New horizons in the reproductive biology of Chinese pangolin (Manis pentadactyla) using the gonadal hormonal profile.

The reproductive uniqueness of pangolins has been documented through diverse biological reports with discernible data discrepancies in gestation, copulation, and pregnancy. These mechanistic reproductive differences have yet to be endocrinologically quantified, which could assist in optimizing natural breeding in zoos to recover endangered species. The present research characterizes the Chinese pangolin's annual seasonal reproductive pattern by measuring immunoreactive estrogens and progestagens in 34 captive females and testosterone in 29 captive males. Our results showed that Chinese pangolins are seasonal breeders, with most births witnessed during Sept-Dec, overlapping with the field records. Females exhibited spontaneous ovulation and post-partum ovulation. Pregnant females exhibited a higher P4 level for ~ 9 months (Jan-Sept) and decreased before parturition (Oct-Dec). The circulating E2 is maintained at the baseline in pregnant females year-round. Contrastingly, in non-pregnant females, P4 is maintained at the baseline, apart from a slight elevation in January, and E2 demonstrates a sudden hike from November and remains elevated until February, suggesting the onset of ovulation. The serum testosterone concentration in males peaked during October, which is in sync with the female ovulation period. As a result that their major reproductive events, ovulation, mating, and parturition, all transpire in November-March. Evidence also supports that Chinese pangolins exhibit signs of postimplantation (pregnancy) ranging only from 5 to 6 months (May-Oct), preceded by possible facultative delay implantation triggered by lactation. The provided data not only fill in the knowledge gap for this critically endangered species but can also assist in making informed decisions, which can directly affect the successful breeding of this species in captivity.

Novel Low-Voltage Electro-Ejaculation Approach for Sperm Collection from Zoo Captive Lanyu Miniature Pigs (Sus barbatus sumatranus).

Semen collection can be achieved via hand penile massage or rectal stimulation using electro-ejaculation methods. Traditional electro-ejaculation procedure applied relatively high voltage of 3-15 volts with a maximum current of 900 mA. However, these manipulations often result in great stress and discomforts in animals. In this study, we showed low-voltage electro-ejaculation procedure using 2-3 volts with a maximum current of 500 mA can efficiently stimulated ejaculations in zoo captive lanyu miniature pigs with a high success rate of 81.3% (13/16). Besides normal semen properties (semen volume, pH, sperm concentration), we demonstrated that low-voltage electro-ejaculation caused less stress in the animals, and sperm cells obtained via low-voltage electro-ejaculation exhibit low abnormality (10.3%), high viability (84.3%), motility (75.7%), progressive motility (63.7%), and acrosome integrity (88%). However, cryopreservation protocol used in the current study requires further optimization, as sperm mitochondrial function was partially compromised during freezing procedures. Taken together, we demonstrated in this study that a low-voltage electro-ejaculation approach can be used to obtain quality sperm cells from zoo captive lanyu miniature pig with less physical stress during electro-ejaculation procedure.

Digesta retention time and recovery rates of ants and termites in Chinese pangolins (Manis pentadactyla).

Pangolins are myrmecophagous mammals whose biology and ecology remain poorly studied. Termite mandibles and ant head capsules are the two primary remains found in pangolin feces. Determining the retention time of insect cuticles is important for understanding the digestive physiology of pangolins, while determining the recovery rate of termites and ants in feces is required to estimate the number of these prey items that are consumed by pangolins. In this study, the authors conducted feeding trials with captive Chinese pangolins (Manis pentadactyla). Sixty grams of the fungus-growing termite Odontotermes formosanus (18,816 individuals) and 15-20 g of the yellow crazy ant Anoplolepis gracilipes (14,400-19,200 individuals) were fed to each pangolin. After feeding, pangolin feces were collected daily for 1 week. The authors also assessed the accuracy of using chromium (III) oxide powder (Cr2 O3 ) as a proxy for determining gut passage time, as has been done in previous studies. The results showed that remaining termite mandibles and ant head capsules in feces peaked at 66 and 90 hr after feeding and their recovery rates were 0.35 ± 0.10 and 0.65 ± 0.04, respectively. In both feeding trials, the retention time of Cr2 O3 was much shorter than that of the termite mandibles and ant head capsules, indicating that Cr2 O3 is not an appropriate indicator for estimating food retention time of myrmecophagous animals. Our results revealed that the ant head capsules were preserved better in feces compared with the termite mandibles, suggesting that termites may be considerably underestimated in the feces of wild pangolins.

