卧龙大熊猫人工育幼技术的研究进展

简要回顾了大熊猫人工育幼的历史，总结了卧龙中国保护大熊猫研究中心在大熊猫人工育幼研究领域的发展。1991年全人工育幼使1只大熊猫弃仔存活160d，但之后连续几年人工育幼失败；1998年成功地将一只弃仔人工哺育2mo后，给不会带仔的母兽培训其带仔。1999年将2个双胞胎及1个三胞胎中的2仔通过人工育幼与母兽交替哺育存活，同时将2只自野外抢救回来的大熊猫幼仔人工哺育存活。创造了1a人工哺育8只幼仔存活的世界记录，大大提高了大熊猫幼仔的存活率。大熊猫人工育幼技术的关键在于；适宜的育幼环境，幼仔免疫力的获得，人工乳的配制，幼兽的疾病防治及正确的饲喂方式。现将卧龙大熊猫庆大熊猫人工育幼方面所取得的突破性进展总结如下。图2表2参6     

食物转化期的大熊猫幼仔肠道细菌结构衍变及其与纤维素酶活性的相关性

大熊猫是具有植食性转化阶段的特殊的食肉动物,但本身对纤维素的消化能力极低,主要依赖于肠道微生物的作用;处于食物转化阶段的大熊猫幼仔是敏感而脆弱的,对纤维素的适应消化能力也是保证其存活的关键.通过分析食物转化期大熊猫幼仔粪便中纤维素酶活性和肠道细菌多样性的变化,探讨大熊猫幼仔食物转化阶段肠道细菌性质和纤维素酶活性的相关性,以期为优化食物转化期大熊猫的饲养技术提供一定的理论指导.结果显示,大熊猫幼仔食物转化阶段纤维素酶活性变化较大,特别是增加竹子的摄入量后,大熊猫个体纤维素酶活性普遍显著增加.大熊猫肠道细菌的结构和数量在食物转化阶段也存在较大变化,且与幼仔消化纤维素密切相关,如Lactobacillus、Romboutsia等典型的肉食动物肠道菌的丰度在大熊猫幼仔肠道中数量减少,而纤维素降解菌属Cellulosiyticum的丰度明显增加,特别是大熊猫肠道优势菌属Streptococcus也表现出与其纤维素消化的密切联系.总体而言,食物转化阶段的大熊猫幼仔对纤维素有一定的适应性消化能力,其和肠道细菌菌群的演变有较大的相关性.(图3表6参30)     

使用冷藏精液进行大熊猫（Ailuropoda melanoleuca）人工授精

使用在4℃冷藏24 h和48 h的精液对5岁的雌性大熊猫妃妃进行了人工授精,以保持圈养大熊猫的遗传多样性. 供精者乐乐是一只患皮肤癌的雄性大熊猫,睾丸体积为(386.1±214) cm3,在2000-02-23～05-11期间,4次人工采精刺激电压5～8 V,射精电压5～8 V,每次采精量(3.2±1.2) mL,精子密度为(1 208.5±564.9)×106/mL-1,每次精子总量(3 908.3±2 546.6)×106,精液平均活力为84.8%±4.1%,活率为87.8%±5.2%,运动状态为2.9±0.25,畸形率为28.3%±11.1%,平均顶体正常率为96%±1%,pH值为7.9±0.3.乐乐的精液用TEST稀释后,在4℃冷藏24 h、48 h和72 h后,精子活力分别是65%,58%和47%,精子的活率分别为73%,73%和68%.用酶免法对雌兽妃妃尿液中雌激素的检测,发现妃妃的雌激素在发情前期开始增加,在发情前期的最后一天达到最大峰值,在发情期(4月3日～5日)迅速下降到正常值,进行人工授精的最佳时间是在雌兽尿液中的雌激素达到最大峰值后的48～72 h之间.妃妃在2000-08-10顺利地产下一仔,雌性,妊娠期127 d,初生幼仔179.3 g. 图1 表3 参19     

