大熊猫(Ailuropoda melanoleuca)生境选择研究进展

在回顾大熊猫生境选择研究历史的基础上,总结了大熊猫生境选择的考察因子、研究方法,概括了大熊猫对对觅食条件、隐蔽条件和气候条件的选择机制,并探讨了大熊猫生境选择的灵活性及大熊猫和与其同域分布的动物种的共存机制.作者认为,野外调查误差大、研究尺度单一等是目前大熊猫生境选择研究中存在的主要问题,应用目前较流行的已被证明比较优良的资源选择函数法、DNA指纹技术、无线电遥测、红外线自动感应照相系统、GIS与遥感成像等先进技术,并融合景观生态学理论,从较大尺度空间来研究大熊猫的生境选择,应是今后的发展方向.     

大熊猫食道梗阻一例报告

首次报道了一例大熊猫患罕见的食道梗阻症的发病经过、临床诊治及解剖病理变化。并对发病原因及预防措施等进行了分析和探讨。     

大熊猫染色体晚复制带研究

以培养的大熊猫外周血淋巴细胞为实验材料,在细胞培养终止前４ｈ加入ＢｒｄＵ（终浓度为１０μｇ／ｍｌ培养基）,对复制的染色体ＤＮＡ进行ＢｒｄＵ标记。掺入ＢｒｄＵ的染色体经吖啶橙（０．０５％）处理、紫外光照射、Ｇｉｅｍｓａ染色后,可在染色体上获得清晰的复制带纹。根据众多分裂相所显示的不同复制带型,可初步确定大熊猫每一染色体独特的晚复制带纹。在雌性个体的两个Ｘ染色体中,一条Ｘ染色体复制明显落后于另一Ｘ染色体,尤其在迟复制Ｘ染色体长臂近着丝粒区显现出较宽的晚复制带纹。     

大熊猫SRY基因的PCR扩增和克隆

本文采用人ＳＲＹ基因的一对引物,通过ＰＣＲ扩增获得了雄性大熊猫ＳＲＹ基因片段。表明大熊猫存在与人ＳＲＹ基因同源的相应基因,将ＰＣＲ产物与载体ｐＵＣ－Ｅｃｏ－Ｔ连接后,用以转化ＪＭ１０９菌,经过与人ＳＲＹ基因探针菌落杂交筛选获得了大熊猫ＳＲＹ苈在克隆,命名为ｐＡＭＹ０．６,其插入片段为相应于人ＳＲＹ基因保守区在内的一段约６０９ｂｐＤＮＡ。此外,还制作和比较分析了人和大熊猫基因片段的限制酶图谱。     

大熊猫精浆蛋白的SDS-PAGE电泳研究

本试验采用SDS-PAGE对大熊猫精浆进行电泳并对电泳条带进行分析,探讨特异条带与大熊猫精液质量的关系。结果显示：大熊猫精浆SDS-PAGE电泳共分离得到12条蛋白谱带,其中6个条带为所有个体大熊猫精浆样品所共有,共有条带的相对量在不同个体之间不存在差异（P〉0．05）。试验中部分大熊猫精浆出现的特异蛋白带和共有带蛋白含量与精液质量无相关性。     

Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells

Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation.     

The thyroid and parathyroid glands in the Giant panda, Ailuropoda melanoleuca

Observations are submitted upon the anatomy of the thyroid and parathyroid glands of Ailuropoda melanoleuca following gland investigation in three specimens. Variations of thyroid configuration and of parathyroid topography are noted, the thyroid vasculature is described in detail and an initial contribution is made to thyroid and parathyroid histology in this genus.     

The faecal flora of the giant panda (Ailuropoda melanoleuca)

The faecal floras of two adult (male and female) and one infant (male) giant panda kept at the Ueno Zoo, Tokyo, Japan were examined and shown to be quite different from those of other animals. The predominant bacteria in the adults were Streptococcus (including Enterococcus ) and Enterobacteriaceae, while obligate anaerobes had minor populations. Fastidious anaerobes were not detected. The predominant bacteria in the suckling infant were Lactobacillus and Streptococcus , followed by Bifidobacterium. After the infant began to eat bamboo leaves the number of Lactobacillus decreased and Bifidobacterium became undetectable, whereas Enterobacteriaceae became one of the most predominant flora. The most dominant streptococcus isolated from the female panda was identified as Streptococcus bovis , but those from the male adult and the weaned infant were not identified as any known species.     