Fatal canine parvovirus-2 (CPV-2) infection in a rescued free-ranging Taiwanese pangolin (Manis pentadactyla pentadactyla).

Carnivore protoparvovirus 1 includes feline parvovirus (FPV), variants of canine parvovirus-2 (CPV-2), mink enteritis virus, and raccoon parvovirus, important pathogens affecting both wild and domestic carnivores. In this report, we described a fatal CPV-2 infection in a rescued Taiwanese pangolin, which provides the first evidence of CPV-2 infection in a non-carnivore. Post-rescue, the Taiwanese pangolin died from complications resulting from a severe panleucocytopenia and bloody diarrhoea. A full autopsy was performed and microscopic examination of the tissues revealed ulcerative, necrotizing, and haemorrhagic glossitis, esophagitis and enteritis. The results of transmission electronic microscopy, polymerase chain reaction and in situ hybridization provided confirmatory evidence that the lesions in the tongue, oesophagus and intestine were associated with a protoparvovirus. Phylogenetic comparison of the whole VP2 gene from the current pangolin protoparvovirus strain showed close clustering with the CPV-2c strains from domestic dogs in Taiwan, China and Singapore. The amino acid sequence of the pangolin protoparvovirus showed 100% identity to the CPV-2c strains from domestic dogs in China, Italy, and Singapore. The current findings highlight that pangolins are susceptible to protoparvoviruses. The potential of cross-species transmission of protoparvoviruses between Carnivora and Pholidota should be considered when housing pangolins in close proximity to carnivores and adopting strict biosecurity measures to avoid cross-species transmission in rescue facilities and zoos.

The effects of type I collagenase on the degelification of chimpanzee (Pan troglodytes) semen plug and sperm quality.

BACKGROUND: Semen from the chimpanzee species becomes a colloidal solid after ejaculation. The formation of this copulatory plug is believed to prevent additional spermatozoa of subsequent mating events from accessing the ova. However, this naturally preserved strategy hampers the processes for sperm preparation. In this study, we investigated whether collagenase can be used to degelify the semen plug and accelerate the semen liquefaction process in zoo captive chimpanzee species (Pan troglodytes). RESULTS: We showed that incubation of chimpanzee ejaculates with 0.1% type I collagenase efficiently and significantly (p < 0.05) releases 2.7-fold more spermatozoa from the coagulated ejaculates, and this degelification process did not alter sperm morphology or viability; nor did it stimulate spontaneous capacitation or an acrosome reaction as assessed by tyrosine phosphorylation and peanut agglutinin stains; moreover, based on computer assisted sperm analysis assay, motility-related parameters remained similar to those of untreated spermatozoa. When collagenase effects were evaluated on cryopreserved sperm samples, we observed post collagenase treatment in which 2.5% glycerol, as a cryoprotectant, preserved sperm acrosome integrity better than 7.8%; however, 7.8% glycerol, as a cryoprotectant, maintained sperm motility better than that of 2.5% glycerol. CONCLUSIONS: Our results demonstrated for the first time that type I collagenase can be used to obtain a significantly higher number of spermatozoa from colloid chimpanzee semen ejaculate without affecting the physiological properties of spermatozoa, and these results are critical for the subsequent gamete development. Our results would benefit sperm preparation processes and cryopreservation efficiency per ejaculate, as more unaffected spermatozoa can be released from the semen plug within a shorter period of time. These results would also benefit the genetic diversity of the chimpanzee species, using sperm cells from less dominant individuals, and for achieving better pregnancy success in primates with significantly higher amounts of sperm for artificial insemination.

