UHPLC-Q/TOF MS结合HPLC-DAD定性和定量分析硫酸黏菌素可溶性粉中的未知添加物

定性和定量分析一批兽药硫酸黏菌素可溶性粉中的未知添加物。照《中国兽药典》2010年版一部对该批检品用微生物检定法进行含量测定时,发现该样品的抑菌圈为虚圈,用薄层色谱鉴别该样品,未显示与标准品溶液一致的主斑点,怀疑该样品中有处方外非法添加物。采用超高效液相色谱-四级杆-飞行时间质谱(UHPLC-Q/TOF MS)对该样品进行筛查,发现疑似添加物,并使用液相色谱-二极管阵列检测(HPLC-DAD)法进行了双重确证和含量测定。该样品中非法添加物确证为磺胺氯达嗪和甲氧苄啶,添加量分别为58.5 mg/g和13.4 mg/g。本研究通过建立筛查方法为监管部门提供技术支撑,通过分析非法添加物的可能原因为打击兽药处方外非法添加提供了思路。     

一株鸭源禽流感病毒（H9N2）全基因序列分析及其排毒规律的研究

为研究一株鸭源H9N2亚型禽流感病毒(JN1株)全基因组的分子特征和遗传进化情况以及感染雏鸭后的排毒规律,对其生物学特性、全基因组序列以及遗传进化进行了分析,通过点眼滴鼻方式感染3周龄健康雏鸭,利用建立的实时荧光定量PCR(RT-PCR)检测鸭的排毒规律,并测定血清抗体效价。JNI株的鸡胚半数感染量为10~(-7.54)/0.2mL,鸡胚最小致死量的平均死亡时间为72h,静脉致病指数为0.266,抗原相关系数为0.47。系统发育分析表明,JN1株的HA与CK/HK/G9/97和DK/HK/Y280/97在同一分支上,NA、M、NP、PA、PB1、PB2基因与SH/F/98同源性较高,NS基因则与H5N1亚型禽流感病毒进化关系较近。HA的裂解位点为PSRSSR↓GL,存在6个潜在糖基化位点,在234位的氨基酸残基为L,NA基因颈部存在缺失。雏鸭在感染该病毒后,饮食正常,精神良好,3~17d均有排毒,咽拭子和泄殖腔棉拭子排毒规律基本一致,分别在第3天和第13天出现排毒高峰。该H9N2亚型禽流感病毒属欧亚大陆分支,为低致病性禽流感,病毒可通过呼吸道和泄殖腔向外界排毒,排毒时间较长。     

Novel reassortant H5N6 highly pathogenic influenza A viruses in Vietnamese quail outbreaks

Avian influenza A H5N6 virus is a highly contagious infectious agent that affects domestic poultry and humans in South Asian countries. Vietnam may be an evolutionary hotspot for influenza viruses and therefore could serve as a source of pandemic strains. In 2015, two novel reassortant H5N6 influenza viruses designated as A/quail/Vietnam/CVVI01/2015 and A/quail/Vietnam/CVVI03/2015 were isolated from dead quails during avian influenza outbreaks in central Vietnam, and the whole genome sequences were analyzed. The genetic analysis indicated that hemagglutinin, neuraminidase, and polymerase basic protein 2 genes of the two H5N6 viruses are most closely related to an H5N2 virus (A/chicken/Zhejiang/727079/2014) and H10N6 virus (A/chicken/Jiangxi/12782/2014) from China and an H6N6 virus (A/duck/Yamagata/061004/2014) from Japan. The HA gene of the isolates belongs to clade 2.3.4.4, which caused human fatalities in China during 2014–2016. The five other internal genes showed high identity to an H5N2 virus (A/chicken/Heilongjiang/S7/2014) from China. A whole-genome phylogenetic analysis revealed that these two outbreak strains are novel H6N6-like PB2 gene reassortants that are most closely related to influenza virus strain A/environment/Guangdong/ZS558/2015, which was detected in a live poultry market in China. This report describes the first detection of novel H5N6 reassortants in poultry during an outbreak as well as genetic characterization of these strains to better understand the antigenic evolution of influenza viruses.     

Effects of concentrate‐to‐forage ratios and 2‐methylbutyrate supplementation on ruminal fermentation,bacteria abundance and urinary excretion of purine derivatives in Chinese Simmental steers

