Isolation and characterization of microsatellite loci for the red panda,Ailurus fulgens

We report on the isolation and characterization of 10 microsatellites in the red panda Ailurus fulgens from genomic DNA‐enriched libraries. Twenty‐one microsatellites were screened from the libraries, and 10 of the screened microsatellites were polymorphic. The number of observed alleles for each locus in 24 individuals ranged from three to 12, and the expected and observed heterozygosity was 0.60–0.90 and 0.50–1.00, respectively. These markers should prove a useful tool for the study of genetic variation in red panda in the future.     

Isolation and characterization of 12 novel microsatellite loci for the red panda (Ailurus fulgens)

The information on dispersal patterns and mating systems of red pandas is quite important for the understanding of the genetic diversity and divergence of this species. And microsatellite marker is an ideal tool to analyze dispersal patterns and mating systems. Thus, we describe in this paper the isolation and characterization of 12 microsatellite loci in the red panda from genomic DNA-enriched libraries. These loci were highly polymorphic with numbers of alleles per locus in 24 individuals ranging from 2 to 14, observed heterozygosity from 0.143 to 0.864 and expected heterozygosity from 0.297 to 0.872. All loci except for RP6 locus followed Hardy–Weinberg expectations. No significant linkage association was found among all these loci. The 12 novel polymorphic microsatellite loci will be of use in studying dispersal patterns and mating systems of red pandas.     

Identification and characterization of ten polymorphic microsatellite loci in the red panda Ailurus fulgens

Ten polymorphic microsatellite loci were characterized from two genomic DNA-enriched libraries of the red panda (Ailurus fulgens). The number of observed alleles among 35 samples of red pandas ranged from five to 12. Observed and expected heterozygosities were 0.286–0.971 and 0.443–0.894, and the mean polymorphic information content was 0.712. All loci followed Hardy–Weinberg expectations except Aifu-14 and Aifu-16, which may due to the presence of inbreeding or null alleles. Three pairs of loci exhibited significant linkage disequilibrium after Bonferroni correction for multiple comparisons. These microsatellites would be useful to strengthen population management, genetic diversity exploration, and demographic history speculation of this species.     

Isolation and characterization of polymorphic tetranucleotide microsatellite loci in the Fire salamander Salamandra salamandra (Amphibia: Caudata)

Ten tetranucleotide and one dinucleotide polymorphic microsatellite loci were cloned and characterized for the Fire salamander (Salamandra salamandra) from 34 populations in Germany. A high genetic diversity (5–22 alleles per locus) and heterozygosity (40.6–95.2%) were observed for these markers. Chord distances for population comparisons of the western evolutionary recolonization lineage in the area near Cologne ranged from 0.139 to 0.366, whereas population comparisons between the western and eastern lineage ranged from 0.541 to 0.670. When compared with classical isolation methods, a sufficient number of polymorphic microsatellites can be obtained for the Fire salamander only from specially enriched sublibraries.     

Scent-marking in captive red pandas (Ailurus fulgens)

Three male and three female captive red pandas (Ailurus fulgens) were observed during and outside of the breeding season to examine changes in scent-marking behavior. Variation in scent-marking was analyzed with respect to site preferences, sex differences, onset of breeding, behavioral correlates, and environmental factors. Both males and females exhibited significant preferences for particular marking sites, those being prominent points in the enclosures. Males showed significantly higher scent-marking frequencies than females, except in one enclosure where increased marking behavior was associated with the presence of young. Sexual dimorphism in the pattern of marking was also observed, with males showing a bidirectional mark and females a unidirectional mark. Female marking was greater during the breeding season. Sniffing and number of waddles per mark were associated with scent-marking. Temperature change was not related to frequency of scent-marking. These results are compared with scent-marking behaviors in other species of carnivores and functional explanations are suggested.     

小熊猫对同性和异性气味的行为反应

食肉目动物的复杂社会行为在种间和种群间变化较大,这种变化是动物对其生态环境的适应表现特征之一。食肉目的动物与嗅觉通讯相关的气味源有肛周腺、肛囊腺、尾下腺、会阴腺、足腺、尿液、唾液和粪便等十几种,产生的气味物质包含有个体、性别和群体的识别信息,在其繁殖调控中发     