Feeding Asian pangolins: An assessment of current diets fed in institutions worldwide.

Pangolins are ant specialists which are under intense threat from the illegal wildlife trade. Nutrition has notoriously been their downfall in captivity and is still an issue in regards to rescue and rehabilitation. We analyzed the nutrient content of diets used by institutions that are successfully keeping Asian pangolins and to assess the variety of the ingredients and nutrients, compared these with the nutritional requirements of potential nutritional model species. We performed intake studies at five institutions and also had data from three other institutions. We also analyzed five different wild food items to use as a proxy of wild diet. We observed two categories of captive diets: those mostly or completely composed of insects and those high in commercial feeds or animal meat. Nutrient values were broad and there was no clear rule. The non-protein energy to protein energy ratio of the diets were much higher than the wild food items, more so for those which receive less insects. The average contribution of carbohydrate, fat and protein energy were also further away from the wild samples the less insects they contained. The previously suggested nutritional model for pangolins is the domestic dog which is supported by our relatively large nutrient ranges of apparently successful diets, however due to their highly carnivorous nature; the upper most nutrient intake data are not consistent with this and favor the feline nutrient recommendations. We are unable to render a conclusion of what model is more appropriate based on our data collected.

Hematologic and serum biochemical parameters of apparently healthy rescued formosan pangolins (Manis pentadactyla pentadactyla).

Natural habitats of pangolins are rapidly deteriorating because of extensive farming, logging, and human construction activities. In addition, the illegal trading of pangolins substantially accelerated the decline of the pangolins' population in southeastern Asia. The maintenance of confiscated pangolins in rescue centers is currently a daunting task for veterinarians and conservation biologists. There is limited information in the literature about the reference values regarding the physiology of pangolins. The purpose of this study is to establish reliable hematologic and serum biochemical reference values for the Formosan pangolin (Manis pentadactyla pentadactyla). Blood samples were collected from 51 apparently healthy pangolins from a population of 117 rescued pangolins at the Taipei Zoo. Sex-related differences were observed in platelet count, alanine aminotransferase level, mean corpuscular hemoglobin concentration, and total protein level. Age-related differences were also noted; juveniles have significantly higher platelet counts and alkaline phosphatase levels than their adult counter parts. The hematologic and serum biochemical reference values for the Formosan pangolin presented in this study can be applied in the medical care of this important species during rescue attempts. It is the first systematic report of blood parameters of apparently healthy pangolins and provides a basis for future investigation of this species. The reference values reported in this study may also be applicable to other pangolin species in the genus Manis.

Meningothelial meningioma in a Malayan sun bear (Helarctos malayanus).

A 24-year-old, spayed female Malayan sun bear (Helarctos malayanus) in the Taipei Zoo (Taipei, Taiwan) showed clinical signs of slowly progressive anorexia, dullness, compulsive pacing, and circling. The animal subsequently developed acute severe stupor and persistent recumbency. Postcontrast study of computed tomography revealed a spheroid, extra-axial mass with strong but heterogeneous hyperattenuation in the left temporal lobe of the cerebrum. At necropsy, a solitary, well-circumscribed intracranial mass measuring 3 cm × 2.5 cm × 2 cm was attached to the left pyriform lobe with compression of the adjacent neuroparenchyma. Cytological examination obtained from the mass revealed large clumps and sheets of cohesive polyhedral cells with round nuclei, wispy cytoplasm, and indistinct cell borders. Microscopically, the mass was composed of densely packed round to polygonal cells arranged in lobules and small nests. Psammoma bodies, xanthomatous change, and cholesterol deposition were also noted. Immunohistochemical staining of the tumor was positive for vimentin, pancytokeratin, cytokeratin (CK)34BE12, neuron-specific enolase, and epithelial membrane antigen, but negative for glial fibrillary acidic protein and S100 protein. The cytological, histological, and immunohistochemical features were compatible with a meningothelial meningioma.

