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Infant call structure should have evolved to elicit maximum maternal attention and investment. Neonates of giant pandas produce three types of vocalizations reported to be vitally important in the context of mother–infant communications. However, how cubs, 0–15 days old, communicate with their mothers to elicit maternal care remains unknown. We analyzed 12 different call parameters of 3475 squawks, 1355 squalls, and 491 croaks from 11 captive giant panda (Ailuropoda melanoleuca) neonates from age 0 to 15 days. In playback experiments, we also tested whether mothers could detect ultrasound. Our results show that neonates use broadband calls with ultrasonic frequencies up to 65 kHz to convey information about their physiological needs and to attract maternal care. In playback experiments, we tested if mothers reacted differently to broadband calls (BBC) than to artificially altered calls that included only frequencies <20 kHz (AUDC) or calls that included only frequencies >20 kHz (USC). Playback confirmed that, although adult females responded significantly less often to USC, BBC than to or AUDC, they could detect USC, BBC and generally made appropriate behavioral responses, indicating a potential benefit for neonates to utilize ultrasonic and broadband frequencies. Our findings provide a new insight into mother–infant communication in giant pandas and will be helpful for reducing the mortality of cubs, younger than 1 month old, in captivity. [ABSTRACT FROM AUTHOR]

This article provides a detailed analysis of antibiotic resistance genes (ARGs) in the gut microbiota of giant pandas. The study identifies specific bacteria species that carry ARGs and reveals that captive pandas have a higher abundance of ARGs compared to wild pandas. The research also uncovers a genetic similarity between ARGs in pandas and those found in the human gut microbiome, suggesting a potential for gene transfer. These findings contribute to our understanding of ARGs in giant pandas and emphasize the importance of further research on resistance gene expression. [Extracted from the article]

Simple Summary: Enterotoxigenic Escherichia coli (ETEC) is an important pathogen that causes diarrhea in both humans and animals, thereby posing a serious threat to public health and animal agriculture. Bacillus subtilis, a probiotic, offers a novel approach to reduce the need for antibiotics and plays a crucial role in treating various intestinal diseases. We previously isolated a strain of B. subtilis HH2 from giant panda feces, which has shown multiple beneficial functions in vitro and in vivo. However, studies on the protective effect of B. subtilis on companion animals with orally administered ETEC have not been reported. Therefore, we explored the effects of B. subtilis HH2 on the fecal microbiota, intestinal barrier integrity, and non-specific immunity in beagles challenged with ETEC. Experimental results showed that B. subtilis HH2 could alleviate diarrhea caused by ETEC, improve non-specific immunity and intestinal barrier integrity, and modulate gut microbiota. Notably, more indicators are needed to determine its protective effect on beagles in future studies. This study evaluated the protective effect of Bacillus subtilis HH2 on beagles orally challenged with enterotoxigenic Escherichia coli (ETEC). We assessed the physiological parameters and the severity of diarrhea, as well as the changes in three serum immunoglobulins (IgG, IgA, and IgM), plasma diamine oxidase (DAO), D-lactate (D-LA), and the fecal microbiome. Feeding B. subtilis HH2 significantly reduced the severity of diarrhea after the ETEC challenge (p < 0.05) and increased serum levels of IgG, IgA, and IgM (p < 0.01). B. subtilis HH2 administration also reduced serum levels of DAO at 48 h after the ETEC challenge (p < 0.05), but no significant changes were observed in D-LA (p > 0.05). Oral ETEC challenge significantly reduced the richness and diversity of gut microbiota in beagles not pre-fed with B. subtilis HH2 (p < 0.05), while B. subtilis HH2 feeding and oral ETEC challenge significantly altered the gut microbiota structure of beagles (p < 0.01). Moreover, 14 days of B. subtilis HH2 feeding reduced the relative abundance of Deinococcus-Thermus in feces. This study reveals that B. subtilis HH2 alleviates diarrhea caused by ETEC, enhances non-specific immunity, reduces ETEC-induced damage to the intestinal mucosa, and regulates gut microbiota composition. [ABSTRACT FROM AUTHOR]

