Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs

The giant panda (Ailuropoda melanoleuca) is one of the world’s most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity.     

育幼经历对圈养成年雌性大熊猫尿液皮质醇含量的影响

为研究育幼期不同的育幼方式对圈养成年雌性大熊猫Ailuropoda melanoleuca福利状况的影响情况,了解圈养育幼模式可能引发成年雌性大熊猫的应激问题,完善育幼期大熊猫的饲养管理和兽舍参数设计,提高育幼过程中圈养大熊猫福利状况,选择成都大熊猫繁育研究基地育幼期采取不同育幼方式管理的成年雌性大熊猫为研究对象,利用...     

大熊猫神经营养素-4基因在大肠杆菌中的表达

本通过PCR技术,直接从大熊猫基因组DNA上克隆得到其神经营养素—4的成熟肽编码序列,通过序列分析发现,该基因在进化上具有较高的保守性。将神经营养素—4成熟肽完整编码序列克隆至pGEX—4T—3表达载体,并经IPTG诱导在大肠杆菌中进行原核生物表达,获得了大熊猫重组蛋白神经营养素—4。重组表达蛋白经纯化后,进行大鼠肾上腺嗜铬瘤细胞神经营养因子的活性鉴定,发现其能够诱导神经细胞分化产生突触,具有预期的生物学活性。对大熊猫神经营养素—4的基因工程研究,为大熊猫癫痫的基因治疗奠定了基础。     

野化培训大熊猫幼仔的食性转换

本文以2010—2020年15只母兽带仔野化培训的大熊猫幼仔为研究对象,基于红外视频监控系统观察和音频颈圈解译获得的行为资料、GPS颈圈跟踪定位采集的粪样数据,分析了野化培训大熊猫幼仔的行为发育进程和食性转换特征。结果表明：随着野化培训大熊猫幼仔的生长发育,与觅食和警戒相关的行为得到充分发育,且具有较强的时间关联性,包括食乳、爬行、走动、玩耍物品、爬树、咬玩竹子、饮水和采食竹子等。8～10月龄的大熊猫幼仔开始取食竹子,其发育性食性转换过程划分为3个阶段：食乳期(1～7月龄)、食母乳—食竹子转换期(8～28月龄)和食竹期(29～39月龄),其中转换期细分为关键期(8～18月龄)和过渡期(19～28月龄)。从统计检验来看,不同食性阶段间差异显著;过渡期的大熊猫幼仔可离开母兽独立生活,此阶段大熊猫幼仔食物组分比例与食竹期相比无显著差异。野化培训大熊猫幼仔的季节性食性转换规律与带仔母兽和野生大熊猫具有相似的格局,即春季主要取食竹笋,夏、秋季则以嫩竹茎和竹叶为食,冬季采食竹叶与竹茎。     

The Microbial Community in the Feces of the Giant Panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>) as Determined by PCR-TGGE Profiling and Clone Library Analysis

Despite having a typical carnivorous digestive tract, the giant panda has a diet consisting exclusively of bamboo, a low-efficiency food source. Given this paradox, we sought to investigate if the giant panda digestive tract is inhabited by organisms indicative of high cellulose diet or their gastrointestinal tract anatomy. The diversity and dynamics of the predominant bacteria in the fecal flora of two adult (male and female) and one young (male) giant panda reared in two different zoos over a 2-year period was studied using 16S rDNA-based approaches. The temperature gradient gel electrophoresis (TGGE) profiles of the 16S rDNA V3 region of the three individuals were highly similar. The structure of their fecal flora remained relatively stable over the 2-year period. Both the most predominant band in TGGE patterns shared by the three pandas and the biggest operational taxonomic unit (OTU) in the clone library were phylogenetically related to Escherichia coli. Gram-negative, facultative bacteria constituted almost 60% of the whole community in the clone library. All the OTUs were related to previously described phylotypes known to reside in the intestine or rumen. The results of our study indicate that the predominant bacterial populations in the intestine of the three pandas were markedly different from that of herbivores. The unbalanced intestinal community structure may play a role in the inefficient digestion of bamboo by the giant pandas.     

