粪便孕酮水平是预测哺乳动物妊娠的重要激素之一,为了真实反映林麝粪便孕酮水平,提高妊娠检测的准确率,以林麝粪便为研究对象,用不同的方法进行抽提,并用酶联免疫吸附分析(ELISA)和高效液相色谱(HPLC)进行检测.结果表明:振荡可显著提高孕酮的溶解效率(P＜0.05);平皿蒸干速度快,但孕酮损耗大(P＜0.05);1 mL甲醇溶解粪便提取物后,再用0.1mL甲醇溶解可显著减少高效液相色谱中的杂带,但不影响其检测效率(P＞0.05).结果证明,改进后的方法可提高ELISA和高效液相色谱检测效率,为后续林麝和其他动物的孕酮检测提供参考.

针对当前林麝养殖管理、遗传育种智能化水平较低的现状,融合养殖管理技术与分子遗传学设计了林麝资源管理平台,支持林麝资源及林麝养殖的日常管理,并可以通过养殖过程数据、林麝遗传谱系数据和有效分子标记数据进行林麝智能选配育种;基于当前主流的"公司+农户"的养殖场景,采用Net Framework和SQL Server构建了 B/S架构的平台软件系统,实现了养殖场管理、个体管理、品系管理、养殖过程管理、系谱分析绘制、亲缘系数计算、分子序列比对、个体/群体选配、可视化数据分析、系统配置等功能模块;通过测试发现,该平台功能完备、实现正确、人机交互界面友好,能够满足林麝日常养殖管理和配种选育需要,也可为改善林麝种质资源提供数据依据.

为了探究泌香期雄麝皮质醇和睾酮的变化规律及其对麝香产量的影响,试验选取6头雄麝,采用酶联免疫吸附法(ELISA)测定雄麝泌香期粪便中的皮质醇和睾酮含量,并对其与麝香产量进行相关性分析.结果 表明:在泌香期三个阶段,皮质醇和睾酮含量都呈现先增高后降低的趋势;皮质醇、睾酮含量与麝香产量相关性不显著(P＞0.05).说明利用粪便样品来研究圈养林麝类固醇激素是一种较为理想的方法,可以开展以粪便类固醇激素为核心的多种试验和推广工作.

林麝(Moschus berezovskii)是典型的林栖偶蹄目动物,被列为国家Ⅰ级保护野生动物.栖息地破坏引起的家域丧失是导致其濒危的重要因素.了解其家域面积,生境偏好等基本信息,是更好保护这一重要珍稀濒危动物的前提.但是,由于林麝生性机警,在野外环境中难以人为追踪,这些数据相对缺乏.2017年6月,我们为8只野化训练后的圈养林麝佩戴GPS项圈,并放归到陕西省平河梁国家级自然保护区.经过一年的监测,共获取6只林麝的野外活动点位,利用固定核空间法计算出家域面积,并分析了其家域特征和迁徙行为.结果 显示:林麝在非繁殖期间各自占据独立的家域,表现出独居的特征;它们倾向于选择植被丰富、接近水源、半阴半阳、坡度陡峭并多悬崖峭壁的区域作为栖息地;一雄一雌两个个体全年家域面积分别为168.85 hm2、64.87 hm2,全年核心家域面积分别为40.08 hm2、15.36 hm2;在交配期间,雄性林麝会主动向雌性靠近,从而发生家域重叠.基于以上数据,本研究提出,林麝的实际家域面积,可能高于此前粗估,并建议在后续保护工作中,需要注重保障其基本生存面积.

为快速准确检测化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌,分别根据化脓隐秘杆菌plo基因、溶血性曼氏杆菌ICEMh1基因和肺炎链球菌pspC基因的特异性序列设计引物和探针,以重组质粒a.p-m.h1-s.p为标准品,通过优化反应条件,建立一种检测化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌三重实时荧光定量PCR方法,对其特异性、灵敏度和可重复性进行测试.结果表明,建立的方法对标准品阳性质粒的最低检测量为20拷贝,标准曲线在1.25×106 copies/μL～1.0×101 copies/μL范围内具有良好的线性关系.对2016年-2018年在陕西省收集的病料中化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌检出率分别为66.67％、5.5％和44.44％.建立的三重实时荧光定量PCR方法能快速、准确和稳定地检测化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌.