The gut microbiome of the giant panda (Ailuropoda melanoleuca) plays a vital role in nutrient acquisition from its specialized bamboo diet. Giant panda cubs harbour significantly different gut microbiota during their growth and development when feeding on milk before switching to bamboo. The fetal gut is sterile, and following birth, mother‐to‐infant microbial transmission has been implicated as a seeding source for the infant gut microbiota. Details of this transmission in giant pandas remain unclear. In this study, faecal samples were collected from seven panda mother–cub pairs when the cubs were 4–16 months old. Additional samples from the cubs' diet, soil and drinking water, and multiple body sites of the mothers were collected. Bacterial 16S rRNA gene sequencing and shotgun metagenomic sequencing were performed to determine the source and potential transmission routes of the cub gut microbiome. Source tracking analysis showed that maternal vagina, milk and faeces were the primary contributory sources of microbes, shaping the cub gut microbiome. Bacterial species from maternal faeces persisted the longest in the cub gut. Bacterial species in the diet contributed to the microbial community. Metagenomics analysis indicated that the predicted metabolic pathways of the gut microbiome also varied at different growth stages. Gut colonization with bacteria from various body sites of the mothers provides a foundational microbial community that is beneficial in fulfilling the evolving dietary needs of the cubs. This study suggests that mother‐to‐cub transmission is indispensable in shaping the gut microbiome of the developing panda cub. [ABSTRACT FROM AUTHOR]

Bamboo is the main food source of the giant panda. To increase bamboo intake in captive giant pandas, we studied factors affecting the bamboo intake. Fourteen healthy captive giant pandas in Dujiangyan Base of China Conservation and Research Center for The Giant Panda ("Dujiangyan Base" for short) were selected as research objects. A bamboo feeding experiment was conducted to study the effects of seasons, bamboo age, slope orientations where bamboo grows and felling-feeding time on bamboo intake of the giant panda. We found that the type of bamboo that captive giant pandas feed on was abundant in spring and summer, but relatively homogeneous in winter. With the increase of bamboo age, the intake of bamboo leaves decreased, while bamboo culms increased. The feed intake of 1-year-old bamboo leaves and 5-year-old bamboo culms reached the highest respectively. The slope orientation also affected the panda's bamboo intake, and the bamboo growing on sunny slopes or semi-sunny slopes was more favored by captive giant pandas. Moreover, the bamboo intake reached the highest when felling-feeding time was less than 24 h. In short, we confirmed that seasons, bamboo age, slope orientations and felling-feeding time were factors affecting bamboo intake for captive giant pandas. This study was expected to provide scientific guidance improving the feeding behavior management of captive giant pandas. [ABSTRACT FROM AUTHOR]

Simple Summary: Giant pandas are key protected animals in China. Baylisascaris schroederi, a parasitic nematode, is one of the main health risks threatening them. We used four anthelmintics—pyrantel pamoate (PYR), mebendazole (MBZ), albendazole (ABZ), and ivermectin (IVM)—on 22 enrolled giant pandas. The fecal egg count reduction (FECR) proportions were calculated using both the Markov chain Monte Carlo (MCMC) Bayesian mathematical model and the arithmetic mean based on fecal egg count data. Anthelmintic resistance (AR) was assessed based on the criteria recommended by the World Association for the Advancement of Veterinary Parasitology (WAAVP). We found that the nematode was suspected to be resistant to PYR. The number of eggs per gram in the feces of giant panda enrolled in the present study was increased near the end of the experiment. Baylisascaris schroederi is one of the main health risks threatening both wild and captive giant pandas. The administration of anthelmintics is a common method to effectively control B. schroederi infection, but there is a notable risk of anthelmintic resistance (AR) after long-term, constant use of anthelmintics. Four anthelmintics—pyrantel pamoate (PYR), mebendazole (MBZ), albendazole (ABZ), and ivermectin (IVM)—were each administered separately at intervals of 2 months to 22 enrolled giant pandas. The fecal egg count reduction (FECR) proportions were calculated by both the Markov chain Monte Carlo (MCMC) Bayesian mathematical model and the arithmetic mean. AR was assessed based on the criteria recommended by the World Association for the Advancement of Veterinary Parasitology (WAAVP). The estimated prevalence of B. schroederi infection was 34.1%. After treatment with PYR, MBZ, ABZ, and IVM, it was determined that MBZ, ABZ, and IVM were efficacious against B. schroederi, while nematodes were suspected to be resistant to PYR according to the fecal egg count reduction (FECR) proportions. [ABSTRACT FROM AUTHOR]

Simple Summary: Giant pandas are considered a national treasure in China. Understanding the changes in immunity and energy metabolism of giant pandas with age might help develop more scientific guidelines for managing the health of giant pandas. Here, 48 giant pandas were collected, and their transcriptome was analyzed. The results showed that the immune system and energy metabolism of giant pandas changed significantly with age. A low reproductive rate coupled with human activities has endangered the giant panda, a species endemic to southwest China. Although giant pandas feed almost exclusively on bamboo, they retain carnivorous traits and suffer from carnivorous diseases. Additionally, their immune system is susceptible to aging, resulting in a reduced ability to respond to diseases. This study aimed to determine the genes and pathways expressed differentially with age in blood tissues. The differentially expressed genes in different age groups of giant pandas were identified by RNA-seq. The elderly giant pandas had many differentially expressed genes compared with the young group (3 years old), including 548 upregulated genes and 401 downregulated genes. Further, functional enrichment revealed that innate immune upregulation and adaptive immune downregulation were observed in the elderly giant pandas compared with the young giant pandas. Meanwhile, the immune genes in the elderly giant pandas changed considerably, including genes involved in innate immunity and adaptive immunity such as PLSCR1, CLEC7A, CCL5, CCR9, and EPAS1. Time series analysis found that giant pandas store glycogen by prioritizing fat metabolism at age 11, verifying changes in the immune system. The results reported in this study will provide a foundation for further research on disease prevention and the energy metabolism of giant pandas. [ABSTRACT FROM AUTHOR]

