The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of intenpecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined within vim development in ligated rabbit oviduct, achieved higher blastocyst development thanin vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the intenpecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not speciesspecific; (ii) there is compatibility between intenpecies somatic nucleus and ooplasm during early development of the reconstructed egg.     

The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of interspecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined with in vivo development in ligated rabbit oviduct, achieved higher blastoeyst development than in vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (     

岷山地区大熊猫(Ailuropoda melanoleuca)生境影响因子连通性及主导因子

岷山地区是大熊猫保护的关键地区。运用图论分析法研究了岷山地区大熊猫生境影响因子,及其相互作用关系。结果表明：在岷山地区,12个影响因子66对组合中,47.0％的影响因子间存在直接的联系,其中89.2％的影响具有增大效应,这说明人类活动对大熊猫生境的影响具有协同增大效应,并占主导地位。研究发现,在影响因子关系集中,存在强连通性的子集K＝{ (TD);(RB);(MI); (HPL);(AP);(AD);(SR);(TH);（FC）},这表明近期进行的旅游景点开发,公路建设,采矿,高压电线走廊建设,以及传统的农业开发, 畜牧业, 薪柴采集等影响因子之间存在着强烈相互作用关系,这些人类活动还会将其他人类活动对大熊猫生境的影响放大,加剧其对大熊猫生境的不利影响,是影响岷山地区大熊猫生境的主导因素。研究还表明如果不能对主导因子进行有效的控制,很难有效地保护岷山地区大熊猫生境。研究表明图论分析法是研究大熊猫生境影响因子的一个有效工具,有助于明确影响熊猫生境的主导和关键因子,为制定有效的大熊猫保护策略提供科学依据。     

岷山、邛崃山山系大熊猫栖息地生态安全综合评价

【目的】岷山与邛崃山系是大熊猫的重要栖息地,但由于该地区干扰、栖息地破碎化等原因,野生大熊猫的生存和种群复壮仍然存在风险。基于大熊猫习性和生存需求来评价其栖息地生态安全,对于更好地保护野生大熊猫种群、明确保护的地理重点,具有重要现实意义。【方法】以岷山山系、邛崃山山系范围内各市、区、县为对象,收集并选择最新的社会、地理、生物三大类目中具有代表性的12个因子作为评价指标,运用层次分析法和主成分分析法相结合的方法对该地区大熊猫栖息的生态安全进行综合评价。【结果】岷山、邛崃山中部的平武县、宝兴县的生态安全指数最高;彭州市、都江堰市和邛崃县等成都的周边地区生态安全指数较低,主要原因是人类活动的影响;都江堰市北部和汶川县域内为大熊猫的重要栖息地,但其生态安全指数较低,需要进一步的保护、管理和恢复;平武中部、松潘、茂县等地区生态安全指数较高,可作为大熊猫潜在栖息地进行保护。【结论】岷山大熊猫栖息地的生态安全指数总体上高于邛崃山系,但都存在明显的空间异质性。本研究结果可为野生大熊猫生态廊道的建设、圈养大熊猫放归地点选择等工作提供科学依据。     

Successful artificial insemination in the giant panda (Ailuropoda melanoleuca) at ueno zoo

Successful breeding of the giant panda (Ailuropoda melanoleuca) following artificial insemination was achieved at the Ueno Zoo in 2 consecutive years (1985 and 1986). The first cub, born in June 1985, unfortunately died 43 hours after birth from being crushed by the mother panda; the second cub, born in June 1986, has been growing in good health. Electroejaculation and artificial insemination procedures were performed after immobilization with diazepam (0.1 mg/kg) and ketamine HCL (4.0–5.0 mg/kg). Semen of the male panda was collected by electroejaculation using a rectal probe with a diameter of 2.0 cm and with eight rings as electrodes. Stimulation of the male was given with 3 V (30–40 mA) over a 5-sec period with 5-sec intervals. The female panda exhibited estrus between late February and early March in 1985 and also between mid-january and early February 1986. Increased excretion of urinary total estrogen showed coincidentally at maximum behavioral estrus, and a gradual rise of pregnanediol level was followed by artificial insemination. The gestational length for the first pregnancy was 110 days and that of the second 121 days.     