Preliminary investigation on iodine nutrition in captive giant pandas

Background/ObjectiveThe giant panda belongs to the family Ursidae and, as a species of bear, still retains the simple digestive system of a Carnivoran. However, under the pressure of a specific habitat they had to adapt to a plant mono-diet consisting of bamboo with different species and growth stages around the year. A plant-based diet has relatively low iodine content with risk of iodine deficiency. Furthermore, bamboo contains cyanogenic glycosides releasing cyanide whose detoxification metabolite the thiocyanate acts as antagonist against iodine uptake and storage in the thyroid. To date very little is known about the iodine nutritional status of the giant panda, thus this study was conducted to receive the first information about the iodine nutrition of captive giant panda.Subjects/MethodsHere we investigated the iodine content of bamboo with different plant parts/vegetation stage and species and further compounds of the captive giant panda diet. Next, the urinary iodine (UI) and urinary thiocyanate (UT) levels of infant, sub-adult, adult and geriatric captive giant pandas was measured during the periods when the pandas consume both bamboo leaves- and culm (bamboo leaf-culm stage). Afterwards, the UI of 19 adult giant pandas was measured again for the different iodine intake during bamboo shoot stage. Finally, in this study part also the fecal iodine concentration was analyzed for calculation of total iodine excretion in relation to the iodine intake.ResultsBamboo leaves had the highest iodine content (453 μg/kg dry matter (DM)), followed by the shoots (84 μg/kg DM, p < 0.05), while bamboo culm had the lowest value (12 μg/kg DM, p < 0.05). During bamboo leaf-culm stage, giant pandas of different age groups had different UI and UT levels (p < 0.05). Furthermore, UI and UT were positively correlated among sub-adult, adult and geriatric giant pandas (p < 0.05). In adult giant pandas during bamboo shoot stage, the iodine excretion in feces was not different from that in urine while their total iodine excretion was less than their iodine intake (p < 0.05). Moreover, during bamboo shoot stage, the UI level of adult giant pandas was much lower than noted during bamboo leaf-culm stage (p < 0.05).ConclusionsOur results indicate that UI of captive giant pandas was related to their age as well as to the vegetation stage/part of bamboo they consumed reflecting a different periodic iodine supply. Thiocyanate and fecal excretion should be emphasized when considering the iodine nutrition of giant pandas.     

野生大熊猫现状、面临的挑战及展望

截至2003年底,我国野生大熊猫种群数量达1596只,分布在陕西、四川和甘肃3省的45个县境内,总栖息地面积达2304991hm^2。与第2次大熊猫调查相比,野生大熊猫生存状况已得到改善,分布范围扩大、栖息地面积增加、种群数量进一步增长。本文在第3次大熊猫调查的基础上,就野生大熊猫种群及栖息地现状进行了分析,指出未来保护大熊猫所面临的3个方面的挑战,即来自物种自身生物学特性的挑战、栖息地破碎化及隔离小种群未来命运的挑战以及大熊猫保护与社区经济发展需求相冲突的挑战。作者还就我国大熊猫保护前景进行了展望,即自然保护区数量将进一步增加,栖息地状况将进一步改善;种群数量在总体保持稳定的基础上将逐步增长,但局部小种群灭绝风险将加剧;圈养种群将形成能自我维持的种群,圈养个体通过培训将逐步放归到隔离野生小种群中以改变其命运。     

Non-invasive giant panda paternity exclusion

Giant panda hair samples obtained by noninvasive methods served as a source of DNA for amplification of seven giant panda microsatellite loci utilizing the polymerase chain reaction. Thirteen giant pandas held in Chinese zoos were tested for identification of paternity. Some males listed as sires have been excluded as the biological father of captive-born giant pandas. Because of the death of some potential sires, paternity is still not assigned for some giant pandas, although there is a high likelihood that paternity assignment could be made if postmortem samples are available for genetic analysis. The DNA microsatellite variation assayed by the test we have developed provides a rapid, highly informative, and noninvasive method for paternity identification in giant pandas. © 1994 Wiley-Liss, Inc.     