Giant pandas feed almost exclusively on bamboo but miss lignocellulose-degrading genes. Their gut microbiome may contribute to their nutrition; however, the limited access to pandas makes experimentation difficult. In vitro incubation of dung samples is used to infer gut microbiome activity. In pandas, such tests indicated that green leaves are largely fermented to ethanol at neutral pH and yellow pith to lactate at acidic pH. Pandas may feed on either green leaves or yellow pith within the same day, and it is unclear how pH, dung sample, fermentation products and supplied bamboo relate to one another. Additionally, the gut microbiome contribution to solid bamboo digestion must be appropriately assessed. Here, gut microbiomes derived from dung samples with mixed colors were used to ferment green leaves, also by artificially adjusting the initial pH. Gut microbiomes digestion of solid lignocellulose accounted for 30–40% of the detected final fermentation products. At pH 6.5, mixed-color dung samples had the same fermentation profile as green dung samples (mainly alcohols), while adjusting the initial pH to 4.5 resulted in the profile of yellow dung samples (mainly lactate). Metaproteomics confirmed that gut microbiomes attacked hemicellulose, and that the panda's alpha amylase was the predominant enzyme (up to 75%). [ABSTRACT FROM AUTHOR]

Helminth diseases have long been a threat to the health of humans and animals. Roundworms are important organisms for studying parasitic mechanisms, disease transmission and prevention. The study of parasites in the giant panda is of importance for understanding how roundworms adapt to the host. Here, we report a high‐quality chromosome‐scale genome of Baylisascaris schroederi with a genome size of 253.60 Mb and 19,262 predicted protein‐coding genes. We found that gene families related to epidermal chitin synthesis and environmental information processes in the roundworm genome have expanded significantly. Furthermore, we demonstrated unique genes involved in essential amino acid metabolism in the B. schroederi genome, inferred to be essential for the adaptation to the giant panda‐specific diet. In addition, under different deworming pressures, we found that four resistance‐related genes (glc‐1, nrf‐6, bre‐4 and ced‐7) were under strong positive selection in a captive population. Finally, 23 known drug targets and 47 potential drug target proteins were identified. The genome provides a unique reference for inferring the early evolution of roundworms and their adaptation to the host. Population genetic analysis and drug sensitivity prediction provide insights revealing the impact of deworming history on population genetic structure of importance for disease prevention. [ABSTRACT FROM AUTHOR]

The pharmacokinetics of levofloxacin mesylate in healthy adult giant panda is unknown. In this study, the pharmacokinetics of levofloxacin after intramuscular administration at a dose of 2 mg/kg and oral administration at a dose of 3 mg/kg in healthy adult giant pandas was determined. Levofloxacin concentrations in plasma were determined using liquid chromatography. In the levofloxacin intramuscular administration group, the absorption and elimination half‐lives of the drug were determined to be 0.123 (range: 0.02) hr and 5.402 (range: 0.70) hr, respectively. In the levofloxacin oral administration group, the absorption and elimination half‐lives were determined to be 0.325 (range: 0.02) hr and 7.143 (range: 0.63) hr, respectively. Furthermore, the blood–drug concentration of levofloxacin was found to be above 1 μg/ml after 8 hr of intramuscular administration and above 0.5 μg/ml after 12 hr of oral administration. In this study, HPLC conditions and pretreatment methods of plasma samples were optimized and a detection method was established. Our results indicated that in healthy adult giant pandas, levofloxacin was rapidly absorbed and slowly eliminated. This study will therefore provide to be a guide in veterinary research and will be helpful in the rational use of levofloxacin in giant panda. [ABSTRACT FROM AUTHOR]

