济宁青山羊多羔性候选基因BMP15的研究

根据绵羊骨形态发生蛋白15（bone morphogenetic protein 15,BMP15）基因序列设计2对引物（P1和P2）,分别扩增随机选取的安哥拉山羊和济宁青山羊各5个个体的BMP15基因外显子1、2并克隆测序。测序结果与绵羊BMP15基因外显子1（AF236078）相比,山羊BMP15基因外显子1在第268-270位插入3个核苷酸CTT（268insCTT）,导致编码的氨基酸第12位插入亮氨酸（12insL）;山羊BMP15基因外显子2序列长度与绵羊（AF236079）一致,但存在7处核苷酸不同,引起4个氨基酸变化,济宁青山羊与安哥拉山羊相比在963位存在核苷酸变化（A963G）。依据A963G变异设计引物P3,利用PCR-SSCP方法检测BMP15基因外显子2在高繁殖力品种（济宁青山羊）、中等繁殖力品种（波尔山羊）和低繁殖力品种（安哥拉山羊和内蒙古绒山羊）中的单核苷酸多态性,分析该基因对济宁青山羊多羔性的影响。结果在济宁青山羊中检测到AA、AG和GG基因型,在波尔山羊中检测到AG和GG基因型,在安哥拉山羊和内蒙古绒山羊中仅检测到AA基因型;测序分析发现GG与AA基因型相比存在1个突变（A963G）,导致第300位的丝氨酸变为甘氨酸（S300G）;济宁青山羊AA、AG和GG基因型频率分别为0.008、0.059和0.933;高繁殖力山羊品种与中、低繁殖力山羊品种之间BMP15基因型分布存在显著（P〈0.05）和极显著（P〈0.001）差异;GG基因型济宁青山羊产羔数最小二乘均值比AG和AA基因型的分别多0.71只（P〈0.05）和1.57只（P〈0.05）,AG基因型比AA基因型的多0.86只（P〈0.05）。本研究结果初步显示BMP15基因可能是影响济宁青山羊多羔性的一个主效基因或是与之紧密连锁的一个标记。     

绵羊和山羊BMP15基因FecXR突变的检测

为了检测骨形态发生蛋白15(BMP15)基因在绵羊和山羊中的突变,本研究采用PCR-SSCP方法在高繁殖力绵羊品种(小尾寒羊和湖羊)、山羊品种(济宁青山羊和波尔山羊)和低繁殖力绵羊品种(多赛特和特克塞尔)、山羊品种(内蒙古绒山羊和安哥拉山羊)中检测BMP15基因FecXR突变,并对小尾寒羊和济宁青山羊扩增片段进行核苷酸和氨基酸序列分析.结果表明,这8个羊品种均没有发生与Rasa Aragonesa绵羊相同的FecXR突变;小尾寒羊核苷酸序列与GenBank中绵羊BMP15序列(AF236079,NM_001114767)完全一致,济宁青山羊与小尾寒羊相比存在3处碱基不同(T529G、C530G和T576C)和2个氨基酸差异(V155G和S171P).可见BMP15基因影响Ra-sa Aragonesa绵羊高繁殖力的突变FecXR对小尾寒羊、湖羊、济宁青山羊和波尔山羊的繁殖力均没有显著影响.     

山羊骨形态发生蛋白2基因外显子克隆及序列分析

设计4对引物,采用PCR-SSCP技术检测骨形态发生蛋白2（bone morphogenetic protein 2,BMP2）基因外显子2、3在济宁青山羊、安哥拉、波尔、内蒙古绒山羊4个品种共191个个体中的单核苷酸多态性,分析该基因对济宁青山羊高繁殖力的影响,并对济宁青山羊BMP2基因2个外显子进行克隆测序。结果表明,4对引物的扩增片段在4个山羊品种中均无多态性;山羊扩增片段核苷酸和氨基酸序列与人、黑猩猩、牛、犬、大鼠、小鼠6个物种的同源性分别为86.5%～91.3%和88.8%～99%;与其他物种相比,山羊BMP2扩增片段推导氨基酸序列存在3处特有突变,分别是位于第131、353、365位的脯氨酸、缬氨酸、亮氨酸变为苏氨酸(P131T)、天冬氨酸(V353D)、脯氨酸(L365P)。可见,哺乳动物BMP2基因外显子2、3序列保守性强,该区域可能不是影响山羊高繁殖力的功能结构域。     