紫色色杆菌感染小熊猫引起肺炎的临床调查

本文报道了福州大熊猫研究中心小熊猫感染紫色色杆菌引起肺炎的群发病例。7 只小熊猫中,出现以呼吸衰竭为主要临床症状的3 只小熊猫在发病3 d内全部死亡,病死率100% 。死亡的小熊猫解剖病变为气管内有白色泡沫样分泌物,肺有化脓性坏死并严重淤血;胸腔,心包积液且胸水浑浊;肝脏呈弥漫性空泡变性,淤血和散在局灶性坏死。通过流行病学调查,病理解剖观察,细菌分离培养和鉴定及动物回归试验,诊断为紫色色杆菌无色变种感染。根据GenBank 数据库提供的紫色色杆菌16S RNA 基因序列,设计一对引物(5’GAG CAAACA GGA TTA GAT ACC 3’;5’TTA CGG TTA CCT TGT TAC GAC 3’),获得目的基因片段739 bp,将核苷酸序列测定的结果与GenBank 数据库提供的7 株紫色色杆菌进行同源性比较。分离的菌株(FJ08A) 与CV09 株、ESBV4400株同源性均为98.8% ,与AY117554 株、EAV2 株、AJ871127 株、LMG3953 株、JS1 株同源性分别为98. 2% 、98.0% 、94.9%、93.1% 、92.8% 。依据调查结果和药物敏感试验,对其余4 只未发病的小熊猫采取肌
肉注射头孢哌酮钠和口服复方新诺明进行预防性治疗,效果良好,未有新病发生。结果表明:(1)紫色色杆菌是革兰氏阴性菌,致病性强,致死率高,腹腔注射分离的紫色色杆菌菌液导致接种小白鼠在2 ～3 d 内全部死亡。小熊猫感染后,发病迅速,致死率高,应引起重视;(2)紫色色杆菌是一种条件致病菌,外伤感染是主要致病因素。     

圈养小熊猫几种病毒的PCR检测

本文采用巢式PCR/ RT-PCR 方法,对我国10 个动物园中无临床症状的圈养小熊猫的71 个肛拭子和61 个唾液拭子样品,进行犬瘟热病毒(CDV)、犬细小病毒(CPV)、犬冠状病毒(CCV)、犬腺病毒(CAV)和犬疱疹病毒(CHV)的检测,以评估我国圈养小熊猫是否面临这几种病毒的威胁。对阳性PCR 结果进行测序分析,并与GenBank 上的序列进行比较。结果,在肛拭子样品中检测到3 个CPV 和6 个CCV 阳性结果,经测序后,与GenBank 上序列的同源性分别达99% 和100% 。而在唾液拭子样品中没有检测到任何阳性结果,且CDV、CAV和CCV 的检测结果均为阴性。从阳性CPV 的肛拭子样品中分离到一株细小病毒毒株,表明圈养小熊猫已受到细小病毒和犬冠状病毒的感染,今后应加强这两种病毒的预防工作。本文所采用的PCR 方法检测病毒性疾病,能检测到微量的病毒模板,可对小熊猫病毒性感染进行早期诊断。     

成体雌性小熊猫卵巢组织学观察

2000 年至2009 年,12 只固定于10% 福尔马林中非生殖系统疾病死亡的小熊猫卵巢组织,按常规组织学技术制作组织切片,HE 染色,光学显微镜观察。结果:(1)不同发情时期卵巢均有原始卵泡、初级卵泡和次级卵泡分布。发情期的卵巢未观察到典型的成熟卵泡和卵母细胞; (2)原始卵泡数量较少,初级卵泡数量较多,多数初级卵泡和大多数的次级卵泡都处在闭锁状态;(3)卵泡腔出现之前,卵母细胞的直径和卵泡直径同时增长;卵泡腔出现之后,卵母细胞直径增长较慢,卵泡直径增长较快; (4)不同发情时期的小熊猫卵巢均存在大量的间质腺细胞;(5)妊娠小熊猫和发情间期无妊娠小熊猫的卵巢均有发育正常的黄体;(6)卵泡细胞发育呈低柱状至柱状时出现透明带。结论:(1) 卵泡闭锁主要发生在初级卵泡阶段,仅少数卵泡能发育至次级卵泡;(2)卵母细胞和卵泡生长呈双相生长的趋势; (3) 不同发情时期的小熊猫卵巢间质腺都发达; (4)发情排卵后,非妊娠黄体与妊娠黄体维持的时间相似,证实了小熊猫存在假孕现象。     

小熊猫繁殖周期血清雌二醇和孕酮含量变化

为研究小熊猫繁殖周期血清雌二醇、孕酮含量变化规律,采用化学发光免疫分析法连续16 次测定了2只成体雌性小熊猫血清雌二醇和孕酮含量变化,历经发情间期、发情期和两次妊娠期;连续9次测定了7只小熊猫妊娠期的孕酮含量变化。结果:(1)发情间期,小熊猫血清雌二醇的水平一直维持在低水平(基础水平),进入发情前期,血清雌二醇水平明显升高,在发情期一直维持高水平,配种后迅速降至基础水平; (2)小熊猫血清孕酮含量在发情间期和发情期均维持在较低水平,直至发情期过后才出现升高,在妊娠期一直维持高水平,峰值出现在5 月;(3)发情的小熊猫不论妊娠与否,在妊娠期内血清孕酮含量均维持在高水平。研究表明:小熊猫血清雌二醇、孕酮含量变化能直接反映其繁殖规律,雌二醇对启动雌性小熊猫季节性繁殖起重要作用;在妊娠期内小熊猫血清孕酮含量升高不能作为判断小熊猫妊娠的标准;雌性小熊猫在妊娠期有假孕现象。     