The giant panda is known worldwide for having successfully moved to a diet almost exclusively based on bamboo. Provided that no lignocellulose-degrading enzyme was detected in panda's genome, bamboo digestion is believed to depend on its gut microbiome. However, pandas retain the digestive system of a carnivore, with retention times of maximum 12 h. Cultivation of their unique gut microbiome under controlled laboratory conditions may be a valid tool to understand giant pandas' dietary habits, and provide valuable insights about what component of lignocellulose may be metabolized. Here, we collected gut microbiomes from fresh fecal samples of a giant panda (either entirely green or yellow stools) and supplied them with green leaves or yellow pith (i.e., the peeled stem). Microbial community composition was substrate dependent, and resulted in markedly different fermentation profiles, with yellow pith fermented to lactate and green leaves to lactate, acetate and ethanol, the latter to strikingly high concentrations (∼3%, v:v, within 3.5 h). Microbial metaproteins pointed to hemicellulose rather than cellulose degradation. The alpha-amylase from the giant panda (E.C. 3.2.1.1) was the predominant identified metaprotein, particularly in reactors inoculated with pellets derived from fecal samples (up to 60%). Gut microbiomes assemblage was most prominently impacted by the change in substrate (either leaf or pith). Removal of soluble organics from inocula to force lignocellulose degradation significantly enriched Bacteroides (in green leaf) and Escherichia / Shigella (in yellow pith). Overall, different substrates (either leaf or pith) markedly shaped gut microbiome assemblies and fermentation profiles. The biochemical profile of fermentation products may be an underestimated factor contributing to explain the peculiar dietary behavior of giant pandas, and should be implemented in large scale studies together with short-term lab-scale cultivation of gut microbiomes. [ABSTRACT FROM AUTHOR]

Reintroduction is a key approach in the conservation of endangered species. In recent decades, many reintroduction projects have been conducted for conservation purposes, but the rate of success has been low. Given the important role of gut microbiota in health and diseases, we questioned whether gut microbiota would play a crucial role in giant panda's wild‐training process. The wild procedure is when captive‐born babies live with their mothers in a wilderness enclosure and learn wilderness survival skills from their mothers. During the wild‐training process, the baby pandas undergo wilderness survival tests and regular physical examinations. Based on their performance through these tests, the top subjects (age 2–3 years old) are released into the wild while the others are translocated to captivity. After release, we tracked one released panda (Zhangxiang) and collected its fecal samples for 5 months (January 16, 2013 to March 29 2014). Here, we analyzed the Illumina HiSeq sequencing data (V4 region of 16S rRNA gene) from captive pandas (n = 24), wild‐training baby pandas (n = 8) of which 6 were released and 2 were unreleased, wild‐training mother pandas (n = 8), one released panda (Zhangxiang), and wild giant pandas (n = 18). Our results showed that the gut microbiota of wild‐training pandas is significantly different from that of wild pandas but similar to that of captive ones. The gut microbiota of the released panda Zhangxiang gradually changed to become similar to those of wild pandas after release. In addition, we identified several bacteria that were enriched in the released baby pandas before release, compared with the unreleased baby pandas. These bacteria include several known gut‐health related beneficial taxa such as Roseburia, Coprococcus, Sutterella, Dorea, and Ruminococcus. Therefore, our results suggest that certain members of the gut microbiota may be important in panda reintroduction. [ABSTRACT FROM AUTHOR]

The gut microbiota diversity of eight panda cubs was assessed during a dietary switch. Gut microbiota diversity of panda cubs significantly decreased after bamboo consumption. Carnivorous species living on a plant-based diet possess low microbial diversity. Mice were fed a bamboo diet but did not display low gut microbiota diversity. Giant pandas have an exclusive diet of bamboo; however, their gut microbiotas are more similar to carnivores than herbivores in terms of bacterial composition and their functional potential. This is inconsistent with observations that typical herbivores possess highly diverse gut microbiotas. It is unclear why the gut bacterial diversity of giant pandas is so low. Herein, the dynamic variations in the gut microbiota of eight giant panda cubs were measured using 16S rRNA gene paired-end sequencing during a dietary switch. Similar data from red panda (an herbivorous carnivore) and carnivorous species were compared with that of giant pandas. In addition, mice were fed a high-bamboo diet (80% bamboo and 20% rat feed) to determine whether a bamboo diet could lower the gut bacterial diversity in a non-carnivorous digestive tract. The diversity of giant panda gut microbiotas decreased significantly after switching from milk and complementary food to bamboo diet. Carnivorous species living on a plant-based diet, including giant and red pandas, possess a lower microbial diversity than other carnivore species. Mouse gut microbiota diversity significantly increased after adding high-fibre bamboo to their diet. Findings suggest that a very restricted diet (bamboo) within a carnivorous digestive system might be critical for shaping a low gut bacterial diversity in giant pandas. [ABSTRACT FROM AUTHOR]

The giant pandas’ (Ailuropoda melanoleuca) reproductive strategy is unique among mammals. Yet there are characteristics of giant panda behaviour we do not fully understand. Probably one of the least understood is the assumption that in captivity virtually all giant panda females rear only one cub when twins are born and abandon the other if given the chance. So far, only two females have raised twins simultaneously, but just with intensive human assistance. This case-study marks the first successful rearing of giant panda twins in captivity entirely by the mother. Using video data for detailed behavioural observations, we provide the first behavioural assessment of a giant panda female raising two cubs simultaneously without direct human assistance or disturbance. We compared the maternal behaviour during the denning period of twin cubs raised in 2016 with two singleton cubs born 2007 and 2010. YANG YANG, the dam, rested less and interacted more with the twins than with the singletons in the first month postpartum and invested a greater part of her daily time budget on rearing the twins. We discuss potential favourable factors for the autonomous twin-rearing of a female giant panda, which could serve as a model for similar efforts elsewhere. [ABSTRACT FROM AUTHOR]

The blood-sucking tick Haemaphysalis hystricis is a common ectoparasite of the giant panda and represents a significant threat to both wild and captive populations. Herein, the complete mitogenome of H. hystricis was sequenced using Illumina sequencing technology. The complete mitogenome sequence was 14,715 bp in size and encoded 37 genes including 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. Phylogeny revealed that two isolates of H. hystricis, regardless of host origins and locations, grouped together and had a closer relationship with Haemaphysalis longicornis than other tick species among the genus Haemaphysalis. The cumulative mitochondrial DNA data provides novel resources for genetic and phylogenetic studies of Haemaphysalis ticks. [ABSTRACT FROM AUTHOR]

The tick Haemaphysalis flava (Acari, Ixodidae) is an obligatory blood-feeding ectoparasite of the giant panda and is also a vector for transmission of pathogenic microorganisms. In this study, the complete mitochondrial genome of this tick was sequenced through Illumina sequencing technology. The genome was 14,699 bp in length and encoded 37 genes including 13 protein-coding genes, 22 transfer RNAs and two ribosomal RNAs. Phylogeny revealed that three isolates of H. flava, regardless of host origins and locations, clustered together and formed a monophyletic relationship with Haemaphysalis japonica, supporting their species validity among the genus Haemaphysalis. These cumulative mitochondrial DNA data provides insights into phylogenetic studies among Haemaphysalis ticks. [ABSTRACT FROM AUTHOR]

Reproductive monitoring for captive breeding in giant pandas is based on behavioural observation and non-invasive hormone analysis. In urine, interpretation of results requires normalisation due to an animal’s changing hydration. Correction of urinary concentrations based on creatinine is the gold standard. In this study, a largely unexplored, easy-to-perform normalisation technique, based on urinary specific gravity (USpG), was examined and compared to creatinine. To this extent, six cycles from two female pandas (SB741(1) and SB569(5)) were monitored through urine analysis for oestrogen, progesterone, ceruloplasmin and 13,14-dihydro-15-keto-PGF2a (PGFM). The Pearson’s correlation between creatinine and USpG was high (r = 0.805–0.894; p < 0.01), indicative for a similar performance of both normalisation methods. However, generally lower values were observed during pro-oestrus and primary (progesterone) rise. This could be associated with huge shifts in appetite, monitored by faecal output (kg) with an averaged > 50% decrease during oestrus and >50% increase during primary progesterone rise. In parallel, respectively highest and lowest creatinine and USpG levels, were measured, with creatinine obviously more affected as a result of linkage with muscle tissue metabolism affected by reproductive hormones. As a consequence, metabolite levels were significantly different between both corrected datasets with significantly higher oestrogen peak levels during oestrus ranging from 2.13–86.93 and 31.61–306.45 ng/mL (USpG correction) versus 2.33–31.20 and 36.36–249.05 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively, and significant lower progesterone levels during primary progesterone rise ranging from 0.35–3.21 and 0.85–6.80 ng/mL (USpG correction) versus 0.52–10.31 and 2.10–272.74 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively. Consequently, USpG correction rendered unbiased profiles, less subject to variation and metabolic artefacts and therefore allowed a more straightforward identification of peak oestrogen and onset of secondary progesterone rise, being potentially advantageous for future studies unravelling key giant panda reproductive events, including (delayed) implantation. The alternative application of USpG as a normalisation factor was further supported by its easy application and environmental and technical robustness. [ABSTRACT FROM AUTHOR]

Reintroducing captive-born individuals of some extirpated flagship animal species is a helpful and remedy measure for promoting biodiversity conservation, and a successful reintroduction relies heavily on public support. However, little information is available on the factors affecting the public support for reintroduction of large carnivore species. In order to evaluate public support and willingness to pay for the Giant Panda Reintroduction Project (GPRP), we conducted 1100 interviews in August 2014 at Huaying city, Sichuan, China, close to where captive-born giant pandas ( Ailuropoda melanoleuca) will be reintroduced. The results showed that local people strongly support this project and that 78.5 % of them are willing to pay an annual contribution. Per capita annual payments averaged USD31, and the potential value of donations to the GPRP in Huaying is about 11 million USD per year. Factors like interviewees' residential area, occupation, education level, liking of wildlife, level of concern for wildlife conservation, the degree of familiarity with giant pandas, and the frequency of visiting captive giant pandas significantly affected their attitudes and willingness to pay for the project. The notion of 'because I love pandas' was the main reason why respondents were willing to pay for the project; respondents' doubts about the appropriate use of donated funds made them be unwilling to pay for the project. The results suggest that the GPRP is highly and socially acceptable amongst locals due to perceived social, economic and ecological benefits of the reintroduction. These findings clearly indicate, for the chances of reintroduction to be most socially accepted, governments should improve management and accountability when using donated funds, and create more opportunities for the public to engage with giant pandas, thereby encouraging people to become involved in conservation work benefiting conservation for the giant pandas and other endangered species. [ABSTRACT FROM AUTHOR]

To study the typical vaginal bacterial flora of giant pandas ( Ailuropoda melanoleuca), we took vaginal swabs for the sake of bacterial isolation, from 24 healthy female giant pandas. A total of 203 isolates were identified, representing a total of 17 bacterial species. The most common bacteria isolated were Lactobacillus spp. (54.2%, 13/24), followed by Staphylococcus epidermidis (41.7%, 10/24) and Escherichia coli (33.3%, 8/24). Some opportunistic pathogenic bacteria, such as Peptostreptococcus spp. , Klebsiella pneumoniae, and Proteus mirabilis, were also isolated but showed no pathology. Antimicrobial susceptibility testing of aerobic bacterial isolates was performed with the disk diffusion method. Of the 152 isolates, resistance was most frequently observed with chloramphenicol (17.8%), followed by tetracycline (14.5%), ciprofloxacin (12.5%), streptomycin (11.8%), and florfenicol (11.8%), whereas 7.2% were multidrug resistant. This is the first report of the normal culturable vaginal bacterial flora of giant pandas and the antimicrobial susceptibility patterns of the isolates. [ABSTRACT FROM AUTHOR]

In the giant panda, adaptation to a high-fiber environment is a first step for the adequate functioning of intestinal bacteria, as the high cellulose content of the gut due to the panda's vegetarian appetite results in a harsh environment. As an excellent producer of several enzymes and vitamins, Bacillus subtilis imparts various advantages to animals. In our previous study, we determined that several strains of B. subtilis isolated from pandas exhibited good cellulose decomposition ability, and we hypothesized that this bacterial species can survive in and adapt well to a high-fiber environment. To evaluate this hypothesis, we employed RNA-Seq technology to analyze the differentially expressed genes of the selected strain B. subtilis HH2, which demonstrates significant cellulose hydrolysis of different carbon sources (cellulose and glucose). In addition, we used bioinformatics software and resources to analyze the functions and pathways of differentially expressed genes. Interestingly, comparison of the cellulose and glucose groups revealed that the up-regulated genes were involved in amino acid and lipid metabolism or transmembrane transport, both of which are involved in cellulose utilization. Conversely, the down-regulated genes were involved in non-essential functions for bacterial life, such as toxin and bacteriocin secretion, possibly to conserve energy for environmental adaptation. The results indicate that B. subtilis HH2 triggered a series of adaptive mechanisms at the transcriptional level, which suggests that this bacterium could act as a probiotic for pandas fed a high-fiber diet, despite the fact that cellulose is not a very suitable carbon source for this bacterial species. In this study, we present a model to understand the dynamic organization of and interactions between various functional and regulatory networks for unicellular organisms in a high-fiber environment. [ABSTRACT FROM AUTHOR]

The captive genetic management of threatened species strives to preserve genetic diversity and avoid inbreeding to ensure populations remain available, healthy, and viable for future reintroduction. Determining and responding to the genetic status of captive populations is therefore paramount to these programs. Here, we genotyped 19 microsatellite loci for 240 captive giant pandas (Ailuropoda melanoleuca) (∼64% of the captive population) from four breeding centers, Wolong (WL), Chengdu (CD), Louguantai (LGT), and Beijing (BJ), and analyzed 655 bp of mitochondrial DNA control region sequence for 220 of these animals. High levels of genetic diversity and low levels of inbreeding were estimated in the breeding centers, indicating that the captive population is genetically healthy and deliberate further genetic input from wild animals is unnecessary. However, the LGT population faces a higher risk of inbreeding, and significant genetic structure was detected among breeding centers, with LGT–CD and WL–BJ clustering separately. Based on these findings, we highlight that: 1) the LGT population should be managed as an independent captive population to resemble the genetic distinctness of their Qinling Mountain origins; 2) exchange between CD and WL should be encouraged because of similar wild founder sources; 3) the selection of captive individuals for reintroduction should consider their geographic origin, genetic background, and genetic contribution to wild populations; and 4) combining our molecular genetic data with existing pedigree data will better guide giant panda breeding and further reduce inbreeding into the future. [ABSTRACT FROM PUBLISHER]

Relocation of captive giant pandas refers to releasing captive-bred giant pandas to the wild with the aim of bolstering and restoring the wild panda population. This method should be one of the key approaches adopted as part of conservation measures implemented for this endangered species. Compared to translocation of wild pandas to a new place, relocation of captive-bred pandas faces more difficulties and challenges. At present, giant panda relocation programs are still at the exploration stage in China. Focusing on captive-bred giant pandas, individual animal traits are discussed here. It is indicated that a well-managed husbandary system which enables the captive pandas to develop normally as wild ones is one of the key factors for relocation programs. Accordingly, based on some latest achievements, some measures to improve husbandary management are presented. This summary provides scientific data concerning ongoing giant panda reintroduction work, particularly on selecting animals for reintroduction and on training animals for release. [ABSTRACT FROM AUTHOR]

Infant giant pandas are highly vocal during the first few weeks of life, producing vocalisations that are characterised by noisy, aperiodic segments. The aperiodic character of many animal vocalisations results from irregular vibratory regimes of the vocal folds, and one proposed function of this so-called nonlinear phenomena ( NLP) in animal vocalisations is to convey information about the caller's arousal state. This hypothesis was tested in the vocalisations of six hand-reared giant panda cubs recorded during handling and feeding procedures that had been categorised into low- and high-arousal contexts based on quantified motor activity. Ninety-three per cent of the vocalisations contained NLP, including deterministic chaos and subharmonics. Vocalisations produced in the high-arousal contexts, however, were characterised by an increase in chaos, as well as increased call duration and [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) is an important component of captive breeding programs for endangered species, such as the giant panda. The panda has been the subject of increasingly successful captive breeding programs involving a compilation of assisted breeding techniques, including AI using cryopreserved spermatozoa. AI implementation is currently hampered by a lack of understanding of the factors that may cause failure. We investigated factors influencing the probability of success of AI for 14 giant panda females housed at the China Center for Research and Conservation of the Giant Panda (CCRCGP) inseminated in a total of 20 instances using cryopreserved spermatozoa from 11 males currently residing in 6 different captive breeding institutions. One of the pandas was the oldest giant panda female to ever successfully conceive from AI (20.5 years old). The success of AI was significantly affected by the timing of AI in relationship to both timing of peak urinary estrogen of the female and percent decline in urinary estrogen between the peak level and the first AI attempt. Our results suggest that the window for successful AI in giant pandas may be narrower than previously suspected, although individual differences in rates of decline in urinary estrogen may reflect some degree of variation in this crucial window across females. Our results are consistent with recent research on pandas and other species that demonstrates the efficacy of cryopreserved spermatozoa for AI and highlights the need for more in-depth analysis of factors related to female physiology that may influence its success. Zoo Biol 31:561-573, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The Baylisascaris schroederi infection rate among wild giant pandas may reach over 50% or even 100%, making it one of the leading causes of death from primary or secondary infection in wild populations. Until now, little was known about how protective immunity to B. schroederi infection could be achieved. The present study was conducted to evaluate the immunogenicity and protective efficacy of recombinant Bs-Ag1 from B. schroederi, by cloning the full-length Bs-Ag1 gene of B. schroederi and expressing it in a heterologous host, Escherichia coli BL21. In mice vaccinated with rBs-Ag1 coupled with Freund's complete adjuvant (FCA), there was a significant reduction (69.2%) in the recovery of challenged B. schroederi L3 compared with either nonvaccinated controls or mice vaccinated with FCA alone. Our study supports the use of Bs-Ag1 as a potential candidate for vaccination against B. schroederi infection and provides basic data for further vaccination trials with mixtures of antigens (with Bs-Ag2 and Bs-Ag3) to B. schroederi. [ABSTRACT FROM AUTHOR]

Abstract: Serum samples from 92 giant pandas in three captive facilities were tested for antibodies against five viruses of carnivores. Antibody titers against canine distemper virus (CDV) in two facilities in which giant pandas were vaccinated were variable. The canine adenovirus (CAV-1) and canine parvovirus (CPV) titers in vaccinated group were both positive, but titers were not high and varied among individual except one vaccinated panda had extremely high CAV-1 titer, indicating infection with the field virus following vaccination. Our results suggest that the vaccines used for these giant pandas do not elicit consistent antibody titers. Antibody titers against CDV, CPV and CAV-1 in unvaccinated giant pandas were highly variable, especially CPV titer. Almost half of sera were CPV antibody positive, and CPV titers were high enough to suggest infection with the virus. Canine coronavirus (CCV) and canine parainfluenza virus (CPIV) titers were not detected in all serum samples. The results of this study emphasize the need for research on infectious diseases of giant pandas and development of suitable vaccines for the species. [Copyright &y& Elsevier]