Use of a cytogenetic whole-genome comparison to resolve phylogenetic relationships among three species: implications for mammalian systematics and conservation biology.

The objective was to apply a novel modification of a genome-wide, comparative cytogenetic technique (comparative genomic hybridization, comparative genomic hybridization (CGH)), to study species belonging to the myrmecophagous (ant/termite eating) mammalian orders/superorders (Pholidota, Tubulidentata, Carnivora, and Xenarthra), as a model for other applications in mammalian systematics and conservation biology. In this study, CGH was applied to high-quality metaphase spreads of pangolin (Pholidota), using probes of sloth and canine (Xenarthra and Carnivora, respectively) genomic DNA labeled with different fluorophores, thereby facilitating analysis of the visible color spectrum on pangolin karyotypes. Our results posited that pholidotes are closer to carnivores than to xenarthrans, which confirmed the current consensus that myrmecophagy in these mammalian lineages was more likely because of homoplasy (convergent evolution) than being an ancestral character. Since the modified CGH technique used is genome-wide, has chromosome-level resolution, and does not need full genome sequencing, it has considerable potential in systematics and other fields.

Monitoring the gestation period of rescued Formosan pangolin (Manis pentadactyla pentadactyla) with progesterone radioimmunoassay.

Eight species of pangolin have been identified in the world. However, understanding of pangolin reproductive biology has been limited to fragmentary records. In this study, the concentration of serum progesterone in three pregnant and two nonpregnant rescued female Formosan pangolins (Manis pentadactyla pentadactyla) was monitored using a commercial progesterone radioimmunoassay kit. During gestation, the serum progesterone of pregnant pangolins A, B, and C remained at 28.5-55 ng/ml (n = 31 samples), 10.9-50.1 ng/ml (n = 34), and 12.4 and 33.5 ng/ml with a peak at 47.6 ng/ml (n = 19), respectively, whereas the serum progesterone of nonpregnant pangolins D and E remained at 1.99 ± 1.62 ng/ml (n = 80) and 2.27 ± 1.64 ng/ml (n = 27), respectively. From this study, it was found that female pangolin weighing as low as 2.14 kg was already capable of reproduction. For pregnant pangolins to give birth to viable offspring, their body weight must increase significantly, 63.89 and 134.0% in the study, from the time of inception or early pregnancy until parturition. In addition, study has found that both viable offspring were born fully developed and exceeded 80 g in weight. The period of gestation was found to be as short as 318 or longer than 372 days. Therefore, the Formosan pangolin should only be able to reproduce once a year. This study is the first insight into hormone assay for determining the gestation period of pangolin. Further investigations on the same subject are necessary to establish criteria for the recognition of reproductive status in pangolins.

Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals.

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.

Establishing the pangolin mitochondrial D-loop sequences from the confiscated scales.

Pangolin scales are encountered in traditional East Asian medicines (TEAM) and the ever increasing demand for these scales has escalated the decline in the numbers of these mammals. The identification of protected pangolin species is necessary to enforce international and national legislation as well as assist with conservation measures. There is limited morphological feature on a pangolin scale thus requiring DNA analysis as a means of identification. We report on the isolation of DNA from pangolin scales and a strategy for obtaining the full length of the mitochondrial D-loop, being 1159 bp. Primer sets creating five overlapping amplicons were designed to amplify sections of this mitochondrial DNA locus. DNA from the blood stain of nineteen Formosan pangolins (Manis pentadactyla pentadactyla) along with 145 scale samples that were suspected to have come from pangolins, was amplified and sequenced; leading to a total of 91 D-loop sequences being obtained. The 19 Formosan pangolin sequences produced 5 haplotypes and 72 of the 145 seized scales provided useable sequence classified as a further 38 haplotypes. The D-loop sequences from those scales suspected to be from a pangolin had a higher similarity to any of the 19 samples taken from M. p. pentadactyla compared to a D-loop sequence from Manis tetradactyla (the only pangolin D-loop sequence in GenBank, NC_004027). These 43 haplotypes were used to establish a local database for the D-loop sequence of pangolins and add to the data of Manis sp. held on GenBank. The PCR amplification strategy development in this study could be used in forensic DNA identification of scales suspected to be from protected pangolin species.