亚成体大熊猫肠道微生物结构的季节差异及其与肠道纤维素酶活性的相关性

大熊猫以低营养高纤维的竹子作为主要食物来源,但其消化系统结构保留了肉食动物的特点,不具备消化纤维素的能力,因此肠道菌群对其消化纤维素具有重要意义.为了解不同季节亚成体大熊猫肠道微生物结构的变化及其与肠道纤维素消化能力的内在联系,通过高通量测序技术分析4只亚成体大熊猫肠道细菌和真菌多样性的季节差异,并检测不同季节的肠道纤维素酶活性.结果发现不同季节亚成体大熊猫肠道细菌、真菌结构和丰富度存在较大差异.复杂的肠道微生物组成,特别是优势细菌结构主要决定了亚成体大熊猫消化利用纤维素的能力.冬季(2月)肠道微生物结构较复杂,微生物丰富度也相对较高,细菌的优势度高于其他季节.秋季(11月)和冬季(2月),亚成体大熊猫的纤维素酶活性较高,冬季纤维素酶活性约为其他季节的5倍.亚成体大熊猫的纤维素酶活与其肠道优势细菌Streptococcus丰度的季节变化具有相似的趋势,相关性系数为0.582,是大熊猫分解利用纤维素的重要贡献者.因此,亚成体大熊猫不同季节的肠道微生物结构演变对其纤维素消化具有重要影响.(图3表6参28)     

基于高通量测序技术研究成年大熊猫肠道菌群

选取6只成年健康大熊猫(Ailuropoda melanoleuca)、1只成年亚健康大熊猫,基于高通量测序技术,测定其肠道内细菌及真菌组成,研究性别和健康状况对成年大熊猫肠道微生物的影响,分析不同年龄段大熊猫肠道菌群的差异.结果表明,在门水平,成年大熊猫肠道内细菌主要为Proteobacteria和Firmicutes,真菌主要为Ascomycota和Basidiomycota.在属水平,成年大熊猫肠道细菌为Escherichia、Streptococcus、Clostridium及Leuconostoc,真菌为Humicola.雌性和雄性大熊猫肠道菌群没有显著性差异(P0.05),健康大熊猫肠道细菌Esherichia、Clostridium(53.33%、1.10%)的含量比亚健康大熊猫(2.48%、0.00%)高,Streptococcus(22.88%)的含量比亚健康大熊猫(48.15%)低,健康大熊猫肠道真菌的多样性高于亚健康大熊猫.与文献比较发现,大熊猫肠道内菌群多样性为成年老年亚成体,随年龄增长,成年时Streptococcus(22.88%)、Leuconostoc(2.97%)和Clostridium(1.10%)的含量升高,Shigella(6.25%)和Citrobater(2.50%)的含量降低,老年时Weissella(16.67%)的含量升高,Leuconostoc(2.97%)和Clostridium(1.10%)的含量降低.本研究结果表明身体健康状况和年龄对大熊猫肠道微生物菌群结构组成有影响,成年大熊猫性别对肠道菌群没有影响.     

佛坪三官庙地区大熊猫种群数量的DNA指纹分析

应用同位素标记大熊猫基因指纹探针Ｆ２ＺＧＰ９６０６０８０１，以秦岭南坡中段佛坪国家级大熊猫自然保护区三官庙范围内采集的大熊猫粪便样品作材料，进行了ＤＮＡ指纹检测．（１）在相同或不同时间、领域采得的粪便样品，显现出相同或不同的ＤＮＡ指纹图谱，达到个体认定的目的，进一步表明了大熊猫的尽量新鲜的粪便，可以作为ＤＮＡ指纹分析材料，进行野生种群数量调查．（２）根据检测２１个粪便样品的结果，无误地认定了三官庙地区有１３只大熊猫个体．其中有３个家系．（３）大熊猫粪便样品的ＤＮＡ指纹图，通过微机识别的个体数，准确可靠，能获得大熊猫在野外的真实个体数量．     

圈养大熊猫排泄的粘液成分分析

收集并测定了8只圈养大熊猫的12个粘液样品的营养成分含量,包括水分、粗蛋白(CP)、粗脂肪(CF)、6种维生素(VA、VE、VD、VB1、VB2和VB6)以及4种微量元素(Fe、Zn、Cu和Mn).结果表明,大熊猫排泄的粘液营养物质含量丰富,尤其是含有很高的蛋白质、丰富的维生素和微量元素.粘液排泄频繁和排泄量大的大熊猫将从粘液排泄中损失很多的营养物质.因此,一方面必须给粘液排泄频繁和排泄量大的大熊猫补充营养,另一方面有必要采取切实可行的措施降低大熊猫的粘液排泄量及其频率.表4参5     

野化培训大熊猫在特定时期采食半枯竹的原因分析

对首次发现的一只野化培训大熊猫“祥祥”采食半枯竹的现象及原因进行研究.作者用4只圈养大熊猫为对照进行试验,从行为观察、营养成分及适口因子、环境气候条件等方面进行了研究.研究发现,圈养大熊猫中也存在采食半枯竹的现象,而且在采食的时间段上和祥祥一致;相对于新鲜竹子,半枯竹中葡萄糖含量有所增加,单宁含量明显降低,并且具有特殊的香甜味,使在一定时期内的半枯竹营养增加,适口性得到改善,这是大熊猫在特定时期更愿意采食半枯竹而不采食新鲜竹子的重要原因;7~9月是全年温湿度最高月份,对引起半枯竹成分中适口因子的改变可能有诱发作用.图3表4参9     

野生大熊猫种群数量两种调查方法对比

准确调查野生大熊猫种群数量是大熊猫保护的基础.目前两种主要的大熊猫种群数量调查方法(距离-咬节区分法与分子生物学方法)在准确性、可操作性、成本等方面尚存在争议.在卧龙自然保护区大熊猫主要分布区域,同时利用距离-咬节区分法与分子生物学的方法估算大熊猫种群数量.结果表明,与分子生物学方法相比较,传统的距离-咬节区分法估算的结果相对保守,估算结果不到前者的50%.结合大熊猫移动模式特征,建议在大熊猫高密度分布区域适当降低距离区分阈值,以提高距离-咬节区分法的灵敏性.     

宝兴县大熊猫栖息地生态系统服务价值评估

生态系统服务价值(ESV)评估对衡量生态保护与经济发展具有指导意义.以大熊猫国家公园体制试点区域中的四川省宝兴县为例,利用当量因子法,评估县域尺度生态系统服务价值,比较宝兴县大熊猫栖息地、蜂桶寨保护区与全县单位面积生态系统服务价值差异,并通过敏感性系数(CS),对评估结果的合理性进行验证.结果显示:(1)2010年宝兴县大熊猫栖息地生态系统服务价值为52.46亿元,占总价值的62.73%;(2)蜂桶寨保护区单位面积生态系统服务价值最高,高出全县平均水平327.95元/hm~2,宝兴县大熊猫栖息地略低于蜂桶寨保护区,为27 221.72元/hm~2;(3)各土地利用类型敏感性系数均小于1,评估结果可信.本研究表明已设立的蜂桶寨保护区覆盖面有限,无法使宝兴县大熊猫栖息地得到最大程度的保护,若以大熊猫栖息地规划大熊猫国家公园范围,以62.03%的县域面积保护全县80.93%的森林资源,将更有利于保护大熊猫栖息地的完整性和原真性;研究结果对其他地区国家公园的规划和建设具有一定参考价值.(图4表4参39)     

圈养大熊猫乳汁营养成分分析及育幼营养对策初探

连续测定了１只１０ａ大熊猫产后１ｍｏ内１２个乳汁样品中水份、粗蛋白（ＣＰ）、粗脂肪（ＥＥ）、粗灰分（ＣＡ）、乳糖（Ｌａｃ）、１７种氨基酸（ＡＡ）、１０种矿物元素、６种维生素的含量．结果表明，大熊猫产后前６ｄ乳汁中多数营养素含量与６ｄ后含量差异明显．产后ｄ１乳汁中ｗ（ＣＰ）＝６．３４％，ｄ５～１５在５％左右；ｗ（ＥＥ）在２．５０％～７．３３％范围内变动；乳糖含量皆在１％左右；大多数维生素和微量元素含量远高于常见的家畜乳和人乳中相应成分的含量．在分析大熊猫乳汁营养成分含量特点的基础上，探讨了育纳营养对策．     

大熊猫与熊类动物性别的分子鉴定

根据已知动物的性别决定因子基因序列设计引物,以非性别特异性的脑源性神经营养因子基因片段（ＢＤＮＦ）作为正对照,用于扩增大熊猫、浣熊、天山棕熊和马来熊个体的ＳＲＹ（ｓｅｘｄｅｔｅｒｍｉｎｉｎｇｒｅｇｉｏｎｏｎＹｃｈｒｏｍｏｓｏｍｅ）基因片段并对其进行凝胶电泳分析,由此建立了一套简单、快速、可靠的动物性别分子鉴定方法．该方法不仅有利于尽快确定大熊猫等珍稀濒危动物的性别,而且也可以用于野外种群的性别比例调查．     

Proposed Conservation Landscape for Giant Pandas in the Minshan Mountains,China

Abstract: The giant panda (Ailuropoda melanoleuca), is one of the world's most endangered species. Habitat loss and fragmentation have reduced its numbers, shrunk its distribution, and separated the population into isolated subpopulations. Such isolated, small populations are in danger of extinction due to random demographic factors and inbreeding. We used least‐cost modeling as a systematic approach to incorporate satellite imagery and data on ecological and behavioral parameters of the giant panda collected during more than 10 years of field research to design a conservation landscape for giant pandas in the Minshan Mountains. We identified 8 core habitats and 4 potential linkages that would link core habitats CH3, CH4, and CH5 with core habitats CH6, CH7, and CH8. Establishing and integrating the identified habitats with existing reserves would create an efficient reserve network for giant panda conservation. The core habitats had an average density of 4.9 pandas/100 km² and contained approximately 76.6% of the giant panda population. About 45% of the core habitat (3245.4 km²) existed outside the current nature reserves network. Total estimated core habitat decreased between 30.4 and 44.5% with the addition of residential areas and road networks factored into the model. A conservation area for giant panda in the Minshan Mountains should aim to ensure habitat retention and connectivity, improve dispersal potential of corridors, and maintain the evolutionary potential of giant pandas in the face of future environmental changes.     

Application of least-cost path model to identify a giant panda dispersal corridor network after the Wenchuan earthquake—Case study of Wolong Nature Reserve in China

With growing levels of human-activity and frequent natural disturbances throughout the world, it is increasingly important that both research and management efforts take into account the widespread landscape fragmentation and its consequences for biodiversity conservation. The magnitude 5.12 Wenchuan earthquake in China caused dramatic impacts on giant panda (Ailuropoda melanoleuca) habitat in the nature reserves within Minshan and Qionglai mountains. With the combined stresses of the natural disaster and the extensive human activities during postquake reconstruction, giant panda habitat in this region may become more fragmented in the future. In order to preserve the giant panda population after the earthquake and protect the species against habitat fragmentation, this article explores a method of identifying giant panda migration corridors involving habitat suitability assessments and a least-cost path model. Focusing on postquake Wolong Nature Reserve, our results demonstrate that it contains 430.3 km² of suitable habitat (21.1% of total area), 463.8 km² of marginally suitable habitat (22.8%) and 1141.9 km² of unsuitable habitat (50.1%). We further show that several giant panda dispersal corridors exist in the reserve, including four corridor groups that cross the provincial highway and five corridors that do not intersect areas of human activity. This study will contribute to management and conservation efforts in Wolong Nature Reserve and beyond after the Wenchuan earthquake.     

Characteristics that make trophy hunting of giant pandas inconceivable

In November 1928, Theodore Jr. and Kermit Roosevelt led an expedition to China with the expressed purpose of being the first Westerners to kill the giant panda (Ailuropoda melanoleuca). The expedition lasted 8 months and resulted in the brothers shooting a giant panda in the mountains of Sichuan Province. Given the concurrent attention in the popular press describing this celebrated expedition, the giant panda was poised to be trophy hunted much like other large mammals around the world. Today, however, the killing of giant pandas, even for the generation of conservation revenue, is unthinkable for reasons related to the species itself and the context, in time and space, in which the species was popularized in the West. We found that the giant panda's status as a conservation symbol, exceptional charisma and gentle disposition, rarity, value as a nonconsumptive ecotourism attraction, and endemism are integral to the explanation of why the species is not trophy hunted. We compared these intrinsic and extrinsic characteristics with 20 of the most common trophy-hunted mammals to determine whether the principles applying to giant pandas are generalizable to other species. Although certain characteristics of the 20 trophy-hunted mammals aligned with the giant panda, many did not. Charisma, economic value, and endemism, in particular, were comparatively unique to the giant panda. Our analysis suggests that, at present, exceptional characteristics may be necessary for certain mammals to be excepted from trophy hunting. However, because discourse relating to the role of trophy hunting in supporting conservation outcomes is dynamic in both science and society, we suspect these valuations will also change in future.     

A Framework for Evaluating the Effects of Human Factors on Wildlife Habitat: the Case of Giant Pandas

Abstract: To address the complex interactions between humans and wildlife habitat, we developed a conceptual framework that links human factors with forested landscapes and wildlife habitat. All the components in the framework are integrated into systems models that analyze the effects of human factors and project how wildlife habitat would change under different policy scenarios. As a case study, we applied this framework to the Wolong Nature Reserve in Sichuan Province (southwestern China), the largest home of the giant panda ( Ailuropoda melanoleuca ). We collected ecological and socioeconomic data with a combination of various methods ( field observations, aerial photographs, government documents and statistics, interviews, and household surveys) and employed geographic information systems and systems modeling to analyze and integrate the data sources. Human population size has increased by 66% and the number of households in the reserve has increased by 115% since 1975, when the reserve was established. During the same period, the quality and quantity of the giant panda habitat dramatically decreased because of increasing human activities such as fuelwood collection. Systems modeling predicted that under the status quo, human population in the reserve would continue to grow and cause more destruction of the remaining panda habitat, whereas reducing human birth rates and increasing human emigration rates would lower human population size and alleviate human impacts on the panda habitat. Furthermore, our simulations and surveys suggested that policies encouraging the emigration of young people would be more effective and feasible than relocating older people in reducing human population size and conserving giant panda habitat in the reserve.     

野化培训大熊猫采食和人为砍伐对拐棍竹无性系种群生物量的影响

采用固定样方法和定位观察法连续测定了2003～2007年间卧龙自然保护区大熊猫野化培训圈内及其附近区域的拐棍竹无性系种群数量和生长发育特性等参数,运用收获法与非破坏性重量估测法建立了不同龄级和残桩的竹子种群和分株生物量估测模型,进而利用最佳模型计算并评估了野化培训大熊猫采食和人为砍伐对拐棍竹无性系种群生物量和植株个体生物量的影响.结果表明:在环境条件、种群密度、生长发育特征和种群生物量等基本相似的基础上,大熊猫采食和人为砍伐不仅降低实验期间的竹子生物生产力,而且影响到后期阶段实验种群的恢复与发展.大熊猫采食样方中的竹子种群生物量虽然较对照样方低,就竹笋生物量而言,约为对照的57.79%,这与野化培训圈的面积较小、竹种单一而使采食比重(67.07%)较大有关,但其各龄级植株个体生物量均能达到大熊猫的取食利用标准(仅2004年生竹除外),具有持续供给大熊猫食物资源的潜力;而人为砍伐措施与大熊猫采食相比,影响效果极为强烈,它严重降低了拐棍竹无性系的种群生物量,尤其是竹笋重量更是如此,仅为对照样方的14.69%,且植株个体鲜重远低于大熊猫的觅食条件.因此,竹笋和无性系植株的生物量是大熊猫采食标准的主要因素.     

Patterns of Genetic Diversity in Remaining Giant Panda Populations

Abstract: The giant panda ( Ailuropoda melanoleuca ) is among the more familiar symbols of species conservation. The protection of giant panda populations has been aided recently by the establishment of more and better-managed reserves in existing panda habitat located in six mountain ranges in western China. These remaining populations are becoming increasingly isolated from one another, however, leading to the concern that historic patterns of gene flow will be disrupted and that reduced population sizes will lead to diminished genetic variability. We analyzed four categories of molecular genetic markers (mtDNA restriction-fragment-length polymorphisms [RFLP], mtDNA control region sequences, nuclear multilocus DNA fingerprints, and microsatellite size variation) in giant pandas from three mountain populations (Qionglai, Minshan, and Qinling) to assess current levels of genetic diversity and to detect evidence of historic population subdivisions. The three populations had moderate levels of genetic diversity compared with similarly studied carnivores for all four gene measures, with a slight but consistent reduction in variability apparent in the smaller Qinling population. That population also showed significant differentiation consistent with its isolation since historic times. From a strictly genetic perspective, the giant panda species and the three populations look promising insofar as they have retained a large amount of genetic diversity in each population, although evidence of recent population reduction—likely from habitat loss—is apparent. Ecological management to increase habitat, population expansion, and gene flow would seem an effective strategy to stabilize the decline of this endangered species.     

Reproductive competition and fecal testosterone in wild male giant pandas (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

The giant panda (Ailuropoda melanoleuca) is notoriously difficult to study in the wild, but its interesting reproductive ecology makes the effort worthwhile. Perhaps more than most species, the panda is energy-limited, which alters the cost/benefit analysis of its reproductive ecology. Using global positioning system/very high frequency radiocollars to locate mating aggregations, we used behavioral observations and fecal testosterone assays to gain insight into male panda reproductive effort and strategies, and test theories relating to reproductive competition. Male pandas initially competed fiercely for access to females that were about to be fertile, but once male competitive status was determined, aggression rates declined. Contact aggression was only observed during the first 2 days of mating aggregations; thereafter, it was replaced with noncontact aggression and avoidance. Agonistic interactions were highly asymmetrical, with contest losers (subordinates) showing less aggression and more avoidance than contest winners (dominants), both before and after contest outcome was established. The competitively superior male displayed mate-guarding tactics and secured all observed copulations. Contrary to theoretical predictions, testosterone levels did not predict aggression levels or contest winners and also were not affected by winning or losing a contest. Body size appeared to be the primary determinant of contest outcome. We discuss our findings in light of theoretical predictions, such as those arising from the “challenge hypothesis,” in the context of the giant panda’s foraging and nutritional ecology.