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.     

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.     

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.     

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.     

Cloning and sequence analysis of FSH and LH in the giant panda (Ailuropoda melanoleuca)

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. It has been proposed that it has a highly specialized reproductive pattern with low fecundity, but little is known about its basic reproductive biology at the molecular level. In this report the genes encoding gonadotropin subunits alpha, follicle-stimulating hormone (FSH) beta and luteinizing hormone (LH) beta of the giant panda were amplified for the first time by RT-PCR from pituitary total RNA, and were cloned, sequenced and analyzed. The results revealed that the open reading region (ORF) of gonadotropin subunits alpha, FSH beta and LH beta are 363, 390 and 426 bp long, respectively. They displayed a reasonably high degree (74-94, 85-93, 75-91%, for alpha, FSH beta and LH beta subunits, respectively) of identity when deduced amino acids were compared with homologous sequences from partial available mammals including human, cattle, sheep, pig, rat, mouse. Three distinct differences were found at the site of 59 aa of the alpha subunit and 55 aa, 68 aa of FSH beta subunit. Our results provide an insight into understanding the mechanism of reproduction regulation and genetic characteristics of giant panda which will make an actual contribution to its conservation. In addition they lay a foundation for a further study towards producing recombinant panda FSH and LH which can be used in artificial breeding aimed to increase its captive reproductive efficiency.     

Genetic diversities of the giant panda (Ailuropoda melanoleuca) in Wanglang and Baoxing Nature Reserves

Genetic variations in the giant panda populations in Wanglang and Baoxing Nature Reserves were evaluated in this study. Panda feces were collected from these two reserves and DNA samples extracted from the feces were genotyped at 13 microsatellite loci. A total of 130 alleles were identified from the 13 microsatellite loci in 63 giant pandas, including 35 private alleles in Wanglang, 53 private alleles in Baoxing, and 42 alleles shared between the two populations. The mean observed heterozygosity, average number of alleles, average number of allelic richness, and average polymorphism information content were 0.488, 6.2, 3.302, and 0.612, respectively for the Wanglang population and 0.553, 7.6, 4.050, and 0.747 for the Baoxing population. A moderate degree of genetic differentiation (F _st = 0.26) and no gene flow were found between these two populations. W. He and L. Lin contributed equally to this work.     

Thirty-three microsatellite loci for noninvasive genetic studies of the giant panda (Ailuropoda melanoleuca)

Limited microsatellite markers useable in noninvasive genetic methods have hampered the studies of dispersal patterns and mating systems of giant pandas. Therefore, we describe in this paper the characterization of 15 novel microsatellite loci from genomic DNA-enriched libraries and 18 redesigned microsatellite loci from published papers on the giant panda. The number of alleles per locus in 60 individuals ranged from 2 to 13, the average observed heterozygosity per locus from 0.168 to 0.800, and the average expected heterozygosity per locus from 0.152 to 0.882. All loci followed Hardy-Weinberg expectations. Four pairs of significant linkage association were found among all these loci. Moreover, the 33 microsatellite loci showed high amplification successes rate in noninvasive samples, which indicated that these loci will be of use in studying dispersal patterns and mating systems of giant pandas using noninvasive genetic methods.     

A reliable, non-invasive PCR method for giant panda (Ailuropoda melanoleuca) sex identification

We developed an inexpensive, fast and reliable PCR method for sex identification of giant panda (Ailuropoda melanoleuca) by using one pair of primers to co-amplify homologous fragments with size polymorphism that located at amelogenin (AMEL) exon 5. In giant panda, a 63 bp deletion in exon 5 of Y-linked allele provides a significant discrimination between AMELX and AMELY, thus the amplification products can be distinguished simply by agarose gel electrophoresis, exhibiting sex-specific banding patterns (male: 237 bp, 174 bp; female: 237 bp). Both blood and feces samples from known-sex giant pandas were successfully amplified. Cross species test also revealed that this method could be applied to other Ursidae species. These authors contributed equally to this work.     

大熊猫胃内纤毛虫检测初报

Twenty three captive giant pandas, fecal specimens and stomach juice from five captive giant pandas(Ailuropoda melanoleuca)were tested for ciliate in 2004. Of them, ciliates of Epdinium spp.and Entodinium spp.were found in the stomach of five giant panda. Density of ciliates in each stomach of giant pandas were 4.21×10 4 ind./ml, 6.98×10 4 ind./ml, 3.05×10 4 ind./ml, 4.46×10 4 ind./ml and 4.38×10 4 ind./ml respectively. But all the fecal specimens were negative.     

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 ...     