Prevalence and antimicrobial susceptibility of salmonellae isolates from reptiles in Taiwan.

Pets, including reptiles, have been shown to be a source of Salmonella infection in humans. Due to increasing popularity and variety of exotic reptiles as pets in recent years, more human clinical cases of reptile-associated Salmonella infection have been identified. However, limited information is available with regard to serotypes in different reptiles (turtles, snakes, and lizards) and antimicrobial resistance of Salmonella in pet reptiles. The current study was thus conducted to determine the prevalence of Salmonella colonization in pet reptiles. Salmonella organisms were isolated from 30.9% of 476 reptiles investigated. The isolation prevalences were 69.7% (23/33), 62.8% (27/43), and 24.3% (97/400) in snakes, lizards, and turtles, respectively. A total of 44 different Salmonella serovars were identified. Compared with S. Heron, Bredeney, Treforest, and 4,[5],12:i:-, S. Typhimurium isolates were resistant to many antimicrobials tested, and notably 61.1% of the isolates were resistant to cephalothin. The results indicated that raising reptiles as pets could be a possible source of Salmonella infection in humans, particularly zoonotic Salmonella serovars such as S. Typhimurium that may be resistant to antimicrobials.

A novel strategy for avian species and gender identification using the CHD gene.

We report on a novel and rapid strategy for the simultaneous identification of both avian species and gender by analyzing a section of the CHD gene. The CHD gene is carried by the avian sex determining chromosomes where a female bird carries both a W and Z chromosome but a cock bird carries two copies of the Z chromosome. Two primer pairs, CHD1F/CHD1R and P2/P8, were used to amplify a part of the CHD gene from 144 samples corresponding to 58 avian species. For all species tested, two fragments were observed at least in one amplification for female samples. All tested species produced species specific size fragments allowing both sex determination and species identification using these primer pairs. However, special care is still warranted as so few samples have been characterised. This novel strategy for avian species and gender identification using the CHD gene was developed for a number of applications from ecology to forensic science.

Ivory identification by DNA profiling of cytochrome b gene.

Ivory can be visually identified in its native form as coming from an elephant species; however, determining from which of the three extant elephant species a section of ivory originates is more problematic. We report on a method that will identify and distinguish the protected and endangered elephant species, Elephas maximus or Loxodonta sp. To identify the species of elephant from ivory products, we developed three groups of nested PCR amplifications within the cytochrome b gene that generate amplification products using highly degraded DNA isolated from confiscated ivory samples dating from 1995. DNA from a total of 382 out of 453 ivory samples were successfully isolated and amplified leading to species identification. All sequences were searched against GenBank and found to match with E. maximus and Loxodonta sp. with at least 99% similarity. The samples that were tested came from eight Asian elephants, 14 African forest elephants (Loxodonta cyclotis), and 360 African savannah elephants (Loxodonta africana). This study demonstrates a high success rate in species identification of ivory by a nested PCR approach within the cytochrome b gene which provides the necessary information for the protection of endangered species conservation.

A novel strategy for avian species identification by cytochrome b gene.

We report a DNA-based test that can be applied to any avian species so that the amplicon can be used in species identification. The need for the test arose from the requirement to enforce the Wildlife Conservation Act in Taiwan where over 150 avian species are protected. It is difficult to enforce the law if no gross morphology is present and hence there is a requirement to develop a DNA test. This study uses a novel strategy for avian species identification by the cytochrome b gene where a series of primer pairs producing amplicons of decreasing size was designed. The test is designed to produce the largest possible amplicon based upon the quality of the DNA in the sample. A total of 331 avian samples were tested representing 40 species. Sequencing of the amplicons revealed limited intraspecies variation and that no DNA sequence was shared by samples from two different avian species. The closest genetic distance among the 40 species was 0.059 which was between Lonchura punctulata and Estrilda melpoda based upon data from the smallest amplicon. A DNA databank including 138 sequence types from 331 samples tested, representing 40 different species, was constructed in this study. A blind test was used to determine the value for this system for forensic applications that successfully identified the species.