[目的]本研究建立了林麝标记辅助选择技术体系,为林麝的品系选育提供理论依据.[方法]通过基因克隆研究林麝胰岛素样生长因子(IGF-1)的基本功能和蛋白质结构,采用PCR-SSCP技术检测IGF-1基因在林麝中的多态性,分析其与产仔数的关联效应.[结果]①IGF-1基因序列全长465 bp,编码153个氨基酸残基,经生物信息学分析,发现该蛋白含有15个磷酸化位点,是一个分泌性蛋白,大部分位于细胞膜上,含有Insulin家族典型的保守结构功能域;②本试验设计IGF-1基因的PCR-SSCP扩增片段长度均为300 bp左右,引物扩增片段用10％的聚丙烯酰胺凝胶进行检测,发现扩增片段没有特异性条带.为证明PCR-SSCP扩增结果,选择其中一条引物对所得的毛发样品进行扩增,测序发现所有PCR产物的序列完全一致,没有多态性.[结论]研究表明不能选择1GF-1基因作为林麝的分子遗传标记,但为林麝产香性能的分子遗传标记选育提供了基本思路.

以圈养林麝为研究对象,采集发情期雄麝粪便,通过比较和评价高效液相色谱(H LPC)和酶联免疫吸附试验(ELISA)对林麝粪便睾酮检出效率;选择检测结果较好的方法对空气中放置不同时间的粪便睾酮水平、发情期睾酮水平、林麝交配质量和麝香分泌量之间的关系,进行初步评价.结果 表明,所有样本经聚合酶链反应(PCR)技术检测均为林麝样本;对睾酮标准品进行检测,HPLC最低检测限为74 ng/mL,ELISA试剂盒检测最低限度为10 pg/mL.两种方法检测粪便样本,发现HPLC没有睾酮信号峰,而ELISA检测样本浓度为2.408 ng/mL.ELISA检测粪便在空气中暴露0、2、4d睾酮水平差异不显著(P＞0.05),但与0d相比,6d的睾酮水平显著下降(P＜0.05).交配能力弱组与交配能力强组麝香产量无显著差异(P＞0.05).结果 证明,HPLC适用于林麝粪便睾酮水平高于74 ng/mL的检测;粪便于室温空气暴露6d,会显著影响粪便检测的睾酮水平(P＜0.05).因此,采集4d前的林麝粪便均可用于睾酮检测;林麝睾酮水平与交配能力相关.

Molecular markers is a quick and efficient method for collecting forest musk deer DNA,and is also the fundament for selection of improved varieties.In this study,feces of forest musk deer was used as object,efficiency of DNA extraction from feces of forest musk deer was compared between normal and improved methods,and two methods were used as control and treated group,respectively.The feces were exposed for 0 d,2 d,4 d,6 d after it was collected from forest musk deer.Two methods with control and treated group were carried out to extract feces DNA,and PCR was used to amplify samples by primer of GAPDH in forest musk deer.Target DNA fragment was separated by agarose gel electrophoresis.Optical density of DNA sample from feces exposed in air for 0 d for reference,and relative optical density was obtained by comparing optical density from that of 2 d,4 d and 6 d with that of 0 d.The results showed that feces were gradually tarnished and roughened with timedependant.DNA was efficiently extracted from forest musk deer feces by both methods in control and treated groups.In treated groups,DNA level significantly increased by comparing 2 d and 4 d with 0 d,and there was no difference at 6 d,compared with 0 d;in control group,DNA level was only obviously elevated at 2 d,compared with 0 d,and DNA level at 4 d and 6 d was declined to that of 0 d.With control groups used as reference,DNA level from treated group was significantly higher than that of control group at all time points.The results demonstrated that DNA could be efficiently obtained from forest musk deer feces from treated groups,which would provide the basis for molecular marker and breeding in forest musk deer.

