGF1-PI3K-Akt signal pathway was first studied in skeletal muscle and liver tissues of Guangxi partridge chickens with slow growth rate and Huashan partridge chickens with moderate growth rate, which would provide a reference for clarifying the regulation mechanism of growth and development of yellow feather broilers. 【Method】SPSS20.0 software was used to compare the differences of growth traits between Guangxi partridge chickens and Huashan partridge chickens. Expression of IGF1, IGF1R and IRS1 was quantified by RT-PCR in the breast muscle (BM), leg muscle (LM) and liver on days 9, 12, and 16 of embryonic development, as well as at 0, 7, 21, 35, 49, and 63 days post-hatching (PH) in the two chicken breeds, and the correlations between the gene expression level and body weight as well as tissue weight were also analyzed by SPSS20.0 software. 【Result】The body weight, skeletal muscle and liver weight of chicken in the early growth development showed significant breed- and age-specificity. The growth of Huashan partridge chickens had far exceeded that of Guangxi partridge chickens from the late embryonic development. The expression patterns of IGF1, IGF1R and IRS1 showed significant differences in breed-, tissue- and age-specific fashion. Overall, the expression levels of the three genes in skeletal muscles of embryonic development were much higher than those in liver; in contrast, the expression levels in liver were higher than those in skeletal muscle at post-hatching development. As for breed, expression levels of the studied genes in skeletal muscle of Guangxi partridge chickens were higher than those in Huashan partridge chickens; in contrast, liver expression levels were higher in Huashan partridge chickens. IGF1 mRNA could be detected as early as on E9 d in the skeletal muscles of both chicken breeds, and the highest level was appeared at this stage in LM, significantly higher than those on the other studied development stages expect on E12 d, and the lowest level was appeared on 0 d at hatching; while in BM, the highest level was appeared on E12 d and lowest level was appeared on 63 d; however, the level of IGF1 mRNA differed between breeds in liver. For Guangxi partridge chickens, the highest level was observed on 21 d and there was no expression during embryonic development, whereas the highest level was found on 49 d and very low level during embryonic development in Huashan partridge chickens. IGF1R mRNA could be detected at the whole studied stages in skeletal muscles and liver of both chicken breeds, and the highest level was appeared on E9 d in both BM and LM, significantly higher than those on the other studied development stages in Guangxi partridge chickens, and significantly higher than those on the other studied development stages expect on E12 d in Huashan partridge chickens. In liver, the highest expression level of IGF1R was detected on 21 d in Guangxi partridge chickens, significantly higher than those on E9 d, E12 d, E16 d and 0 d; while the highest level was found on 63 d and significantly higher than those on the other studied development stages. IRS1 mRNA could also be detected at the whole studied stages in skeletal muscles and liver of both chicken breeds. In skeletal muscles, expression of IRS1 were higher on E9 d or E12 d than any other age, and then decreased with age and kept relative low level during PH development in both breeds. The liver IRS1 expression pattern was consistent with IGF1R in both breeds. Significant positive relationships were observed for the expression of studied genes in BM, LM and liver tissues of both chicken breeds. Meanwhile, the skeletal muscle expression of these three genes were all showed significant negative relationships with body weight and breast (leg) muscle weight, whereas the liver expression of these three genes were all showed significant positive relationships with body weight, breast (leg) muscle weight and liver weight. 【Conclusion】These results implied that the expression of selected genes that comprise the IGF1-PI3K-Akt pathway existed identical trends, and differential expression between breeds and tissues might be one of the main reasons for the different growth rate of the different types of yellow feather broiler. Table and Figures | Reference | Related Articles | Metrics

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Comparison Analysis on Eggshell Quality, Biochemical Index of Calcium Metabolism and Calcium Binding Protein CaBP-D28k mRNA Expression Between Langya Chicken and Its Synthetic Lines ZHANG NingBo,HAN ZhaoQing,JIN TaiHua,ZHUANG GuiYu,LI JiongKui,ZHENG QuanSheng,LI YongZhu Scientia Agricultura Sinica    2021, 54 (9): 2017-2026.   DOI: 10.3864/j.issn.0578-1752.2021.09.017 Abstract （350）   HTML （38）    PDF （552KB）（866）       Knowledge map    Save 【Objective】The aim of this research was to provide a theoretical basis for the conservation of Langya Chicken breed resources and the development of its synthetic lines based on the comparative study on eggshell quality and calcium metabolism of Langya Chicken and its two synthetic lines. 【Method】Each of one hundred and eighty 240-day-old birds of Langya Chicken and its two synthetic lines (Light linen and Dark linen synthetic lines), were randomly divided into 6 repeats, each with 30 replicates. When the chicken were raised to 300 days of age under the same breeding conditions, thirty eggs and six birds were randomly collected from each repeat, blood samples were taken from the veins under the wings, and tissue samples from the left leg tibia and duodenum, egg shell glands, and kidneys were collected after slaughter. Calcium- and phosphorus-related indicators and the expression levels of calcium binding protein CaBP-D28k mRNA were detected. 【Result】The results showed that the egg weight of the light linen synthetic line was significantly lower than that of the dark linen synthetic line (P<0.05), and the eggshell weight was lower than other lines (P>0.05). In addition, the eggshell thickness and eggshell ratio were significantly higher than those of the linen line (P<0.05), the strength of eggshell was significantly higher than that of other lines (P<0.05). The calcium content of egg yolk, eggshell, and tibia in light linen synthetic line was significantly higher than that in dark linen synthetic line (P<0.05). The content of phosphorus in the tibia was significantly higher than that of other lines (P<0.05), and the ash content in eggshells and tibia was the highest in the light linen synthetic line, followed by the Langya Chicken and the dark linen synthetic line (P<0.05). The body weight and tibia length of light linen synthetic line was significantly lower than those of dark linen synthetic line (P<0.05). The contents of plasma calcium and calcitonin in the light linen synthetic line were significantly lower than those in other lines, while the plasma phosphorus, alkaline phosphatase, and parathyroid hormone contents in the light linen synthetic line were significantly higher than those in other lines, and the differences between the lines were significant (P<0.05); the calcium binding protein content of the light linen synthetic line was significantly lower than that of the dark linen synthetic line (P<0.05). While the osteocalcin of the synthetic lines was significantly higher than that of the Langya Chicken (P<0.05). The expression level of calcium binding protein CaBP-D28k mRNA in duodenum of light linen synthetic line was significantly higher than that of other lines (P<0.05), and the expression level in eggshell gland was significantly higher than that of dark linen synthetic line (P<0.05). The expression level of calcium binding protein in kidney was significantly higher than that of Langya Chicken (P<0.05). 【Conclusion】The above results showed that the plasma phosphorus, alkaline phosphatase, parathyroid hormone, osteocalcin and other related active substances and calcium-binding protein CaBP-D28k mRNA expression level in duodenum, kidney, and shell glands of light linen synthetic lines was all higher than other lines during the 240-300 day-old laying period. which could promote the absorption of calcium and phosphorus in the upper small intestine and the release and transport of bone calcium, affect the deposition of minerals in egg yolk and eggshell, and improve the quality of eggshell and tibia. Table and Figures | Reference | Related Articles | Metrics

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Curcumin Alleviates H2O2-Induced Oxidative Stress in Bovine Mammary Epithelial Cells Via the Nrf2 Signaling Pathway JIANG ChunHui,SUN XuDong,TANG Yan,LUO ShengBin,XU Chuang,CHEN YuanYuan Scientia Agricultura Sinica    2021, 54 (8): 1787-1794.   DOI: 10.3864/j.issn.0578-1752.2021.08.017 Abstract （391）   HTML （42）    PDF （766KB）（1507）       Knowledge map    Save 【Objective】The aim of this study was to investigate whether curcumin alleviated oxidative stress in bovine mammary epithelial cells induced by H2O2 via the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. 【Method】Bovine mammary epithelial cells MAC-T cells were treated with H2O2 (500 μmol·L-1) for 24 h, followed by incubation of curcumin (0, 5, 15 or 30 μmol·L -1) for an additional 3 h; the MAC-T cells were transfected with Nrf2 siRNA for 48 h, followed by incubation of H2O2 (500 μmol·L-1) for 24 h and then treated with curcumin (30 μmol·L -1) for an additional 3 h. Real-time quantitative PCR (qPCR), Western blot (WB) were used to detect the protein abundance of Nrf2 and the mRNA and protein abundance of NAD(P)H quinone oxidoreductase 1 (NQO1) and Heme oxygenase 1 (HO-1), the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and the content of malondialdehyde (MDA). 【Result】 (1) Compared with the control group, H2O2 treatment significantly increased MDA content (P<0.01), while it decreased the activity of SOD, GSH-Px, and CAT (P<0.01). Compared with the H2O2 group, the content of MDA in MAC-T cells in the 15 μmol·L-1 or 30 μmol·L -1 curcumin with H2O2 treatment groups was significantly decreased (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly increased (P<0.05, P<0.01). (2) Compared with the control group, H2O2 treatment significantly decreased Nrf2 protein abundance (P<0.01) and decreased HO-1 and NQO1 their mRNA and protein abundance (P<0.01). However, compared with the control group, curcumin treatment significantly increased Nrf2 protein abundance (P<0.01) and increased HO-1 and NQO1 mRNA and protein abundance (P<0.01). Compared with the H2O2 group, H2O2+curcumin treatment significantly increased Nrf2 protein abundance (P<0.01) and increased HO-1 and NQO1 mRNA and protein abundance (P<0.01). (3) Compared with the control group, si-Nrf2 treatment group significantly decreased Nrf2 mRNA abundance (P<0.01). Compared with si-Control+H2O group, the content of MDA was significantly decreased in si-Control+H2O2+curcumin treatment group (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly increased (P<0.01). However, compared with si-Control+H2O2+curcum group, the content of MDA was significantly increased in si-Nrf2+H2O2+curcumin treatment group (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly decreased (P<0.01). 【Conclusion】These results suggested that curcumin could alleviate the oxidative stress of bovine mammary epithelial cells induced by H2O2 through increasing the expression of Nrf2 and inducing the transcription of downstream antioxidant molecules. This study provided a theoretical basis for the prevention and treatment of oxidative damage of mammary epithelial cells caused by metabolic disorderd in perinatal dairy cows. Table and Figures | Reference | Related Articles | Metrics