山羊BMP15基因FecX-L突变的检测

采用PCR-SSCP方法在高繁殖力山羊品种（济宁青山羊、波尔山羊和文登奶山羊）和低繁殖力山羊品种（内蒙古绒山羊、安哥拉山羊和辽宁绒山羊）中检测骨形态发生蛋白15(bone morphogenetic protein 15,BMP15)基因的FecX-L突变,同时研究该基因突变对济宁青山羊高繁殖力的影响。结果表明这6个山羊品种都没有发生与Lacaune绵羊相同的FecX-L突变（C53Y）。可见BMP15基因中影响Lacaune 绵羊高繁殖力的突变位点对济宁青山羊的高繁殖力没有显著影响。     

山羊催乳素基因PCR-SSCP分析

设计5对引物，采用PCR SSCP技术检测催乳素（prolactin，PRL）基因外显子1~外显子5在高繁殖力山羊品种（济宁青山羊、波尔山羊、文登奶山羊）和低繁殖力山羊品种（内蒙古绒山羊）中的单核苷酸多态性，同时研究该基因对济宁青山羊高繁殖力的影响。通过克隆测序首次获得了山羊PRL基因外显子全序列。结果表明PRL基因在研究的4个山羊品种中都不存在单核苷酸多态性，提示检测的PRL基因座位对济宁青山羊高繁殖力没有显著影响。     

小尾寒羊高繁殖力候选基因RARG的研究

以视黄酸受体γ（retinoic acid receptor-gamma，RARG）基因为候选基因，采用PCR—SSCP技术分析了RARG基因在高繁殖力绵羊品种（小尾寒羊、湖羊）以及低繁殖力绵羊品种（特克塞尔、多赛特、萨福克）中的单核苷酸多态性，同时研究这个基因对小尾寒羊高繁殖力的影响。结果表明：RARG基因引物1扩增片段在5个绵羊品种中存在PCR—SSCP多态性，AA基因型只出现在湖羊中，AB和BB基因型均出现在5个绵羊品种中；BB基因型小尾寒羊平均产羔数比AB基因型多0．41只，但差异不显著（P〉0．05）。RARG基因引物2扩增片段在5个绵羊品种中存在PCR—SSCP多态性，CC和CD基因型均出现在5个绵羊品种中，5个绵羊品种中都没有检测到DD基因型；CC基因型小尾寒羊平均产羔数比CD基因型多0．55只（P〈0．05）。     

4个山羊品种BMP6基因部分片段的多态性分析

根据GenBank发表的绵羊骨形态发生蛋白6（bone morphogenetic protein 6,BMP6）基因部分序列所包含的外显子5、6、7和小鼠BMP6基因外显子5、6、7序列设计3对引物,采用PCR SSCP技术检测BMP6基因外显子5、6和7在济宁青山羊、安哥拉山羊、波尔山羊、内蒙古绒山羊4个山羊品种250个个体中的单核苷酸多态性。结果发现这3对引物的扩增片段在检测的4个品种中均无多态性,说明所检测的BMP6基因外显子5、6、7序列比较保守。     