This study evaluated the effects of dietary concentrate levels and 2‐methylbutyrate (2MB ) supplementation on performance, ruminal fermentation, bacteria abundance, microbial enzyme activity and urinary excretion of purine derivatives (PD ) in steers. Eight ruminally cannulated Simmental steers (12 months of age; 389 ± 3.7 kg of body weight) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement. Moderate‐concentrate (400 g/kg diet [MC ]) or high‐concentrate (600 g/kg diet [HC ]) diets were fed with or without 2MB (0 g/day [2MB ?] or 15.0 g/day [2MB +]). Dry matter intake and average daily gain increased, but feed conversion ratio decreased with the HC diet or 2MB supplementation. Ruminal pH decreased, but total volatile fatty acid increased with the HC diet or 2MB supplementation. Molar proportion of acetate and acetate‐to‐propionate ratio decreased with the HC diet, but increased with 2MB supplementation. Propionate molar proportion and ruminal NH ₃‐N content increased with the HC diet, but decreased with 2MB supplementation. Neutral detergent fibre degradability decreased with the HC diet, but increased with 2MB supplementation. Crude protein degradability increased with the HC diet or 2MB supplementation. Abundance of Ruminococcus albus , Ruminococcus flavefaciens , Fibrobacter succinogenes and Bufyrivibrio fibrisolvens as well as activities of carboxymethyl cellulase, cellobiase, xylanase and pectinase decreased with the HC diet, but increased with 2MB supplementation. However, abundance of Prevotella ruminicola and Ruminobacter amylophilus as well as activities of α‐amylase and protease increased with the HC diet or 2MB supplementation. Total PD excretion also increased with the HC diet or 2MB supplementation. The results suggested that growth performance, ruminal fermentation, CP degradability and total PD excretion increased with increasing dietary concentrate level from 40% to 60% or 2MB supplementation. The observed diet × 2MB interaction indicated that supplementation of 2MB was more efficacious for improving growth performance, ruminal fermentation and total PD excretion with promoted ruminal bacteria abundance and enzyme activity in the MC diet than in the HC diet.     

Based serum metabolomics analysis reveals simultaneous interconnecting changes during chicken embryonic development

Metabolic disorder is a major health problem and is associated with a number of metabolic diseases. Due to native hyperglycaemia and resistance to exogenous insulin, chickens as a model had used in the studies of adipose tissue biology, metabolism and obesity. But no detailed information is available about the comprehensive changes of serum metabolites at different stages of chicken embryonic development. This study employed LC/MS‐QTOF to determine the changes of major functional metabolites at incubation day 14 (E14d), 19 (E19d) and hatching day 1 (H1d), and the associated pathways of differential metabolites during chicken embryonic development were analysed using Metabolite Set Enrichment Analysis method. Results showed that 39 metabolites were significantly changed from E14d to E19d and 68 metabolites were significantly altered from E19d to H1d in chicken embryos. Protein synthesis was promoted by increasing the concentrations of L‐glutamine and threonine, and gonadal development was promoted through increasing oestrone content from E14d to E19d in chicken embryos, which indicated that serum glutamine, threonine and oestrone contents may be considered as the candidate indicators for assessment of early embryonic development. 2‐oxoglutaric acid mainly contributed to enhancing the citric cycle, and it plays an important role in improving the growth of chicken embryos at the late development; the decreasing of L‐glutamine, L‐isoleucine and L‐leucine contents from E19d to H1d in chicken embryonic development implied their possible functions as the feed additive during early posthatch period of broiler chickens to satisfy the growth. These results provided insights into understand the roles of serum metabolites at different developmental stages of chicken embryos, it also provides available information for chicken as a model to study metabolic disease or human obesity.     

Identification of Suspected Stem/progenitor Cells in Bovine Mammary Epithelial Cells Culture System

To develop the potential function of dairy cow mammary stem cells (DCMECs) in regulation of lactation,we identify putative DCMECs which were BrdU label retaining epithelial cells,at the same time,analysis the location of two new mammary stem cells molecular marks FNDC3B and PROCR to verify the feasibility of them to indicate DCMECs.The mRNA levels of prolactin,growth hormone,insulin-like growth factor-1 and their receptors were detected along with cell passage by Real-time quantitative PCR.The results showed that the proportion of BrdU label-retaining epithelial cells was nearly 0.4% after 25 d continuous culture (passaged 4 times) and few cells were positive for FNDC3B or PROCR.Moreover,we observed the BrdU labelled epithelial cells by asymmetric division.The mRNA levels of prolactin,growth hormone,insulin-like growth factor-Ⅰ and their receptors in primary and passage cells were extremely significant difference(P<0.01).DCMECs would rapidly lose some physiological characteristics and the ability of milk synthesis when not under the condition of induction of lactation differentiation,but a certain percentage of mammary stem/progenitor cells will be retained,whose potential effects on the regulation of lactation and mammary acinar remodeling were worthy of attention.     