圈养大熊猫食谱组成与营养成分分析

为了探讨圈养大熊猫主食竹食谱组成及主要影响因素,并为圈养单位提供科学合理的食物营养配比和饲养管理建议。本研究通过对成都大熊猫繁育研究基地１０ 只成年大熊猫４ 年（２００９. ０３ －２０１３.０２）的饲养数据,３只成年大熊猫１ 年（２０１２. ０７ －２０１３. ０６）的觅食行为观察数据,以及投饲的３ 种竹笋和５ 种主食竹常规养分和总黄酮含量的统计分析。结果发现,基地圈养大熊猫偏好竹笋,喜食巴山木竹叶、白夹竹茎和苦竹茎,随机采食箬竹叶和刺竹茎;基地大熊猫根据投饲竹种类和季节的变化,形成了较为稳定的食谱组成;对投饲竹各部位成分比较分析发现,投饲竹养分含量的变化呈现出一定的规律性：其中粗蛋白、粗脂肪、蛋白质和能量比都以竹叶最高,竹枝次之,竹茎最低;干物质和粗纤维的含量则以竹茎最高,竹枝次之,竹叶最少;竹茎和竹枝呈现钙少磷多的现象,而竹叶则是钙多磷少;各投饲竹不同部位总黄酮含量以叶中最高,枝中其次,茎中最低。研究结果表明,圈养大熊猫主食竹食谱的选择遵循能量和营养学的规律,选择适口性强、营养价值高的食物假说。     

Population genetics reveals high connectivity of giant panda populations across human disturbance features in key nature reserve

The giant panda is an example of a species that has faced extensive historical habitat fragmentation, and anthropogenic disturbance and is assumed to be isolated in numerous subpopulations with limited gene flow between them. To investigate the population size, health, and connectivity of pandas in a key habitat area, we noninvasively collected a total of 539 fresh wild giant panda fecal samples for DNA extraction within Wolong Nature Reserve, Sichuan, China. Seven validated tetra‐microsatellite markers were used to analyze each sample, and a total of 142 unique genotypes were identified. Nonspatial and spatial capture–recapture models estimated the population size of the reserve at 164 and 137 individuals (95% confidence intervals 153–175 and 115–163), respectively. Relatively high levels of genetic variation and low levels of inbreeding were estimated, indicating adequate genetic diversity. Surprisingly, no significant genetic boundaries were found within the population despite the national road G350 that bisects the reserve, which is also bordered with patches of development and agricultural land. We attribute this to high rates of migration, with four giant panda road‐crossing events confirmed within a year based on repeated captures of individuals. This likely means that giant panda populations within mountain ranges are better connected than previously thought. Increased development and tourism traffic in the area and throughout the current panda distribution pose a threat of increasing population isolation, however. Maintaining and restoring adequate habitat corridors for dispersal is thus a vital step for preserving the levels of gene flow seen in our analysis and the continued conservation of the giant panda meta‐population in both Wolong and throughout their current range.     

圈养大熊猫血清微量元素含量的测定与分析

正微量元素包括Fe、Cu、Cr、Mn和Zn等,虽含量极微,但具有极重要的生理功能,涉及机体的生长发育、免疫代谢等生命活动过程(Shenkin,2008; Subrahmanyam et al.,2015),也是反映机体新陈代谢及生理功能状况的重要指标(Carpene et al.,2017)。例如Fe元素参与机体氧的运输和贮存(Abbaspour et al.,2014),血清铁被认为是动物营养评估的生化指标(Ganapathy et al.,2011); Cu元素是多种氧化酶的重要组分,参与动物新陈代谢、生长发育等(Desai et al.,2014)。     