西农莎能奶山羊和波尔山羊GDF9基因多态性与产羔数的相关分析

为了研究西农萨能奶山羊和波尔山羊生长分化因子9(GDF9)基因遗传多态性及其与产羔数和生长体重的相关性。根据绵羊GDF9基因序列设计4对引物,采用PCR-SSCP技术检测GDF9基因的多态性,同时用最小二乘法研究了其多态性与产羔数和生长体重的关系。所研究的西农萨能奶山羊群体和波尔山羊群体P1扩增片段存在多态性,分别定义为AA、AG和GG3种基因型;P2、P3和P4扩增片段均不存在多态性,只出现一种带型。通过测序发现:GG型与AA型相比在外显子2的792 bp处有1个G→A的单碱基突变,结果导致第396位氨基酸由缬氨酸变为异亮氨酸。在西农莎能奶山羊群体中,AA基因型产羔数最小二乘均值均极显著高于AG基因型和GG基因型;AG基因型显著高于GG基因型。此外,在西农莎能奶山羊中还发现AA基因型的个体各个生长阶段的体重和GG型和AG型的相比差异均不显著。在波尔山羊群体中,AA基因型产羔数最小二乘均值均极显著高于AG基因型和GG基因型,AG基因型的高于GG型,但没达到显著水平。结果表明,GDF9基因对西农莎能奶山羊和波尔山羊的产羔数均有显著影响,因此认为GDF9基因外显子2第792 bp处G→A的突变可作为西农莎能奶山羊和波尔山羊多胎性状的有效分子标记。     

山羊AMH和AMHR2基因多态性及其与产羔数关联分析

本研究旨在分析抗苗勒氏激素(AMH)和抗苗勒氏激素II型受体(AMHR2)基因单核苷酸多态性(SNPs)与山羊产羔性状的关联性。通过MassARRAY~?SNP分型技术对AMH基因和AMHR2基因SNPs位点进行分型,检测其在云上黑山羊(n=544)、济宁青山羊(n=133)和辽宁绒山羊(n=91)3个群体中的遗传学特征,并将其多态性与产羔性能(产羔数、初生窝重、断奶窝重)进行关联分析。群体遗传学结果表明,AMH基因g.89172108TG和AMHR2基因g.26334739AG在3个群体中为中度多态(0.25PIC0.5);AMHR2基因g.26335365CT在3种山羊中为低度多态(PIC0.25)。卡方检验显示,AMH基因g.89172108TG在济宁青山羊中处于Hardy-Weinberg不平衡状态(P0.05),在云上黑山羊和辽宁绒山羊中处于Hardy-Weinberg平衡状态(P0.05);AMHR2基因g.26334739AG在辽宁绒山羊、g.26335365CT在济宁青山羊中处于Hardy-Weinberg不平衡状态(P0.05),其余位点均处于Hardy-Weinberg平衡状态(P0.05)。关联分析结果表明,云上黑山羊中,AMH基因和AMHR2基因候选位点均对产羔数无显著影响,候选位点对初生窝重和断奶窝重均无显著影响。综上所述,AMH基因和AMHR2基因的候选位点不适合作为山羊产羔数选择的潜在分子标记,也不适合窝重选择。     

山羊抑制素-α第1外显子的PCR-SSCP分析

采用PCR-SSCP技术检测了抑制素α(inhibin-α,INHA)基因第1外显子在高繁殖力山羊品种(黄淮山羊和长江三角洲白山羊)以及低繁殖力品种(波尔山羊)中的单核苷酸多态性,并研究了该基因对黄淮山羊产羔数的影响.设计4对引物扩增山羊INHA基因外显子1序列,只有引物1在3个山羊品种中检测到多态性,出现了3种基因型(AA、AB、BB),测序结果表明,BB型和AA型相比在INHA基因第1外显子1 006 bp处都发生了碱基突变(C→A),导致脯氨酸改变为苏氨酸.黄淮山羊突变纯合型(BB)平均产羔数比野生型(AA)少0.65只(P＜0.05).研究结果初步表明:INHA基因可能是影响黄淮山羊繁殖力的一个主效基因或是与之存在紧密遗传连锁的一个分子标记.     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

乳酸杆菌作为益生菌广泛用于人和动物。本文综述了乳酸杆菌改善宿主健康的机制。乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道。文章重点讨论了乳酸杆菌表面成分（表面蛋白、脂磷壁酸和肽聚糖）与肠道受体（Ｃ型凝集素受体、Ｔｏｌｌ样受体和 Ｎｏｄ样受体）,阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.     

Mechanism of Action of Probiotic Function of Lactobacilli

乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis.