Novel environmental enrichment may provide a tool for rapid assessment of animal personality: a case study with giant pandas (Ailuropoda melanoleuca)

Historically, the assessment of nonhuman animal personality has included a variety of methods--from direct behavioral observations in a variety of test situations to assessments provided by animal caretakers or trainers. Careful observation of how animals in zoos interact with novel enrichment may provide reliable insight into their personality. This study sought to describe a process for evaluating whether different methods of assessing personality result in similar conclusions. The study exposed 4 giant pandas at the Smithsonian National Zoological Park and Zoo Atlanta to 10 novel enrichment items and recorded their behavior. Keepers also rated each panda on 23 behavioral characteristics on a survey. The study obtained individual behavior profiles for each panda. Significant differences across individuals in both the novel enrichment trials and keeper surveys formed the basis for the profiles. These methods also provided some insight into differences between the sexes that--based on the natural history of giant pandas--are qualitatively similar to what would be expected. The study found some consistency between assessment methods. However, there is a need for further study to validate these measures in a larger sample of giant pandas.     

基于高分辨率网络的大熊猫姿态估计方法

对圈养大熊猫 (Ailuropoda melanoleuca) 开展长期行为监测能及时了解其所处生理周期和健康状况,有助于繁殖饲养机构迅速采取相应繁育保护措施提高饲养管理水平,但目前无法对大熊猫进行24 h监控并及时地获得相应的行为信息。准确的动物姿态估计是动物行为研究的关键,也是诸多下游应用的基础。了解大熊猫的姿态可以促进大熊猫行为研究并提升保护管理水平。为了提高复杂环境下大熊猫姿态估计的准确率,本文以高分辨率网络 (High resolution net, HRNet) 为基础网络架构提出了一种大熊猫姿态估计方法：针对大熊猫不同部位尺度差异较大的问题,在HRNet-32中引入了空洞空间金字塔池化 (Atrous spatial pyramid pooling, ASPP) 模块,在提升特征感受野的同时捕获多尺度信息;同时对大熊猫身体关键点进行分组,引入基于部位的多分支结构来学习特定于每个部位组的表征。多次对比实验结果表明本文所用模型具有较高的检测精度：在PCK@0.05中所用模型精度达到了81.51%。本文提出的方法可为大熊猫的行为分析和健康评估提供技术支撑。     

野生大熊猫行为谱及PAE编码系统

行为谱是动物行为学研究的基础。大熊猫(Ailuropoda melanoleuca)是生物多样性保护中的旗舰物种,其行为生态的研究备受关注,然而由于大熊猫数量稀少且警惕性强,很难在野外进行观察,有关其行为谱等基础行为研究结果主要是基于圈养大熊猫的观察研究。为了促进野生大熊猫行为学的深入研究,2015年1月—2016年6月,2018年8月— 2019年4月我们在卧龙自然保护区基于红外相机图片和视频数据,利用PAE编码系统建立了野生大熊猫行为谱和PAE编码系统。研究共计在14种环境背景中记录到大熊猫12种姿势、52种动作、56种行为。一方面,本研究结果基本涵盖了大熊猫的主要行为,并发现了一些未在圈养大熊猫行为谱中记录到的行为,如野生大熊猫利用植物的茎进行自我除雪行为;另一方面丰富了观察大熊猫行为的环境因素。以上结果为深入研究野生大熊猫行为生态学提供了基础信息。       

基于发育网络识别模型的大熊猫面部识别

个体识别是动物行为学与生态学研究工作的基础,也是制定珍稀野生动物保护政策的重要依据。为了丰富大熊猫个体识别和种群数量调查的方法,我们于2017年7月分别在四川省雅安市碧峰峡大熊猫基地和四川省汶川县耿达镇的中华大熊猫苑共计拍摄18只大熊猫个体,每只大熊猫拍摄6~13张高质量面部照片（共计131张）,利用发育网络（Developmental Network）建立大熊猫面部识别模型。利用此模型对存在部分背景的大熊猫面部照片进行识别检测,得到的个体识别率为79.41%,对完全去除背景的大熊猫面部照片进行识别检测,得到的个体识别率为58.82%。研究表明,发育网络具有足够的大熊猫个体识别能力,不同背景比例的照片对大熊猫个体识别的实际结果具有较大的影响。随着发育网络识别模型的发展,我们建议更多的野生动物保护研究者结合这一技术深入地开展珍稀野生动物（如大熊猫）个体识别研究,逐步提高识别准确度,并应用到关键区域大规模的动物调查中。     

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...     

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。     

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。     

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。     

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.     

Epigenomic profiling indicates a role for DNA methylation in the postnatal liver and pancreas development of giant pandas

Giant pandas are unique within Carnivora with a strict bamboo diet. Here, the epigenomic profiles of giant panda liver and pancreas tissues collected from three important feeding stages were investigated using BS-seq. Few differences in DNA methylation profiles were exhibited between no feeding and suckling groups in both tissues. However, we observed a tendency toward a global loss of DNA methylation in the gene-body and promoter region of metabolism-related genes from newborn to adult. Correlation analysis revealed a significant negative correlation between the changes in methylation levels within gene promoters and gene expression. The majority of genes related to nutrition metabolism had lost DNA methylation with increased mRNA expression in adult giant pandas. The few galactose metabolism and unsaturated fatty acid metabolism related genes that were hypomethylated and highly-expressed at early stages of giant panda development may meet the nutritional requirement of this species' highly altricial neonates.     

基于双模型融合的大熊猫头部图像分割

在大熊猫(Ailuropoda melanoleuca)的迁地保护和种群饲养管理中,及时、快速地进行个体识别和行为监测,对其健康管理具有至关重要的作用。圈养大熊猫健康状况通常由专门的饲养人员肉眼观测,人力成本高、效率低并且缺乏时效性。基于图像的动物个体识别与行为分析技术效率高、时间成本低,已经成为新的监测发展趋势。已有研究提出,通过大熊猫面部图像的检测和分析,可实现个体识别和行为分类。但该方法依然存在检测精度不足导致识别准确率难以提升的问题。本文提出一种基于YOLOv3和Mask R-CNN的双模型融合方法,实现了大熊猫头部图像分割和精准检测。包含3个部分：YOLOv3完成头部检测,Mask R-CNN完成大熊猫轮廓分割,然后将两个模型的输出进行交并比融合。结果显示,头部检测准确率为82.6%,大熊猫轮廓分割准确率为95.2%,总体头部轮廓分割准确率为87.1%。该方法对大熊猫头部图像的识别率和分割准确率高,为大熊猫的个体识别、性别分类提供了帮助,为行为分析提供了技术参考。     

No evidence for MHC‐based mate choice in wild giant pandas

Major histocompatibility complex genes (MHC), a gene cluster that controls the immune response to parasites, are regarded as an important determinant of mate choice. However, MHC‐based mate choice studies are especially rare for endangered animals. The giant panda (Ailuropoda melanoleuca), a flagship species, has suffered habitat loss and fragmentation. We investigated the genetic variation of three MHC class II loci, including DRB1, DQA1, and DQA2, for 19 mating‐pairs and 11 parent‐pairs of wild giant pandas based on long‐term field behavior observations and genetic samples. We tested four hypotheses of mate choice based on this MHC variation. We found no supporting evidence for the MHC‐based heterosis, genetic diversity, genetic compatibility and “good gene” hypotheses. These results suggest that giant pandas may not use MHC‐based signals to select mating partners, probably because limited mating opportunities or female‐biased natal dispersal restricts selection for MHC‐based mate choice, acknowledging the caveat of the small sample size often encountered in endangered animal studies. Our study provides insight into the mate choice mechanisms of wild giant pandas and highlights the need to increase the connectivity and facilitate dispersal among fragmented populations and habitats.     

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。     

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.     

大熊猫视力的定量化

有大量的研究提出,大熊猫具有发达的嗅觉和听觉系统但是视力很差。 虽然有些关于大熊猫视力和色觉方面的研究,但是至今我们对大熊猫的具体视力值方面的内容知之甚少。为了定量化大熊猫的视力值,我们观察并记录了8只圈养大熊猫（雌:雄=4:4）对于移动不同水平黑白相间条纹宽度的纸片的行为反应。我们发现,在测量距离为50 cm的情况下,大熊猫能够区分大约为0.46mm宽的黑白相间条纹。这个研究结果为大熊猫视网膜结构的解剖学研究提供了行为学支持,也为今后研究大熊猫的通讯和认知提供一定的参考。     

野生大熊猫种群数量调查方法研究进展

大熊猫(Ailuropodamelanoleuca)是中国特有珍稀野生动物,被誉为野生动物保护领域的"旗舰物种"。大熊猫种群数量的变化可以实时反应大熊猫种群动态,提供大熊猫分布区域、栖息地质量等最直接的信息,是制定大熊猫保护方案的基础,也是有效实施大熊猫保护措施的前提。综述了几种传统野生大熊猫种群数量调查方法(包括直接计数法、数学模型法、距离-咬节分析法及分子生物学方法),以及近年来最新应用于野生动物种群数量调查的红外相机技术、足迹鉴定法,讨论了传统方法中可能存在的问题、分析新方法的应用前景,并针对今后的野生大熊猫种群数量调查提出了一些建议与展望。     

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.     

Synergistic effects of anthropogenic disturbances on giant pandas

Livestock grazing and the collection of bamboo shoots are the main threats to giant panda (Ailuropoda melanoleuca) habitat in the Liangshan Mountains in China. It is important to clarify the effect of these disturbances to the giant panda to formulate targeted management policies. Based on species distribution models and daily activity models, we investigated the effects of livestock grazing and bamboo shoot collection on giant pandas from May 2021 to July 2022. Our results indicated the giant panda's suitable habitat in the reserve covered 51.83 km² (15.02% of the reserve area). Grazing and bamboo shoot collection led to losses of 19.08 km² and 7.68 km² of suitable habitat, respectively. Together, the 2 activities resulted in a loss of 28.35 km² of suitable habitat, which was more than half of the area of panda habitat. The areas of suitable habitat for giant pandas significantly overlapped with the areas affected by both disturbances. Giant pandas did not show significant differences in daily activity rhythms under a single disturbance, but the daily activity rhythms of giant pandas differed when we compared the area combining the 2 disturbances with the undisturbed area. Our study reveals that the anthropogenic disturbances in the reserve have varying effects on the suitable habitat range and daily activity rhythm of giant pandas and evidence of a synergistic effect. Therefore, when formulating relevant conservation policies, it is important to fully evaluate the extent and characteristics of anthropogenic disturbances in shaping the population distribution and habitat preferences of the giant panda and other wildlife to enhance the efficacy of conservation management practices.     

Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system

Background

The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda.

Results

By screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What’s more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system.

Conclusion

The microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1268-z) contains supplementary material, which is available to authorized users.     

Impact of livestock on giant pandas and their habitat

Livestock production is one of the greatest threats to biodiversity worldwide. However, impacts of livestock on endangered species have been understudied, particularly across the livestock–wildlife interface in forested protected areas. We investigated the impact of an emerging livestock sector in China's renowned Wolong Nature Reserve for giant pandas. We integrated empirical data from field surveys, remotely sensed imagery, and GPS collar tracking to analyze (1) the spatial distribution of horses in giant panda habitat, (2) space use and habitat selection patterns of horses and pandas, and (3) the impact of horses on pandas and bamboo (panda's main food source). We discovered that the horse distribution overlapped with suitable giant panda habitat. Horses had smaller home ranges than pandas but both species showed similarities in habitat selection. Horses consumed considerable amounts of bamboo, and may have resulted in a decline in panda habitat use. Our study highlights the need to formulate policies to address this emerging threat to the endangered giant panda. It also has implications for understanding livestock impacts in other protected areas across the globe.