不同温度下圆叶决明降解过程红壤可溶性氮及氮水解酶动态变化研究

为了探明圆叶决明（Chamaecrista rotundifolia）降解过程果园红壤供氮水平的变化规律，本研究采用模拟培养试验，研究15℃和25℃培养果园红壤硝态氮、铵态氮、可溶性总氮、可溶性有机氮含量的变化及脲酶、蛋白酶、天冬酰胺酶活性的变化。结果表明，圆叶决明降解过程果园红壤4种可溶性氮含量均显著提高，且在培养140 d达到最大值，25℃培养的效果更佳。其中，可溶性总氮、可溶性有机氮和硝态氮含量随时间的动态变化可用三次曲线方程来拟合。圆叶决明降解过程还能显著提高脲酶、蛋白酶和天冬酰胺酶的活性，其中蛋白酶活性和脲酶活性的变化可用指数方程和三次曲线方程来拟合。本研究认为圆叶决明降解过程可提高果园红壤可溶性氮含量，因此可通过翻压圆叶决明提高土壤的供氮水平。     

南果梨周年干物质与氮磷钾积累动态

【目的】明确南果梨干物质积累特征和氮磷钾养分周年动态积累规律,为南果梨优化施肥量和施肥时期提供依据。【方法】以12年生南果梨树为试材,采用田间采样和树体分解方法,分别于萌芽后10 d(萌芽期)、 30 d(花期)、 65 d(幼果膨大期)、 100 d(果实膨大或新稍停止生长期)、 130 d(果实着色前)、 155 d(果实采收期)、 185 d(采收后)、 210 d(落叶前)8个生育期,选干周和树高一致的3株树,将树体连根从土壤中挖出,分出果实、 叶片、 枝条、 主干、 主根、 侧根、 须根,各器官单独称重,并取200 g左右的鲜样按清水、 洗涤剂、 清水、 1%盐酸、 3次去离子水冲洗、 杀青、 烘干后,电磨粉碎过0.15 mm筛,测定样品中氮、 磷、 钾含量。【结果】1)南果梨周年生育期内,树体干物质当年净积累量为18.4 kg/plant,干物质累积速率出现两次累积高峰,分别是幼果膨大期(0.15 kg/d)和采收期(0.11 kg/d)。2)南果梨树体总氮周年积累量为154.0~301.0 g,新生器官为0~116.2 g,果实膨大期达到最高;多年生器官氮积累量为154.0~194.8 g,落叶前达到最高。3)南果梨树体总磷周年积累量为17.1~37.2 g,果实着色前最高。其中新生器官为13.7 g,果实采收期最高;多年生器官为17.1~24.9 g,果实转色期最高。4)南果梨树体总钾周年积累量为27.9~174 g。新生器官钾为97.3 g,采收期最高;多年生器官钾为27.6~76.6 g,落叶前最高。5)产量大约为17 t/hm2的12年生南果梨从萌芽到落叶前树体当年氮磷钾的单株净累积量分别为146.2、 20.1、 146.1 g,折合1000 kg果实经济产量需吸收氮(N)、 磷(P)、 钾(K)5.4、 0.7、 5.4 kg。【结论】南果梨周年干物质单株积累总量为41.4 kg,当年净积累量为19.7 kg。南果梨干物质积累主要集中在花期到果实膨大期和果实转色到落叶前,分别占47.3% 和47.5%。南果梨从萌芽到落叶前氮、 磷、 钾的单株净累积量分别为146.2、 20.1、 146.1 g,每1000 kg果实经济产量需吸收氮(N)、 磷(P)、 钾(K)5.4、 0.7、 5.4 kg。从开花到果实膨大期和从果实着色到采收后30天对氮吸收分别占总氮累积量的39.0%和49.0%,而磷、 钾的累积从萌芽到开花较快,到果实膨大期磷的累积达67.4%,钾的累积达65.1%,果实膨大期是干物质和氮磷钾积累的关键时期。     

Effect of Different Concentrate-roughage Ratios on Rumen Degradability of Cassava Stems and Leaves Total Mixed Ration in Goat

The rumen degradations of TMR were studied in this experiment,in which cassava stems and leaves were used as roughage and formulated with concentrate in different proportions.The purpose of this study was to formulate the available TMR for finishing goat,and provide a theoretical basis for the applications of cassava stems and leaves in goat production.Six Hainan Black goats with permanent rumen fistulas were selected,the rumen degradabilities of DM,CP,NDF and ADF of TMR with 6 different concentrate to forage ratio（2:8,3:7,4:6,5:5,6:4 and 7:3）were tested using nylon bag method.The results showed that,the DM effective degradabilities at the ratio of 4:6,5:5,6:4,7:3 were significantly higher than that of 2:8 and 3:7（P<0.05).CP effective degradabilities at the ratio of 2:8,3:7,4:6 were significantly higher than that of 5:5,6:4 and 7:3（P<0.05).The NDF effective degradation at the ratio of 5:5 was significantly higher than that in the other groups（P<0.05).The ADF degradation at the ratio of 3:7 was significantly higher than that of 4:6,5:5, 6:4 and 7:3（P<0.05).The results indicated that,the nutrient degradation in rumen were higher when the concentrate to forage（cassava stems and leaves）ratio in TMR were 3:7,4:6 and 5:5,and the recommended ratio range were 4:6 to 5:5 in production practice.