雄性小熊猫粪便中睾酮水平的变化与繁殖周期的关系

1999 年12 月至2000 年9 月, 使用放射免疫分析法, 测定了成都动物园和成都大熊猫繁育研究基地的4 只雄性小熊猫粪样中睾酮水平变化情况, 同时对其行为进行了观察。研究结果表明, 雄性粪样中睾酮水平在繁殖期(12 月～3 月) 显著高于非繁殖期(4 月～9 月) (P < 0.01) , 且睾酮水平变化与繁殖行为密切相关。在非繁殖期,雄性小熊猫的睾酮水平处于基础的较低水平时, 其繁殖行为如蹭阴、舔阴、舔标记物和咩叫等几乎没有发生或很少发生; 而在繁殖期, 雄性小熊猫的睾酮水平明显升高, 这些繁殖行为频繁发生。以上结果说明, 睾酮是调节雄性小熊猫繁殖行为季节性变化的生理因子。     

蜂桶寨自然保护区小熊猫巢域初步研究

2002年5~11月,在蜂桶寨自然保护区利用无线电遥测技术对6只小熊猫的巢域利用进行了初步研究。结果表明,6只戴颈圈个体M1、M2、M3、F1、F2、F3的巢域面积分别为330·26hm~2、135·18hm~2、190·67hm~2、98·23hm~2、141·60hm~2、204·80hm~2;雄性个体平均巢域面积为218·70hm~2,雌性个体为148·21hm~2。小熊猫个体间巢域重叠普遍,平均重叠率达25·33%,其中雄性个体之间为26·00%,雌性个体之间为23·67%,两性个体之间为25·67%。可能受人为干扰的影响,M1在6只监测个体中巢域面积、日均移动距离均为最大。     

日粮中霉菌毒素引发小熊猫中毒性肝炎的临床调查

本文报道了小熊猫霉菌毒素中毒性肝炎的群发病案。35 只小熊猫中,21 只发病,5 只死亡。通过日粮成分调查,饲料中霉菌毒素检测,病原微生物检查,血液参数测定,病理学检查和动物试验,确诊为呕吐毒素(DON)和玉米赤霉烯酮(F-2)毒素中毒。依据调查结果对小熊猫进行治疗,小熊猫预后良好。结论:(1)小熊猫对霉菌毒素(DON 和F-2 毒素)极其敏感,应予以高度重视;(2)DON 和F-2 毒素存在相互增强作用,对小熊猫的毒性作用主要为肝毒性和肾毒性,并可导致小熊猫机体免疫功能降低;(3)小熊猫对霉菌毒素的耐受性与环境的适应性有关,应激因素能提高小熊猫对霉菌毒素的敏感性。      

Influences of mating groups on the reproductive success of the Southern Sichuan Red Panda (Ailurus fulgens styani)

The reproduction of female red pandas under three different husbandry conditions (monogamy, polygamy, and polyandry) was studied at the Chengdu Research Base for Giant Panda Breeding, and Chengdu Zoological Park, China. We monitored the fecal steroid profiles of seven females by radioimmunoassay in order to understand how the different mating groupings affect the animals' reproductive success. Of the seven females studied, all gave birth except the one that was kept in the polyandrous group. Although fecal estradiol metabolites exhibited several peaks during estrus, only one coincided with mating behavior. Fecal progesterone metabolites did not display a sustained rise in the absence of mating behavior, even though estradiol displayed a peak of secretion. This suggests that the red panda may be an induced ovulator. During gestation, fecal progesterone metabolites were maintained at a comparatively high level, and were much higher than those of nonpregnant females. The female in the polyandrous group showed an abnormal estradiol metabolite profile, with a level that was four to five times higher than those of the females in the polygynous groupings. It was observed that this female always escaped and hid in the trees to avoid courtships with males, and did not give birth. This phenomenon suggests that polyandrous groupings may be unsuitable for successful reproduction in captivity. Zoo Biol 24:169–176, 2005. © 2005 Wiley‐Liss, Inc.     