为探索林麝粪便在空气中暴露不同时间与孕酮水平的关系,以妊娠期林麝粪便为研究对象,通过高效液相色谱(HPLC)和酶联免疫吸附试验(ELISA)两种方法检测孕酮水平,选择合适的检测方法评价不同新鲜度粪便孕酮水平,比较妊娠期和非妊娠期林麝孕酮水平.结果发现,高效液相色谱检测孕酮水平的最低检测浓度为32μg/m L,用粪便样本检测时未见色谱峰,酶联免疫吸附试验检测孕酮水平为460 ng/m L,已显著低于高效液相色谱分析的浓度.随粪便在空气中暴露时间延长,孕酮水平降低,其中空气暴露后4d和6 d的粪便孕酮水平显著低于0 d(P<0.05);妊娠期林麝粪便孕酮水平显著高于非妊娠个体((P<0.05).结果表明,与ELISA相比,HPLC要求样本浓度高,粪便长时间空气暴露可导致孕酮水平降低,孕酮可作为预测林麝妊娠的候选激素,研究工作可为后续林麝的妊娠诊断和选育提供参考.

麝(Moschus)是亚洲东部特有的小型反刍动物,是珍稀濒危资源动物.因麝机警灵敏,胆怯性急,耐受力差,传统的取样方法会造成不同程度的伤害,不利于保护野生种群和可持续利用麝资源.收集粪便和毛发样品是在不触及动物或低干扰动物正常活动的情况下进行的,有助于麝资源的保护.文章系统总结了麝科动物粪便和毛发的取样方法及其在麝科动物遗传多样性、疾病、应激反应、生殖生理等方面的应用研究,并对粪便和毛发取样法在麝科动物的应用前景进行展望.

The study was aimed to establish a specific and sensitive TaqMan Real-time PCR assay for detection of Arcanobacterium pyogenes(A.pyogenes).Based on the conservative sequence of pyolysin(PLO)gene of A.pyogenes published in GenBank,specific primers and TaqMan probes were designed.The TaqMan Real-time PCR assay was established,and the specificity and sensitivity were tested.The specificity test results showed that only A.pyogenes exhibited typical curves.The detection sensitivity of this assay was 77.6fg genomic DNA per 20μL reaction,and63CFU/mL for pure cultures.16 out of 23 clinical samples were positive detected by the TaqMan Real-time PCR assay,which were consistent with API Coryne identification.The TaqMan Real-time PCR assay developed in this study was specific and sensitive for detection of A.pyogenes,and it could be used for identification and epidemiological investigation of A.pyogenes.

根据GenBank已公布的化脓隐秘杆菌溶血素(（Pyolysin,PLO）基因设计6条引物,建立一种灵敏的环介导等温扩增方法（loop-mediated isothermal amplification,LAMP）用于化脓隐秘杆菌（Arcanobacterium pyogenes）的快速检测,并对检测方法的特异性和灵敏度进行验证。对化脓隐秘杆菌、鼠伤寒沙门氏菌、非O1霍乱弧菌、单增李斯特氏菌、表皮葡萄球菌、金黄色葡萄球菌、嗜水气单孢菌、副溶血性弧菌、蜡样芽孢杆菌等9株实验菌株进行的特异性试验表明,建立的LAMP方法仅对化脓隐秘杆菌的检测结果为阳性;该方法的检测灵敏度在DNA水平上可达112 fg。采用本研究建立的方法检测20份林麝临床病例样品,共鉴定出10株化脓隐秘杆菌,与API Coryne生化鉴定方法的符合率为100%。

从林麝Access数据库建立、数据查询及时性与准确性等方面总结提出了陕西省林麝Access数据库检索系统的应用体会.