降低圈养大熊猫排粘液频率的措施初探

大熊猫(Ailuropoda melanoleuca)是中国特有的珍稀动物,在其整个消化道内膜上,都分布有丰富的粘液腺。在野外,这些粘液腺的分泌物包裹于大熊猫摄食的竹茎、竹叶和竹笋表面而排出体外;圈养条件下,常常见到这些分泌物积聚到一定数量而成团排出,这即被称为大熊猫的排粘液现象(王平     

岷山地区大熊猫(Ailuropoda melanoleuca)生境影响因子连通性及主导因子

岷山地区是大熊猫保护的关键地区.运用图论分析法研究了岷山地区大熊猫生境影响因子,及其相互作用关系.结果表明:在岷山地区,12个影响因子66对组合中,47.0%的影响因子间存在直接的联系,其中89.2%的影响具有增大效应,这说明人类活动对大熊猫生境的影响具有协同增大效应,并占主导地位.研究发现,在影响因子关系集中,存在强连通性的子集K={(TD);(RB);(MI);(HPL);(AP);(AD);(SR);(TH);(FC)},这表明近期进行的旅游景点开发,公路建设,采矿,高压电线走廊建设,以及传统的农业开发,畜牧业,薪柴采集等影响因子之间存在着强烈相互作用关系,这些人类活动还会将其他人类活动对大熊猫生境的影响放大,加剧其对大熊猫生境的不利影响,是影响岷山地区大熊猫生境的主导因素.研究还表明如果不能对主导因子进行有效的控制,很难有效地保护岷山地区大熊猫生境.研究表明图论分析法是研究大熊猫生境影响因子的一个有效工具,有助于明确影响熊猫生境的主导和关键因子,为制定有效的大熊猫保护策略提供科学依据.     

Sequencing,annotation and comparative analysis of nine BACs of the giant panda(Ailuropoda melanoleuca)

A 10-fold BAC library for the giant panda was constructed and nine BACs were selected to generate finish sequences.These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of the giant panda newly generated by Illumina GA sequencing technology.Complete Sanger sequencing,assembly,annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb.Homologue search and de novo prediction methods were used to ...     

甘肃白水江国家级自然保护区林缘社区饲养犬只对大熊猫时空节律的影响

人类对于野生动物的影响复杂多样, 其中家养动物所产生的干扰往往被忽视, 例如保护区林缘社区饲养的家犬。半散放的家犬(Canis familiaris)进入野生动物栖息地, 对保护区内以大熊猫(Ailuropoda melanoleuca)为代表的野生动物可能产生重要影响。为明确家犬进入林区活动范围对大熊猫的影响, 本研究采用红外相机技术和GPS项圈无线追踪技术对甘肃白水江国家级自然保护区内家犬对大熊猫潜在栖息地范围和时间节律的影响进行研究, 利用MaxEnt模型对家犬活动范围及大熊猫潜在栖息地进行预测。结果表明: 甘肃白水江国家级自然保护区内大熊猫潜在栖息地面积为885.8 km², 约占保护区总面积的48.2%; 家犬活动面积为861.2 km², 约占保护区总面积的47.6%。家犬活动范围与大熊猫潜在栖息地重叠的面积约占保护区面积的28.2%, 且多集中在低海拔的常绿阔叶林。大熊猫在有家犬出现的位点和没有家犬出现的位点日活动节律具有显著差异。本研究揭示了家犬进入林区对大熊猫栖息地潜在影响的空间范围, 为保护区开展生物多样性管理提供了关键信息, 对于其他保护区具有借鉴意义。     

大熊猫源肺炎克雷伯菌耐药性和分子分型研究

【目的】对大熊猫源肺炎克雷伯菌进行耐药性及分子分型分析,掌握肺炎克雷伯菌在大熊猫圈养种群中的耐药和流行情况,指导临床用药。【方法】对2018–2019年收集到的178株大熊猫源肺炎克雷伯菌使用纸片扩散法(K-B法)分析耐药表型,使用Wafergen Smartchip超高通量荧光定量PCR法分析其耐药基因和可移动遗传元件,使用多位点序列分型(MLST)法分析其序列类型(ST)。【结果】178株大熊猫源肺炎克雷伯菌对多西环素耐药率最高(15.2%),而且2019年分离到的肺炎克雷伯菌对头孢噻肟、亚胺培南和阿奇霉素的耐药性显著高于2018年(P<0.05);检出耐药基因106种(106/227),可移动遗传元件11种(11/19),涉及的耐药机制主要为外排泵(42.0%)、抗生素失活(41.8%)和改变作用靶位(16.2%);MLST分型显示大熊猫源肺炎克雷伯菌主要分为42个不同的ST型,且ST型与耐药性具有一定的相关性。【结论】从2018年到2019年大熊猫源肺炎克雷伯菌对β-内酰胺类和大环内酯类抗生素耐药率有所增加,耐药机制以外排泵和抗生素失活为主。ST17、ST23和ST400...     