Genetic structuring and recent demographic history of red pandas (Ailurus fulgens) inferred from microsatellite and mitochondrial DNA

Clarification of the genetic structure and population history of a species can shed light on the impacts of landscapes, historical climate change and contemporary human activities and thus enables evidence‐based conservation decisions for endangered organisms. The red panda (Ailurus fulgens) is an endangered species distributing at the edge of the Qinghai‐Tibetan Plateau and is currently subject to habitat loss, fragmentation and population decline, thus representing a good model to test the influences of the above‐mentioned factors on a plateau edge species. We combined nine microsatellite loci and 551 bp of mitochondrial control region (mtDNA CR) to explore the genetic structure and demographic history of this species. A total of 123 individuals were sampled from 23 locations across five populations. High levels of genetic variation were identified for both mtDNA and microsatellites. Phylogeographic analyses indicated little geographic structure, suggesting historically wide gene flow. However, microsatellite‐based Bayesian clustering clearly identified three groups (Qionglai‐Liangshan, Xiaoxiangling and Gaoligong‐Tibet). A significant isolation‐by‐distance pattern was detected only after removing Xiaoxiangling. For mtDNA data, there was no statistical support for a historical population expansion or contraction for the whole sample or any population except Xiaoxiangling where a signal of contraction was detected. However, Bayesian simulations of population history using microsatellite data did pinpoint population declines for Qionglai, Xiaoxiangling and Gaoligong, demonstrating significant influences of human activity on demography. The unique history of the Xiaoxiangling population plays a critical role in shaping the genetic structure of this species, and large‐scale habitat loss and fragmentation is hampering gene flow among populations. The implications of our findings for the biogeography of the Qinghai‐Tibetan Plateau, subspecies classification and conservation of red pandas are discussed.     

圈养小熊猫育幼行为的初步观察

Nursing behaviors of the captive md pandas were quantitatively studied by focal sampling methods in Chengdu Research Base of Giant Panda Breeding from Jldy to August, 2000. The results indicated that frequencies of activity and rest were low in the first three days after birth, and became higher as time went by. The frequency of licking cub was higher in the first day after birth and deserting cub was only observed in the seventh day. Frequencies of some behaviors, such as rest, licking cub, cherishing cub,sniffing cub, returning to shed and deserting cub, differed sioaificantly in different phases of the first month after birth. Frequencies of licking cub, cherishing cub, sniffing cub and returning to shed were significantly higher in the pre-nursing period than in the midnursing and post-nursing periods. However, frequencies of rest and deserting cub were sioaificantly higher in the post-nursing period than in the per-nursing and mid-nursing periods. The degree of maternal behaviors can be inferred from degree that the mother exposes her babies to the envim~unent and the time that the mother leaves her shed.     

一种筛选微卫星位点的改进方法及其在小熊猫微卫星基因文库构建上的应用

微卫星（Simple Sequence Repeat,SSR）基因位点是在各个遗传学领域被广泛使用的分子标记。而对于首次研究的物种,必须筛选出可以应用的微卫星位点。对于分离微卫星位点来说由于只有大约0.5％-2%的重组体含有微卫星位点,所以标准的基因组文库需要至少产生5 000个重组体。传统的筛选方法就是通过检测大量重组体来定位这些少量的位点。近几年来出现了一些新的筛选方法,采用“富集”微卫星序列的技术将包含微卫星位点的重组体比例提高了10-100倍。这样就降低了筛选工作的时间和劳动强度,大大提高了筛选微卫星位点的效率。本文在结合了几种筛选微卫星位点技术路线的基础上,采用生物素标记探针杂交、富集和PCR筛选技术,对原有技术路线进行了改进。主要试验步骤为：1）基因组DNA的提取和消化;2）生物素标记探针富集;3）克隆建库;4）“PCR”筛选;5）引物设计和多态性检测。新的方法在小熊猫微卫星基因文库的构建中取得成功,获得了10个具有多态性的(CA)n重复序列微卫星位点。     

小熊猫圈养种群遗传多样性及其影响因子

利用寡核苷酸指纹探针ＬＺＦ－Ｉ检测了成都大熊猫繁育研究基地仿生态圈养场小熊猫种群全部２２只个体的ＤＮＡ指纹,检测结果表明其遗传多样性参数如下：⑴各体间ＤＡＮ指纹图的相似性为０．１７２;个体Ａ的指纹谱带在个体Ｂ中出现的概率为０．１８０６。⑵圈养种群的等位基因频率为０．０９４８,杂合率为９０．５２％,⑶种群中指名亚种亲本个体的引进是影响该种群遗传多样性的主要因子。     

An ARMS-based technique for sex determination of red panda (Ailurus fulgens)

Molecular sexing is a key component in the investigation of wild populations. In this study, we developed a fast, accurate and reliable amplification refractory mutation system (ARMS) technique for sex determination of red panda based on the exon 4 of the ZFX/ZFY gene. The amplicons were distinguished simply by agarose gel electrophoresis, exhibiting one fragment in females (X: 300 bp) and two in males (X: 300 bp, Y: 166 bp). Robustness of this ARMS system was confirmed by testing both 43 captive red pandas using DNA samples with known-sex and 10 wild red pandas using faecal DNA samples with unknown sex.