Sex identification of owls (family Strigidae) using oligonucleotide microarrays.

Molecular sexing of the diversified avian family Strigidae is difficult. Sex identification using the intron length difference between W and Z chromosomal CHD1 genes, as visualized by agarose gel electrophoreses, often produces ambiguous results. Here we describe a simple method for sexing a variety of Strigidae species using oligonucleotide microarrays, on which several sex-specific probes operated complementarily or in concert. The sex of 8 owl species was identified clearly on the microarrays through sequence recognition. This sequence-directed method can be easily applied to a wider range of Strigidae species.

Simultaneous detection and differentiation of Newcastle disease and avian influenza viruses using oligonucleotide microarrays.

Newcastle disease (ND) and avian influenza (AI) are two of the most important zoonotic viral diseases of birds throughout the world. These two viruses often have a great impact upon the poultry industry. Both viruses are associated with transmission from wild to domestic birds, and often display similar signs that need to be differentiated. A rapid surveillance among wild and domestic birds is important for early disease detection and intervention, and is the basis for what measures should be taken. The surveillance, thus, should be able to differentiate the diseases and provide a detailed analysis of the virus strains. Here, we described a fast, simultaneous and inexpensive approach to the detection of Newcastle disease virus (NDV) and avian influenza virus (AIV) using oligonucleotide microarrays. The NDV pathotypes and the AIV haemagglutinin subtypes H5 and H7 were determined at the same time. Different probes on a microarray targeting the same gene were implemented in order to encompass the diversified virus strains or provide multiple confirmations of the genotype. This ensures good sensitivity and specificity among divergent viruses. Twenty-four virus isolates and twenty-four various combinations of the viruses were tested in this study. All viruses were successfully detected and typed. The hybridization results on microarrays were clearly identified with the naked eyes, with no further imaging equipment needed. The results demonstrate that the detection and typing of multiple viruses can be performed simultaneously and easily using oligonucleotide microarrays. The proposed method may provide potential for rapid surveillance and differential diagnosis of these two important zoonoses in both wild and domestic birds.

Intestinal adenocarcinoma with pancreas and lymph node metastases in a captive cotton-top tamarin (Saguinus oedipus).

A case of intestinal adenocarcinoma with metastases to the pancreas and regional lymph node was found in a 9-year-old, captive female cotton-top tamarin (Saguinus oedipus) with intermittent diarrhea. At necropsy, the tumor mass was located in the ileo-cecal junction causing circumferential thickening and annular stenosis. Microscopically, the lesions at primary and metastatic sites showed typical features of mucinous adenocarcinoma as seen in humans, including intracellular and extracellular mucin production and characteristic appearance of a signet ring of the tumor cells. The diagnosis was confirmed by histological evaluation, positive cytokeratin immunostain, and mucin production demonstrated by PAS and Alcian blue stain. It is speculated that the development of intestinal carcinoma was partly attributable to the excessive absorption of a diet of refined food, unbalanced nutrition, and the nature of these animals to develop stress easily.

Fibrosarcoma with lung and lymph node metastases in an Asian elephant (Elephas maximus).

A case of fibrosarcoma with lung and lymph node metastases in a 54-year-old female Asian elephant (Elephas maximus) is described. After pododermatitis of 2 years duration in the right forefoot, a mass developed in the lateral toenail. At postmortem, metastasis to the right axillary lymph node and both lungs was noted. Microscopic examination of primary and metastatic sites revealed infiltrating bundles of spindle cells, with fairly distinct cell borders, variable amounts of eosinophilic cytoplasm, and elongate or oval nuclei. Tumor cells were often arranged in interwoven bundles and herringbone patterns. Mitotic figures were numerous and frequently bizarre. The diagnosis of fibrosarcoma with lung and lymph node metastases was made on the basis of histologic features and positive immunohistochemical staining for vimentin.