Cloning, sequence, and function analyses of giant panda (Ailuropoda melanoleuca) CD9 gene

CD9 is a member of the tetraspanin family proteins and has recently been shown to be essential for sperm-oocyte fusion in mice. The giant panda (Ailuropoda melanoleuca) CD9 (gpCD9) cDNA was amplified for the first time by RT-PCR from ovary total RNA and cloned, sequenced and analyzed. The result revealed that the open reading frame (ORF) of gpCD9 was 681 bp, which has the same length as that of mouse. Sequence analysis and structure prediction displayed that the amino acid sequence of gpCD9 is over 80% identity to those of mammals with the conserved structures, including the four transmembrane domains (TM) and certain characteristic residues. The results of sperm-egg fusion experiments demonstrated that giant panda CD9 large extracellular loop (LEL) significantly inhibited (P < 0.05) the mouse gamete fusion when the recombinant protein was added. However, when three amino acid residues TVT (173-175) of the gpCD9 were mutated to AAA, the large extracellular loop (LELM) of mutated protein was rarely inhibiting the gamete fusion of mice. Our results may be useful in improving an insight into understanding the potential mechanism of gamete fusion and genetic characteristics of giant panda.     

秦岭大熊猫种群扩散格局及研究方法

种群扩散格局是研究种群扩散规律和机制的关键信息,也是制定物种保护对策的重要基础。大型动物种群扩散格局研究方法为扩散生态学研究的薄弱领域,并制约扩散生态学的发展。以秦岭大熊猫为研究对象,根据2000年以来的种群调查数据,基于大熊猫领域的特性,利用GIS的扩展区分析功能和景观分析方法研究了大熊猫种群分布区及动态;基于聚集的特性,利用GIS的核密度分析功能对大熊猫种群多度和聚集状况及空间变化进行了分析。发现2012年的秦岭大熊猫种群分布区较2000年增加5.5%(即15307.8hm~2),高密度种群聚集区从2处变成1处,种群聚集程度进一步增加、聚集格局的完整性大大提升,尤以中密度聚集区增长最显著,种群格局呈明显的分布区扩张、聚集度增加的态势。表明基于物种的生物学特性,立足于种群分布和多度格局变化,通过长期调查和监测可以有效掌握物种的种群扩散格局;大型动物可根据其生物学特性探索可行的方法与量化种群扩散的参数来研究其扩散格局,从而促进大型动物种群扩散研究的开展。     

Hopes and challenges for giant panda conservation under climate change in the Qinling Mountains of China

One way that climate change will impact animal distributions is by altering habitat suitability and habitat fragmentation. Understanding the impacts of climate change on currently threatened species is of immediate importance because complex conservation planning will be required. Here, we mapped changes to the distribution, suitability, and fragmentation of giant panda habitat under climate change and quantified the direction and elevation of habitat shift and fragmentation patterns. These data were used to develop a series of new conservation strategies for the giant panda. Qinling Mountains, Shaanxi, China. Data from the most recent giant panda census, habitat factors, anthropogenic disturbance, climate variables, and climate predictions for the year 2050 (averaged across four general circulation models) were used to project giant panda habitat in Maxent. Differences in habitat patches were compared between now and 2050. While climate change will cause a 9.1% increase in suitable habitat and 9% reduction in subsuitable habitat by 2050, no significant net variation in the proportion of suitable and subsuitable habitat was found. However, a distinct climate change‐induced habitat shift of 11 km eastward by 2050 is predicted firstly. Climate change will reduce the fragmentation of suitable habitat at high elevations and exacerbate the fragmentation of subsuitable habitat below 1,900 m above sea level. Reduced fragmentation at higher elevations and worsening fragmentation at lower elevations have the potential to cause overcrowding of giant pandas at higher altitudes, further exacerbating habitat shortage in the central Qinling Mountains. The habitat shift to the east due to climate change may provide new areas for giant pandas but poses severe challenges for future conservation.     