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Hc-hrg-2 of Haemonchus Contortus Rescues the Growth of Heme Deficient Yeast Strain ZHOU JingRu,WU Fei,CHEN XueQiu,HUANG Yan,SHI HengZhi,DU AiFang,YANG Yi Scientia Agricultura Sinica    2021, 54 (8): 1795-1804.   DOI: 10.3864/j.issn.0578-1752.2021.08.018 Abstract （313）   HTML （18）    PDF （2902KB）（488）       Knowledge map    Save 【Objective】In previous study, we have identified a heme responsive gene Hc-hrg-2 in Haemonchus contortus (H. contortus), with a high transcriptional level in the presence of high concentration of heme. However its function in heme regulation is still lacking research. To verify that Hc-hrg-2 was involved in the intracellular heme transport, a hem1 gene knockout strain of Saccharomyces cerevisiae, which was heme deficient, was constructed by homologous recombination technique and then exogenously expressed Hc-hrg-2 of H. contortus to rescue the growth of the knockout strain. 【Method】The genomic DNA of S. cerevisiae BY4741 was used as a template to obtain the upstream and downstream homology sequences of hem1 (SGD: S000002640) gene. The plasmid pYES2-CT was used to obtain the screening marker URA3 sequence. Two overlapping PCR techniques were used to sequentially connect upstream homology sequence, URA3, and downstream homology sequence to form the knockout components which was purified and then transformed into BY4741 competent cells by lithium acetate transformation method, and the transformants were selected on SD /-URA plates supplemented with 250 μmol·L -1 5-aminolevulinic acid (ALA). PCR identification using multiple primer pairs was further performed to verify the correctness of Δhem1 strain. The Hc-hrg-2 sequence (GenBank: MK371241) of Zhejiang strain and its functional domain deleted sequence Hc-hrg-2(Δgst-n) and Hc-hrg-2(Δgst-c) were amplified from the plasmids and inserted into the yeast expression vector pESC-LEU through a seamless cloning kit. The expression vectors, which were identified and sequenced to be correct, were then transformed into Δhem1 competent cells and selected on SD/-URA/-LEU (containing 250 μmol·L -1 ALA) plates. PCR identification was performed to verify the positive exogenous expression strain. By comparing the growth of Δhem1 strain and its exogenous expression strains in SD/-URA/-LEU liquid medium with or without 250 μmol·L -1 ALA, the phenotype of the knockout strain were further verified and the effects of expression vectors on phenotype were excluded. The exogenous expression strains were induced by 2% w/v galactose and then sonicated to identify the protein expression by Western Blot. The induced strains were resuspended to an OD600 of 0.2 and 4 μL of 5-fold serial dilutions of each induced strain was spotted onto 2% w/v galactose plates supplemented with either 250 μmol·L-1 ALA or different concentrations of heme for 2 to 3 days at 28 ℃ to compare the growth of the strains. 【Result】The hem1 gene knockout strain was successfully obtained. Compared with wild strain, Δhem1 cannot synthesis heme in vivo and requires ALA (250 μmol·L -1) or heme (≥10 μmol·L-1) for growth. The phenotypes of the exogenous expression strains were consistent with that of the knockout strain. Western Blot results indicated that 2% w/v galactose could successfully induce the expression of Hc-hrg-2 and its functional domain deleted gene in the knockout strain. Hc-hrg-2 expression allowed S. cerevisiae to import heme from the environment, and rescued the growth of Δhem1 strain. Notably, the deletion of two signature domains of Hc-hrg-2, a thioredoxinlike (GST-N) and a glutathione S-transferase C-terminal domain-like (GST-C), could reduce the effect of rescue. 【Conclusion】Hc-hrg-2 could facilitate heme uptake in cells and its functional domains, GST-N and GST-C, played an important role in this process. This study laid a solid foundation for further exploring heme transport mechanism of H. contortus. Table and Figures | Reference | Related Articles | Metrics