The faecal flora of the giant panda (Ailuropoda melanoleuca)

The faecal floras of two adult (male and female) and one infant (male) giant panda kept at the Ueno Zoo, Tokyo, Japan were examined and shown to be quite different from those of other animals. The predominant bacteria in the adults were Streptococcus (including Enterococcus) and Enterobacteriaceae, while obligate anaerobes had minor populations. Fastidious anaerobes were not detected. The predominant bacteria in the suckling infant were Lactobacillus and Streptococcus, followed by Bifidobacterium. After the infant began to eat bamboo leaves the number of Lactobacillus decreased and Bifidobacterium became undetectable, whereas Enterobacteriaceae became one of the most predominant flora. The most dominant streptococcus isolated from the female panda was identified as Streptococcus bovis, but those from the male adult and the weaned infant were not identified as any known species.     

In vitro maturation of follicular oocytes of the Giant Panda (Ailuropoda melanoleuca): a case report

The Giant Panda is an endangered species that would benefit from biotechnological assistance in reproduction. However, because there are only a few of these animals left in the world, scientists hesitate to use them for research procedures. We were fortunate to obtain ovaries from a Giant Panda that died of hepatic cirrhosis during the nonbreeding season. Oocytes were harvested within 4 h of death by dissecting the ovarian cortex in physiological saline and collecting the cumulus-oocyte complexes from the fluid, and then were classified into large (> 125 microns) and small (100 to 124 microns) follicular oocytes and placed in TCM199 supplemented with FSH (10 micrograms/mL) and LH (20 micrograms/mL). After culture for 22 h at 37 degrees C in air with 5% CO2, response was evaluated by growth of oocytes and presence of the first polar body. Of the 26 large follicular oocytes that were harvested, 12 were considered suitable for IVM, and 14 were degenerated, had a broken zona pellucida or had lost some cytoplasm. Of the 12 cultured oocytes, all grew to a mean diameter of 141.1(SD = +/- 6.7, n = 12), and 4 released the first polar body. None of the small follicular oocytes showed growth or other signs of maturation. We conclude from our preliminary results that it is possible to obtain functional Giant Panda oocytes from ovaries obtained post mortem during the nonbreeding season.     

大熊猫生长分化因子9(GDF-9)基因克隆与序列分析

生长分化因子9(GDF-9)是转化生长因子β(TGF-β)超家族的一个新成员,主要表达于卵母细胞,调节卵泡早期发育. 本文以大熊猫基因组DNA为模板,克隆了大熊猫GDF-9基因.序列分析显示:大熊猫GDF-9基因含两个外显子,编码一个由453个氨基酸组成的蛋白前体.该结果为进一步分析大熊猫GDF-9基因生物学功能奠定了基础.     

大熊猫与黑熊显带染色体的比较研究

以体外培养的大熊猫（Ａｉｌｕｒｏｐｏｄａｍｅｌａｎｏｌｅｕｃａ）与黑熊（Ｓｅｌｅｎａｒｃｔｏｓｔｈｉｂｅｔａｎｕｓ）外周血淋巴细胞为实验材料,应用ＢｒｄＵ复制带显示技术,研究了大熊猫和黑熊染色体晚复制带带型。通过对大熊猫与黑熊显带染色体带型的比较,发现黑熊部分具端着丝粒的染色体与大熊猫部分具中,亚中,或亚端着丝粒的染色体的整个短臂或整个长臂有明显的带型相似性,在黑熊具中,亚中着丝粒染色体中,仅３３     

Artificial insemination in the Giant panda (Ailuropoda melanoleaca)