In order to study the growth and development law of the musk deer,the body weight and size of 10 semi-free male musk deer from0 to 12 months were measured.The results showed that the weight cumulative growth curve increased from intense to gently.The 0- 4 and 10- 12 months absolute growth rate has an increasing trend and the absolute growth peak was between 0 to1 months old,hereafter slowed down gradually.The growth intensity in 0- 3 months was higher than that of other months.The growth intensity of 3- 6 months has a significant linear downtrend,which increases slowly after the age of 8 months.The increase of the body ruler index was significant before the age of 6months and a downtrend existed between 6 to 12 months.The length and shoulder height of 8- 12 months changed fewer.The results revealed the growth development law of musk deer,which can provide reference for breeding and semi-free management in the future.

随着人工养殖林麝数量的增加和营养水平的提高,母麝孕期营养充分,林麝胎儿有增大趋势.同时由于圈养场地狭小,母麝活动范围太小、运动量不足,所以难产现象时有发生.仔麝蹄子锋利且四肢修长,难产时往往腿部卡在子宫,造成助产催产困难,强行拉扯胎儿会损伤阴道甚至子宫,此时应果断采取剖腹产.此前国内尚无关于林麝剖腹产的报道,笔者整理了凤县康宁林麝救助中心实施的36例林麝剖腹产手术经验,总结如下.

Based on determinating ingredients of plant leaves,used a simple linear correlation,nutritive type and cluster analysis to classify separately according to nutritive ingredients and partial minerals contents.This study expected to provide references of mixed ration for rasing forest musk deer according to plant leaves nutrient trait.The results indicated that crude fiber satisfy the nutrition demand of the forest musk deer,crude protein contents of Broussonetia papyrifera,Humlus scandens and Morus alba were also satisfied the demand.The calcium content surpassed the demand,while some kinds of plant leave phosphorus content surpassed the demand.The ratios of calcium to phosphorus of Eucommia ulmoids,Picrasma quassiodes,H.scandens and Litsea pungens were less than 7.00,the iron and manganese contents surpassed the demand,the copper and zinc canno satisfy.ASH and CP had significant positive correlation(r=0.610 8),ASH and NEF had significant negative correlation(r=0.684 2),CF and NEF had significant negative correlation(r=0.600 4),CP and NEF had significant negative correlation(r=0.793 2).The nutrition ratio(C/N) of Dipteronia sinensis and M.alba,L.pungens was lower than 3.75,belonged to the N type.The nutrition ratio(C/N)of Acer grosseri was 7.27,belonged to the C-N type,Ampelopsis bodinieri,H.scandens and B.papyrifera were belonged to ash-N type,Lonicera mackii,Schisandra propinqua and Celastrum rosthornianus belonged to NC-ash type,the rests belonged to N-C type.The plant leaves have been divided into 6 kinds according to the conventional nutrient content,and also may be divided into 7 kinds according to the mineral content.

This research enacted the grades of musk deer by observing the coat colour,mental status,breath,excrement and urine of them.In order to make effective measures,we combined with breeding and muskiness secretion.The results could be used for disease prevention,breeding and muskiness secretion.These measures were helpful for increasing the development and the level of musk deer cultivation.

某养麝场的2只林麝1只鼻镜发生脓肿,另一只右颌下发生脓肿,对患脓肿病的林麝无菌采取其两处脓汁进行细菌培养并鉴定。经细菌形态、培养特性和生化特性分析,鉴定为化脓棒状杆菌(Corynebacterium pyogenes)。药敏试验表明:该菌对克林霉素、头孢曲松、头孢噻肟、万古霉素、氯霉素、红霉素和四环素均敏感。

陕西省人工养麝历经半个世纪取得了重要进展,现有林麝的存栏量已达2 500余只,约占全国人工养麝数量的50%以上,主要分布在凤县、镇坪、陇县、华阴市、眉县和留坝等地.由于受到养麝经济效益的制约,近50 a来陕西省养麝业的发展特点为:养麝场规模不大,养麝的散户多且各户麝存栏量小,种源质量问题突出,养麝技术仍不够成熟,饲养管理模式属于粗放型,疾病防控能力和麝的产香量有待提高.基于此,今后应从政府、农户、科研、公司四方面努力,共同促进养麝业健康持续发展.