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MicroRNA-Mediated Cross-Kingdom Regulation of Apis mellifera ligustica Worker to Nosema ceranae DU Yu,FAN XiaoXue,JIANG HaiBin,WANG Jie,FENG RuiRong,ZHANG WenDe,YU KeJun,LONG Qi,CAI ZongBing,XIONG CuiLing,ZHENG YanZhen,CHEN DaFu,FU ZhongMin,XU GuoJun,GUO Rui Scientia Agricultura Sinica    2021, 54 (8): 1805-1820.   DOI: 10.3864/j.issn.0578-1752.2021.08.019 Abstract （342）   HTML （35）    PDF （12391KB）（365）       Knowledge map    Save 【Objective】Nosema ceranae infects Apis mellifera ligustica and causes microsporidiosis. In this study, to reveal the mechanism of miRNA-mediated cross-kingdom regulation of A. m. ligustica worker to N. ceranae, prediction, GO and KEGG database annotation as well as regulatory network analysis of N. ceranae mRNAs and differentially expressed mRNAs (DEmRNAs) targeted by differentially expressed miRNAs (DEmiRNAs) of A. m. ligustica workers’ midguts were conducted by bioinformatic approaches based on previously gained miRNA and mRNA omics data. 【Method】Significant host DEmiRNAs were screened out by comparison of miRNA omics data from A. m. ligustica workers’ midguts at 7 d and 10 d post N. ceranae infection (AmT1, AmT2) and corresponding uninfected midguts (AmCK1, AmCK2). DEmRNAs of pathogen were screened out through comparison of mRNA omics data from N. ceranae infecting A. m. ligustica worker’s midgut (NcT1 and NcT2) and pure fungal spores (NcCK). mRNAs and DEmRNAs of N. ceranae targeted by significant host DEmiRNAs were predicted using TargetFinder software. GO and KEGG database annotations of aforementioned targets were conducted using related bioinformatics tools. On basis of our previous findings, pathogen DEmRNAs associated with spore wall protein, polar tube protein, ricin B lectin, ATP/ADP translocase, ABC transporters and glycolysis/gluconeogenesis, and their target significant DEmiRNAs of host were filtered out, followed by construction and investigation of regulatory network. 【Result】In AmCK1 vs AmT1 comparison group, 48 significantly up-regulated miRNAs and 36 significantly down-regulated miRNAs could respectively target 1 345 and 1 046 mRNAs of N. ceranae; additionally, 47 significantly up-regulated miRNAs and 34 significantly down-regulated miRNAs of host could target 584 significantly down-regulated mRNAs and 265 significantly up-regulated mRNAs in NcCK vs NcT1; these targets were involved in 19 and 22 functional terms as well as 66 and 64 pathways. In AmCK2 vs AmT2 comparison group, 56 significantly up-regulated miRNAs and 51 significantly down-regulated miRNAs could respectively target 1 260 and 1 317 mRNAs of N. ceranae, additionally, 52 significantly up-regulated miRNAs and 49 significantly down-regulated miRNAs could target 587 significantly down-regulated mRNAs and 336 significantly up-regulated mRNAs in NcCK vs NcT2, which were engaged in 20 and 23 functional terms as well as 64 and 65 pathways. Further, eight common significantly up-regulated miRNAs and one common significantly down-regulated miRNA in AmCK1 vs AmT1 and AmCK2 vs AmT2 comparison groups could respectively target 144 common significantly down-regulated mRNAs and 10 common significantly up-regulated mRNAs in NcCK vs NcT1 and NcCK vs NcT2 comparison groups, which could be annotated to 18 and 13 functional terms as well as 38 and seven pathways. Moreover, host significantly up-regulated miRNAs in AmCK1 vs AmT1 and AmCK2 vs AmT2 could target pathogen significantly down-regulated mRNAs in NcCK vs NcT1 and NcCK vs NcT2, associated with RNAi, virulence factors such as polar tube protein, spore wall protein and ricin B lectin, glycolysis/gluconeogenesis and MAPK signal pathway. 【Conclusion】Complex target binding relationship and potential cross-kingdom regulatory relationship exist between host DEmiRNAs and pathogen DEmRNAs during the infection of A. m. ligustica worker with N. ceranae; host DEmiRNAs are likely to inhibit or degrade pathogen DEmRNAs associated with RNAi, virulence factor/infection factor, glycolysis/gluconeogenesis pathway, ATP/ADP translocase, ABC transporters, and MAPK signal pathway to affect N. ceranae infection and proliferation. Table and Figures | Reference | Related Articles | Metrics

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The Temperature-Humidity Index Estimated by the Changes of Surface Temperature of Broilers at Different Ages YuYan YANG,YaoWen LI,Shuang XING,MinHong ZHANG,JingHai FENG Scientia Agricultura Sinica    2021, 54 (6): 1270-1279.   DOI: 10.3864/j.issn.0578-1752.2021.06.016 Abstract （455）   HTML （52）    PDF （556KB）（401）       Knowledge map    Save 【Objective】The present study was conducted to estimate the temperature-humidity index (THI) based on the variations of surface temperature (ST) of broilers raised at different relative humidity (RH) levels and increasing ambient temperature (AT). 【Method】At day of 28, 35, 42 and 49, thirty AA broilers were raised in two controlled climate chambers. The RH of two chambers was set at 50% and 80%, respectively, and the AT in two chambers was set at a same procedure with increasing gradually by one degree per 0.5 h from 18 ℃ to 33 ℃. The ST of broilers, as well as the AT in two chambers was recorded at 2 min intervals using mini temperature data loggers. The wet-bulb temperature of two chambers was recorded at 10 min intervals. The THI model as as follow: THI = a*Tdry-bulb + (1-a)* Twet-bulb, the ‘a’ was weighting coefficient of Tdry-bulb, which was calculated when the coefficient of correlation between THI and ST reach to the maximum. 【Result】When the AT exceeded 24 ℃, the ST of broilers increased linearly with the AT and was affected by the RH. The present study estimated the THI for broilers at different ages by using the data when AT exceeded 24 ℃. The THI models for broilers at different age were as follow: THId28=0.82Tdb+0.18Twb; THId35=0.69Tdb+0.31Twb; THId42=0.67Tdb+0.33Twb; THId49=0.61 Tdb+0.39Twb. The linear correlation coefficients between THI and ST reached more than 0.96. In two independent experiments, it was verified again that there was a linear relationship between THI and ST, and the predicted ST by THI model was basically consistent with the actual measured results. 【Conclusion】The present results indicated that THI model had a good linear relationship with ST and was suitable for the evaluation of warm environment when the AT exceeded 24℃. The THI models for broilers at different ages were different, and the weighting coefficient of wet bulb temperature in THI models were increasing with the increase of broiler age. Table and Figures | Reference | Related Articles | Metrics

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Based on PK15 Cell Line for PCV2 Fully Suspension Culture Process JiaQi WANG,YuHong DONG,JuLing JIANG,JianNing QIAN,WenTao WEI,GuoLiang SONG,JinBo JIAO,XinXin GUAN,GuoBiao JI,YeXin ZHANG Scientia Agricultura Sinica    2021, 54 (6): 1280-1287.   DOI: 10.3864/j.issn.0578-1752.2021.06.017 Abstract （672）   HTML （40）    PDF （1051KB）（648）       Knowledge map    Save 【Objective】Selected one suspension PK15 cell line which is suitable for PCV2 virus , and then developed the production process for PCV2 vaccines(source of virus, MOI and harvest time ), to provide basic theory and guarantee for large-scale production use suspension culture instead of adherent culture. 【Method】The PK15 primary cells were diluted using the limiting dilution method and seeded in 96-well plates. The cell growth and morphology were observed every 2 days. After 90% of the cells were overgrown, the cells were gradually expanded from 96-well plates to 24-well plates,to 12-well plates, to 6-well plate, and finally to the square flask, three PK15 clones with good morphology that can grow adherently were selected. Three PK15 clones with good morphology that can grow adherently were selected. Three clones (PK15-1C8, 2F11, 1E5) were directly seeding in PK15 serum-free medium at a density of 1×106cells/mL and placed in a shaker incubator at 37 ℃, 5% carbon dioxide, and 120 r/min to continue culture. Monitor the cell density and viability every day, passage every 3 days, make the cells gradually adapt to the suspension environment, the PK15 clone can culture as a fully suspension cell line and can growth well in serum free media. After suspension cells stability passage and cell bank, compared the PCV2 virus content with three suspension clone cells, one clone cell was selected for PCV2 production. For different kinds of viruses (source from adherent cells or suspension cells), explore the infection MOI (0.1, 0.2, 0.5) and harvest time (48, 72, 96, 120h), determine the best production process of PCV2. 【Results】(1)The results showed that when the adherent cells passage to second generation in serum media CD PK15 259, the cells can suspended growth, continuous passage for eleven generation, suspension cells can growth stable, when seeding with 1×106 /mL cells, cells can reach 10×106 /mL cells when growth at 72h, viability rate is above 95%, and the doubling time is about 20h; (2) The three suspension cells use the same condition to infection PCV2 virus, the virus content of PK15-1C8 cloned cells can reach 106.4TCID50/mL, clone PK15-2F11 (105.5TCID50/mL), PK15-1E5 (105.6TCID50/mL), the virus content of the three cloned cells was higher than that of the original clone (104.7TCID50/mL), however, the virus content of PK15-1C8 cloned cells is higher and more stable, so it is determined as a cell for follow-up research; (3) After infecting PK15-1C8 cloned cells with seed virus (106.4TCID50 /mL) from adherent cells, the optimal process is 1×106/mL cell density with 0.1MOI and harvest at 72h the virus content can reach 106.5TCID50 /mL. After infecting PK15-1C8 cloned cells with seed virus (106.3TCID50 /mL) from suspension cells, the optimal process is 1×106/mL cell density with 0.2MOI and harvest at 72h the virus content can reach 107.3TCID50 /mL. 【Conclusion】Through the monoclonal screening of PK15 cells, adapted to suspension cells compared the PCV2 virus content of 3 suspension cells, a suspension cell with the highest virus content was determined, and based on this suspension cell, explored the process of PCV2 virus, and then established the PCV2 fully suspension, serum-free culture process. The SV15-1C8 cell proliferation PCV2 process of full suspension serum-free culture was established. This process used suspension cells to amplify PCV2 virus for seed poisoning for the first time. The highest virus content can reach 107.3TCID50/mL, which can be used for factory vaccine production. According selection sensitive clones and optimized the process for PCV2, can increase the virus titer, and realizes full suspension culture without serum, improved the production process of PCV2, improve the production efficiency, reduce the cost and improve the quality of the production. This process first use suspension cells to amplify the PCV2 virus. The highest virus content can reach 107.3TCID50/mL, which can be used for large-scale PCV2 virus production. Table and Figures | Reference | Related Articles | Metrics

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Improvement of Nosema ceranae Genome Annotation Based on Nanopore Full-Length Transcriptome Data HuaZhi CHEN,YuanChan FAN,HaiBin JIANG,Jie WANG,XiaoXue FAN,ZhiWei ZHU,Qi LONG,ZongBing CAI,YanZhen ZHENG,ZhongMin FU,GuoJun XU,DaFu CHEN,Rui GUO Scientia Agricultura Sinica    2021, 54 (6): 1288-1300.   DOI: 10.3864/j.issn.0578-1752.2021.06.018 Abstract （363）   HTML （32）    PDF （7681KB）（590）       Knowledge map    Save 【Objective】The objective of this study is to improve gene sequence and functional annotation of current reference genome of Nosema ceranae using previously obtained Nanopore full-length transcriptome dataset. 【Method】TransDecoder software was used to predict open reading frames (ORFs) of N. ceranae and corresponding amid acids. Comparison between full-length transcripts and transcripts annotated in reference genome was performed using gffcompare software to extend upstream sequences or downstream sequences of annotated genes’ untranslated regions and correct genes’ boundaries. MISA software was used to explore simple sequence repeat (SSR) loci within transcripts with a length above 500 bp, including single nucleotide repeat, dinucleotide repeat, trinucleotide repeat, tetranucleotide repeat, pentanucleotide repeat, hexanucleotide repeat and mixed SSR. By using Blast tool, novel genes and novel transcripts were aligned to Nr, KOG, eggNOG, GO and KEGG databases to gain functional annotations. 【Result】A total of 2 353 complete ORFs were predicted, and those ORFs with a length distribution among 0-100 aa were the predominant, reaching a ratio of 72.12% among total ORFs. Additionally, structures of 2 340 N. ceranae genes were optimized; 5′ ends of 1 182 genes and 3′ ends of 1 158 genes were respectively prolonged. Moreover, 1 658 SSRs were identified, and the numbers of single nucleotide repeat, dinucleotide repeat, trinucleotide repeat, tetranucleotide repeat were 1 622, 23, seven and six, respectively. The density of single nucleotide repeat was the highest (182.32/Mb), followed by those of mixed SSR, dinucleotide repeat and trinucleotide repeat, reaching 6.90, 2.78 and 0.73/Mb, respectively. Further, 954 novel genes were identified, among them 951, 333, 371, 422 and 321 were respectively annotated to Nr, KOG, eggNOG, GO and KEGG databases. In addition, 6 164 novel transcripts were identified, among them 6 141, 2 808, 2 932, 3 196 and 2 585 were annotated to the aforementioned five databases, respectively. The species annotated by the highest number of new gene and new transcript was N. ceranae followed by Nosema apis. 【Conclusion】Our results well improve sequences and functional annotations of annotated genes in current reference genome of N. ceranae, and supplement and annotate a number of unannotated novel genes and transcripts. Lots of SSR sites were provided for research on molecular markers, information of genes and transcripts on reference genome were supplemented. Table and Figures | Reference | Related Articles | Metrics

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Molecular Epidemiological Investigation of Porcine Group A Rotavirus in Sichuan from 2017 to 2019 Qun ZHOU,XiaoFei CHEN,RuiCi KAN,Yu LI,Hui CAO,YanLing PENG,Bin ZHANG Scientia Agricultura Sinica    2021, 54 (5): 1063-1072.   DOI: 10.3864/j.issn.0578-1752.2021.05.017 Abstract （422）   HTML （20）    PDF （3472KB）（741）       Knowledge map    Save 【Objective】The aim of this study was to investigate the prevalence and molecular characterization of porcine group A rotavirus (RVA) in large-scale pig farms in Sichuan, to provid a theoretical basis for the development for porcine RVA vaccine. 【Method】 From 2017 to 2019, the 303 samples were collected from 40 pig farms in 14 regions of Sichuan province. Prevalence of RVA was detected by real-time RT-PCR method, and RVA positive samples were typed by RT-PCR. At the same time, the whole genomes of RVA VP4 and VP7 gene were amplified from some positive samples. The genotypes of the corresponding strains were determined by RotaC2.0 classification tool. Sequence homology was analyzed by MegAlign. Phylogenetic tree was constructed by Neighbor-Joining method through MEGA 7.0. SimPlot and RDP4 softwares were applied for recombination analysis. 【Result】 Of the 303 samples examined, 32.34% (98/303, 95%CI=27.1%-37.9%) were positive for RVA. Among the 39 G types, G9 (41%) was the dominant genotype, while G4, G5, G26, and G3 were detected in 23%, 28.2%, 5.1% and 2.7%, respectively. The P[13] genotype (40.7%) was dominant among the 59 P types, followed by P[6], P[23] and P[1] in 30.5%, 23.7% and 5.1%, respectively. Furthermore, the 30 strains successfully identified the G/P combination genotype, and the dominant combination genotype was G9P[23] (23.3%), the other combinations were G4P[6] (16.7%), G9P[13] (13.3%), G5P[23] (10%), G5P[13] (10%), G9P[6] (6.7%), G26P[13] (6.7%), G4P[13] (6.7%), G4P[23] (3.3%) and G3P[13] (3.3%). Notably, the G5P[23], G4P[13], G9P[6], G26P[13] and G4P[23] were first identified in China. In addition, the recombination analysis showed that four strains had recombination on VP7 or VP4 genes. 【Conclusion】 The results demonstrated that the prevalence of RVA in diarrhea piglet feces was high and the genotypes were complex in Sichuan. The dominant genotype of RVAs was G9P[23]. The results of this study enriched the epidemiological data of RVA and provided an important reference for the prevention and control of porcine RVA in Sichuan province. Table and Figures | Reference | Related Articles | Metrics

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