Molecular characterization of major histocompatibility complex class I genes from the giant panda (Ailuropoda melanoleuca)

The major histocompatibility complex class I genes play crucial roles in the adaptive immune system of vertebrates against intracellular pathogens. To date, no class I genes from the giant panda (Ailuropoda melanoleuca) has been reported, even none from species of Ursidae. In this study, we successfully identified three class I genes from a giant panda bacterial artificial chromosome library and designated them as Aime-128, 152, and 1906, respectively. Pairwise sequence alignments revealed that (1) the Aime-1906 always possessed the lowest identities (52–86%) in different regions compared with the Aime-128 and 152 and (2) the Aime-128 also varied from the Aime-152 in the regions of 5′ untranslated region (UTR), 3′ UTR, and exon1, whose similarities were 83%, 87%, and 91%, respectively. Comparison of structure characteristics indicated that the Aime-128 possessed all conserved amino acids important to the function of antigen presentation while the Aime-152 and 1906 presented two and five mutated residues. Analysis of phylogenetic trees demonstrated that the Aime-128, 152, and 1906 were clustered into three different branches with 99% or 100% bootstrap values. As a result, these three kinds of evidence supported that the Aime-1906, 152, and 128 should be derived from different loci. Furthermore, in view of a prestop codon in the exon 7 and patterns of amino acid replacement within alleles, the Aime-1906 gene is predicted to be a nonclassical locus, which is most closely related to dog leukocyte antigen 79 in the phylogenetic tree constructed with various mammalian class I loci. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Composition and oligosaccharides of a milk sample of the giant panda,Ailuropoda melanoleuca

A milk sample from a captive giant panda (Ailuropoda melanoleuca), obtained at 13 days postpartum, contained 7.1% protein, 1.6% carbohydrate, 10.4% lipid and 0.9% ash. The ratio of casein to whey proteins was 5.0:2.1. Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) of the whey protein fraction showed the presence of at least two major proteins other than alpha-lactalbumin and beta-lactoglobulin. SDS-PAGE and urea-gel electrophoresis showed that alphas-casein is not a major component. The proportions of triacylglycerol, cholesterol, cholesterol esters and phospholipid were 90.5, 5.3, 0.96 and 3.1%, of the total lipid, respectively. The dominant saccharide in the panda milk was Gal(alpha1-3)Gal(beta1-4)Glc (isoglobotriose). The milk contained, in addition, lesser amounts of lactose, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc (fucosyl isoglobotriose), Neu5Ac(alpha2-3)Gal(beta1-4)Glc (3'-N-acetylneuraminyl-lactose), Neu5Ac(alpha2-6)Gal(beta1-4)Glc (6'-N-acetylneuraminyl-lactose) and Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc.     

cDNA cloning of growth hormone from giant panda (Ailuropoda melanoleuca) and its expression in Escherichia coli

A cDNA encoding Ailuropoda melanoleuca growth hormone (AmGH) was isolated from pituitary total RNA using RT-PCR and expressed in Escherichia coli. This is the first report of a GH nucleotide and amino acid (aa) sequence from giant panda. The open reading frame of AmGH (651 bp) encodes a precursor of 216 aa comprising a 26 aa signal peptide and a 190 aa mature protein with four cysteine residues similar to the typical primary structure of mammalian GH precursor. AmGH shares a high degree of identity (54-98.9%) with that of mammals, birds and amphibians, but a very low identity with bony fish GH (only 20-30%). The mature AmGH exhibits striking similarity to that of putative ancestral GH with a difference of only two residues, indicating a very slow basal rate of molecular evolution. The DNA fragment encoding mature AmGH was then subcloned into the pGEX-4T-1 expression vector and highly expressed in E. coli host BL21 with IPTG induction. The expressed proteins fused to GST were found to be sequestered into inclusion bodies and therefore the NaOH method was employed to solubilize the inclusion bodies; the proteins were further purified by Glutathione Sepharose 4B affinity chromatography. The production and purification of GST-AmGH reported here provide a basis for further studies on the biological activity of AmGH.     

Dietary vitamin and mineral concentrations of two juvenile female giant pandas (Ailuropoda melanoleuca)

During a 7 mo exhibit loan, diets of two juvenile female giant pandas (Ailuropoda melanoleuca) were monitored. Mineral [calcium (Ca), copper (Cu), phosphorus (P), potassium (K), zinc (Zn)] and vitamin (beta-carotene and total tocopherol) concentrations in the diet were quantified as well as in a serum sample obtained from one of these animals. Diets consumed by the pandas contained (dry matter basis): 0.82% Ca, 0.62% P, 1.05% K, 14.3 mg/kg Cu, 31.8 mg/kg Zn, 12.2 mg/kg beta-carotene, and 12 mg/kg total tocopherol. Serum values for these vitamins and minerals were within reported normal limits for the spectacled bear (Tremarctos ornatus). This diet appeared to provide adequate nutrition for maintenance and growth.