Characteristics and in vitro fertilizing ability of giant panda (Ailuropoda melanoleuca) frozen‐thawed epididymal spermatozoa obtained 4 hours postmortem: A case report

When Chu‐Lin, a male giant panda (studbook #249), died at Madrid Zoo, his reproductive tract was removed 4 hr postmortem and the epididymal spermatozoa were collected. Extended sperm were kept at 5°C for 4 hr, loaded into straws, and frozen for 7 min in liquid nitrogen vapor before the straws were plunged into liquid nitrogen. Two straws were thawed and evaluated. Sperm motility was assessed in fresh, refrigerated, and thawed spermatozoa (75%, 60%, 35%, respectively). Sperm viability and acrosome status were estimated using a triple‐stain technique (TST). The results showed 33% live sperm with intact acrosomes after thawing. A hypoosmotic swelling (HOS) test demonstrated the retention of membrane integrity in 72% of thawed sperm. To evaluate the in vitro fertilizing ability of thawed sperm, a sperm penetration assay (SPA) was performed. The values obtained for the percentage of penetration and the penetration index were 62% and 1.78 sperm/oocyte, respectively. The results obtained demonstrate that epididymal sperm recovered from a giant panda postmortem can be successfully cryopreserved. The sperm fertilizing ability demonstrated in vitro after thawing may provide a final opportunity for this male to contribute to the currently small germplasm reserves of this endangered species, and to reproduce in the future through assisted reproductive technology. Zoo Biol 23:279–285, 2004. © 2004 Wiley‐Liss, Inc.     

海拔对岷山大熊猫主食竹营养成分和氨基酸含量的影响

缺苞箭竹(Fargesia denudata)是岷山山系大熊猫的主食竹,其可食性在大熊猫食物营养质量评价中具有重要意义。为揭示海拔梯度上温差对大熊猫主食竹营养成分及其含量的影响,以自然生长在岷山山系核心区四川王朗国家级自然保护区不同海拔(2600、2850、3100 m)的缺苞箭竹为对象,对其笋、秆和叶的营养成分和氨基酸含量进行了研究。结果表明:(1)同一海拔缺苞箭竹的粗蛋白和粗脂肪含量均表现为叶笋秆,且笋、秆和叶的营养成分含量有显著差异(P0.05);(2)粗蛋白和氨基酸总量随海拔升高而显著增加(P0.05),而对粗脂肪和粗纤维的影响不显著(P0.05);(3)除笋中苏氨酸、甘氨酸、半胱氨酸,秆中丝氨酸、蛋氨酸以及叶中半胱氨酸外,海拔对其余氨基酸含量均有显著影响(P0.05);(4)海拔对粗蛋白/粗脂肪、粗蛋白/粗纤维有显著影响(P0.05),在笋和秆中表现为海拔3100 m最高,而海拔对纤维素/粗纤维、粗脂肪/粗纤维的影响不显著(P0.05)。缺苞箭竹叶、笋的营养成分含量明显高于秆;高海拔有利于缺苞箭竹各器官粗蛋白和氨基酸的积累,不利于粗纤维的积累,这可能使大熊猫的取食选择发生改变,更倾向于取食高海拔的笋和叶。     

凉山山系大熊猫和黑熊适宜生境预测及重叠分析

研究同域物种的分布格局及重叠状况对物种的区域整合保护管理及区域生物多样性保护具有重要实践价值。本研究基于全国第四次大熊猫调查及长期野外调查数据, 利用MaxEnt模型预测了凉山山系两种同域分布的熊科动物——大熊猫(Ailuropoda melanoleuca)和黑熊(Ursus thibetanus)的适宜生境, 基于适宜生境预测结果, 分析了两个物种的生境需求因子、生境破碎化现状及重叠状况。结果显示: (1)大熊猫和黑熊的适宜生境分布格局相似, 主要分布在凉山山系的山脊地带, 适宜生境面积分别为1,383.84 km²和2,411.49 km²; (2)两个物种的适宜生境都较为破碎, 且存在一些隔离分布区, 相较而言, 黑熊适宜生境的连通性要优于大熊猫; (3)两个物种生态位重叠度较高(D = 0.654, I = 0.901), 适宜生境重叠面积为958.29 km², 分别占大熊猫和黑熊适宜生境总面积的69.25%和39.74%; (4)两个物种对环境因子的选择和响应表现出了相似性和差异性。相似性在于对两个物种生境分布影响最大的两个因子均为距居民点距离和海拔; 差异性在于对大熊猫生境分布影响次之的因子是植被类型和最冷季均温, 而黑熊的是年最大EVI指数和距道路距离。为了更有效地保护两个物种, 应加强对人类干扰的控制和植被的恢复, 对栖息地实行连通管理, 并建立多物种保护规划。     

Sequencing,annotation and comparative analysis of nine BACs of giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.