大熊猫粪便乳酸菌的分离鉴定

大熊猫作为国家保护动物,其健康问题备受瞩目。为了维护大熊猫的肠道健康,本研究从大熊猫肠道内分离出适宜于大熊猫肠道环境的乳酸菌菌株,有望将其制成熊猫肠道微生物制剂,从而改善大熊猫肠道菌群环境。从雅安市宝兴县蜂桶寨自然保护区选取圈养与野生大熊猫的粪便,通过体外培养分离出9个菌株。分离菌株经过革兰氏染色镜检、过氧化氢产气、菌落形态观察等方法与技术初步鉴定为乳酸菌。对这9株乳酸菌进行耐酸试验、耐胆盐试验、抑菌能力试验和产酸能力等测试,筛选出了3个适应性较强,有望制成调节大熊猫肠道内环境平衡作用的微生态菌剂的菌株。16S rRNA基因序列分析表明:分离菌株J1、J2和J4分别为融合魏斯氏菌（Weissella confusa）,海氏肠球菌（Enterococcus heynei）和非解乳糖链球菌（Streptococcus alactolyticus）,有望被应用于大熊猫肠道微生态制剂的研究。     

Characterizing the spatial distribution of giant pandas (Ailuropoda melanoleuca) in fragmented forest landscapes

Aim To examine the effects of forest fragmentation on the distribution of the entire wild giant panda (Ailuropoda melanoleuca) population, and to propose a modelling approach for monitoring the spatial distribution and habitat of pandas at the landscape scale using Moderate Resolution Imaging Spectro‐radiometer (MODIS) enhanced vegetation index (EVI) time‐series data. Location Five mountain ranges in south‐western China (Qinling, Minshan, Qionglai, Xiangling and Liangshan). Methods Giant panda pseudo‐absence data were generated from data on panda occurrences obtained from the third national giant panda survey. To quantify the fragmentation of forests, 26 fragmentation metrics were derived from 16‐day composite MODIS 250‐m EVI multi‐temporal data and eight of these metrics were selected following factor analysis. The differences between panda presence and panda absence were examined by applying significance testing. A forward stepwise logistic regression was then applied to explore the relationship between panda distribution and forest fragmentation. Results Forest patch size, edge density and patch aggregation were found to have significant roles in determining the distribution of pandas. Patches of dense forest occupied by giant pandas were significantly larger, closer together and more contiguous than patches where giant pandas were not recorded. Forest fragmentation is least in the Qinling Mountains, while the Xiangling and Liangshan regions have most fragmentation. Using the selected landscape metrics, the logistic regression model predicted the distribution of giant pandas with an overall accuracy of 72.5% (κ = 0.45). However, when a knowledge‐based control for elevation and slope was applied to the regression, the overall accuracy of the model improved to 77.6% (κ = 0.55). Main conclusions Giant pandas appear sensitive to patch size and isolation effects associated with fragmentation of dense forest, implying that the design of effective conservation areas for wild giant pandas must include large and dense forest patches that are adjacent to other similar patches. The approach developed here is applicable for analysing the spatial distribution of the giant panda from multi‐temporal MODIS 250‐m EVI data and landscape metrics at the landscape scale.     

唐家河自然保护区大熊猫种群数量及栖息地的移动

本文根据大熊猫季节性活动规律特点,应用海拔高度路线调查法,全面收集唐家河自然保护区大熊猫的比较新鲜粪便,计获１２４个粪样材料,提取ＤＮＡ作ＤＮＡ指纹图,经微机检测,保护区有大熊猫３７只,个体数量与粪样采集量之比为１∶３３５１４,有６个家系,雌兽２１只,雄兽１６只。并搞清了保护区生态环境质量,箭竹遭受病虫害的程度,洪水对栖息地的影响,特别对引起大熊猫数量减少及栖息地移动等原因,作了较为详细的分析。     