Artificial insemination was carried out on three adult captive Giant pandas (Ailuropoda melanoleuca). Oestrus in these animals was detected by behavioural observations and by the rise in urinary oestrogen metabolites. Semen was collected from a mature male by electroejaculation. Changes in the quality of semen and the size of the testes indicated a seasonal fluctuation in sperm production. Spermatozoa were capable of penetrating zona-free hamster oocytes and remained viable for up to five days at 15–20°C in a modified Tyrode's medium. After freezing and thawing in a cryopreserving diluent, approximately 50% of spermatozoa recovered progressive motility and displayed a similar velocity to spermatozoa prior to freezing. Unusual protrusions of the acrosome of the panda spermatozoon were noted.
Following insemination, one female conceived and gave birth, after a gestation of 159 days, to two cubs, one of which subsequently died. Another female exhibited a rise in circulating progesterone but pregnancy was not confirmed.     

A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China

Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60 × 3.99 μm (n = 50). The isolate was genetically analyzed using the partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype.     

Sixteen novel microsatellite loci developed for the giant panda (Ailuropoda melanoleuca)

Sixteen novel microsatellite DNA loci were developed from the giant panda (Ailuropoda melanoleuca) using a magnetic-bead capture method. A total of 115 alleles were obtained for these markers, ranging from 4 to 12 alleles per locus (average 7.188). These loci exhibited high levels of polymorphic information content and expected heterozygosity, 0.558–0.855 (average 0.729) and 0.628–0.885 (average 0.778), respectively. Therefore, the allelic polymorphism and heterozygosity show that the giant pandas raised in China Research and Conservation Center possess abundant genetic variation. In addition, if the three markers showing null alleles were excluded, the remaining 13 microsatellite loci still presented extremely low non-exclusion probabilities of parentage (0.002), paternity (0.000) and identity (0.000). As a result, this new suit of microsatellite markers would be a very informative tool for the genetic and conservation studies of giant pandas.     

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.     

Major histocompatibility complex class II variation in the giant panda (Ailuropoda melanoleuca)

Habitat destruction and human activity have greatly impacted the natural history of the giant panda (Ailuropoda melanoleuca). Although the genetic diversity of neutral markers has been examined in this endangered species, no previous work has examined adaptive molecular polymorphisms in the giant panda. Here, the major histocompatibility complex (MHC) class II DRB locus was investigated in the giant panda, using single-strand conformation polymorphism (SSCP) and sequence analysis. Comparisons of DNA samples extracted from faecal and blood samples from the same individual revealed that the two materials yielded similar quantities and qualities of DNA, as well as identical SSCP patterns and allelic sequences, demonstrating the reliability of DNA isolation from panda faeces. Analysis of faecal samples from 60 giant pandas revealed relatively low number of alleles: seven alleles. However, the alleles were quite divergent, varying from each other by a range of 7-47 nucleotide substitutions (4-25 amino acid substitutions). Construction of a neighbour-joining tree and comparisons among DRB alleles from other species revealed that both similar and highly divergent alleles survived in the bottlenecked panda populations. Despite species-specific primers used and excellent faecal DNA isolated, a lower level of heterozygosity than expected was still observed due to inbreeding. There were three types of evidence supporting the presence of balancing selection in the giant panda: (i) an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions; (ii) trans-species evolution of two alleles between the giant panda and other felids; and (iii) a more even distribution of alleles than expected from neutrality.     

Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca)

It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.     

The complete mitochondrial genome and phylogenetic analysis of the giant panda (Ailuropoda melanoleuca)

The complete mitochondrial genome sequence of the giant panda, Ailuropoda melanoleuca, was determined by the long and accurate polymerase chain reaction (LA-PCR) with conserved primers and primer walking sequence methods. The complete mitochondrial DNA is 16,805 nucleotides in length and contains two ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes and one control region. The total length of the 13 protein-coding genes is longer than the American black bear, brown bear and polar bear by 3 amino acids at the end of ND5 gene. The codon usage also followed the typical vertebrate pattern except for an unusual ATT start codon, which initiates the NADH dehydrogenase subunit 5 (ND5) gene. The molecular phylogenetic analysis was performed on the sequences of 12 concatenated heavy-strand encoded protein-coding genes, and suggested that the giant panda is most closely related to bears.