从野外大熊猫的粪便估计年龄及其种群年龄结构的研究

本文对野外大熊猫粪便中的竹秆咬节及切缘与竹叶残片量度和破碎状况的研究,并以此对已知年龄个体的牙齿切片和齿冠磨损程度进行验证,发现通过粪便分析,可将野外大熊猫种群,大体划分为4个年龄组。     

大熊猫胃肠道中消化酶活力的分析

为了研究大熊猫对食物的化学性消化特点和机制,测定了9只大熊猫唾液和3只大熊猫胃肠道中主要消化酶的活力,并与其他动物进行了比较.结果显示,大熊猫唾液呈碱性,蛋白酶和淀粉酶等消化酶活力低;肠道中淀粉酶活力高,而脂肪酶活力明显低于棕熊.大熊猫小肠粘膜中存在显著量的蔗糖酶、乳糖酶和麦芽糖酶活力.另外,在1只大熊猫胃和直肠液中检测到了少量纤维素酶活力.研究结果提示,大熊猫唾液直接参与食物消化的作用可能很弱;大熊猫对淀粉类食物有很好的消化能力,但对脂肪类食物消化能力相对不高.大熊猫胃肠道消化酶的活力特点适应其消化天然食物中的营养物质.     

大熊猫与同域动物在海拔分布上的生态位分化

大熊猫（Ailuropoda melanoleuca）是中国的特有物种,其成功放归可能受放归地气候、栖息地、同域动物等多种因素的影响。本研究在野外调查的基础上,基于生态位理论等方法,探讨了栗子坪国家级自然保护区大熊猫与其大中型同域动物的空间关系。调查结果表明,该保护区内大熊猫同域动物共20种,分别属于2纲5目,其中东洋型分布物种占优势（30%）。大熊猫同域动物分布海拔显著低于大熊猫的分布海拔（P＜0.05）,大熊猫分别与灵长目等5个目的动物在海拔分布上均存在显著的生态位分化（P＜0.001）。灵长目动物在空间分布上与大熊猫分布相异,而其他目的动物与大熊猫空间分布类似。偶蹄目动物痕迹数量占比最高（54.14 %）,而食肉目的小熊猫与大熊猫样线共同遇见率达到了43.75%。本研究结果表明,该地大熊猫同域动物较为丰富,在海拔分布上与大熊猫存在生态位分化。该研究可为圈养大熊猫的放归提供参考依据。     

基于线粒体12S对中国两大山系大熊猫西氏贝蛔虫种群遗传多样性的分析

大熊猫是中国特有的珍稀濒危物种,而西氏贝蛔虫是大熊猫体内最为常见的肠道寄生虫,对野生和圈养大熊猫危害极大。考虑到大熊猫因分布区域的差异而形成了不同的亚种以及寄生虫与宿主间广泛的协同进化关系,西氏贝蛔虫是否也存在与大熊猫相适应的亚种分化一直是野生动物学家极其关注和热议的话题。为此,本文选择中国两大山系（岷山和邛崃）大熊猫种群体内共计34株西氏贝蛔虫虫体样本进行种群遗传多态性研究。利用PCR技术扩增出了岷山（14株）和邛崃（20株）西氏贝蛔虫的线粒体12S基因全序列并对其做了遗传多样性分析。结果表明：（1）34个样本包含9个单倍型,呈现出一个高单倍性多样性和低核苷酸多样性的特点;（2）负的Tajima's D和Fu's Fs中性检验值及“多峰型”的种群歧点分布图暗示种群不久前曾经历过突增长的现象;（3）低的种群间的分化系数和高的基因流表明两个地理种群间未形成显著的遗传分化;（4）系统发育树和单倍型网络图表明两山系种群分布无区域特异性。因此,岷山和邛崃山系的大熊猫体内的西氏贝蛔虫种群遗传变异性较低,分化不明显。该发现不仅暗示了西氏贝蛔虫与其宿主（大熊猫）的进化不同步,而且还为不同区域大熊猫西氏贝蛔虫病的监控提供了理论依据。     

Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls

Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.