Wildlife conservation is often challenged by a lack of knowledge about the reproduction biology and adaptability of endangered species. Although monitoring steroids and related molecules can increase this knowledge, the applicability of current techniques (e.g. immunoassays) is hampered by species-specific steroid metabolism and the requisite to avoid invasive sampling. This study presents a validated steroidomics method for the (un)targeted screening of a wide range of sex and stress steroids and related molecules in urine using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). In total, 50 steroids (conjugated and non-conjugated androgens, estrogens, progestogens and glucocorticoids) and 6 prostaglandins could be uniquely detected. A total of 45 out of 56 compounds demonstrated a detection limit below 0.01 ng μL−1. Excellent linearity (R2 > 0.99), precision (CV < 20 %), and recovery (80–120 %) were observed for 46, 41, and 39 compounds, respectively. Untargeted screening of pooled giant panda and human samples yielded 9691 and 8366 features with CV < 30 %, from which 84.1 % and 83.0 %, respectively, also demonstrated excellent linearity (R2 > 0.90). The biological validity of the method was investigated on male and female giant panda urine (n = 20), as well as pooled human samples (n = 10). A total of 24 different steroids were detected with clear qualitative and quantitative differences between human and giant panda samples. Furthermore, expected differences were revealed between female giant panda samples from different reproductive phases. In contrast to traditional biomonitoring techniques, the developed steroidomics method was able to screen a wide range of compounds and provide information on the putative identities of metabolites potentially important for reproductive monitoring in giant pandas. These results illustrate the advancements steroidomics brings to the field of wildlife biomonitoring in the pursuit to better understand the biology of endangered species. [Display omitted] • A validated steroidomics method in urine using UHPLC-HRMS. • Method allows targeted and untargeted screening of steroids and related molecules. • Method validation revealed excellent linearity, precision and recovery. • Biological validity proven with giant panda and human urine samples. • Aimed to be applicable on different wildlife species for conservation research. [ABSTRACT FROM AUTHOR]

To explore the effects of the mutations of ATP6 and ATP8 genes on energy metabolism and genetic structure, we sequenced the ATP6 and ATP8 genes of Ailuropoda melanoleuca. Our results showed that ATP8 is a conserved gene and ATP6 gene is positively selected during the evolution of the giant panda population with a low genetic diversity. Population expansion was observed in the giant panda group. The T179C mutation on Haplotype7 made the production of a potential phosphorylation site. This non-synonymous mutation may occur at the post-translational modification site that have a potential effect on the function of ATP synthase, related to the maintenance of body temperature of pandas at low metabolic rates. [ABSTRACT FROM AUTHOR]

Female giant pandas show complex reproductive traits, being seasonally monoestrus, displaying a variable length embryonic diapause and exhibiting pseudopregnancy. Currently, there is no confirmatory non-invasive biomarker of blastocyst implantation or pregnancy. This study aimed to monitor urinary estrogens across gestation in pregnancy (n = 4), pseudopregnancy (n = 4) and non-birth cycles (n = 5) in the giant panda. A pregnancy-specific profile of estrogens corrected for urinary specific gravity was identified during the gestation period. Pregnant females showed increasing concentrations of estrogens for 29 days until birth, no increase was observed during pseudopregnancy and the two profiles were distinguishable from each other for the final 2 weeks of the cycle suggesting the estrogens are of placental origin. This allowed a nomogram, starting at a known fixed point during the cycle, to be created and tested with cycles of known outcome, and cycles which were inseminated but did not result in a birth. Non-birth profiles showed deviations from that of pregnancy. We believe these deviations indicate the point of failure of the placenta to support a developing cub. Non-invasive longitudinal monitoring of estrogen concentrations therefore has the potential to be developed as a panda pregnancy test to predict viable cub development. [ABSTRACT FROM AUTHOR]

The independent dietary shift from carnivore to herbivore with over 90% being bamboo in the giant and the red pandas is of great interests to biologists. Although previous studies have shown convergent evolution of the giant and the red pandas at both morphological and molecular level, the evolution of the gut microbiota in these pandas remains largely unknown. The goal of this study was to determine whether the gut microbiota of the pandas converged due to the same diet, or diverged. We characterized the fecal microbiota from these two species by pyrosequencing the 16S V1-V3 hypervariable regions using the 454 GS FLX Titanium platform. We also included fecal samples from Asian black bears, a species phylogenetically closer to the giant panda, in our analyses. By analyzing the microbiota from these 3 species and those from other carnivores reported previously, we found the gut microbiotas of the giant pandas are distinct from those of the red pandas and clustered closer to those of the black bears. Our data suggests the divergent evolution of the gut microbiota in the pandas. [ABSTRACT FROM AUTHOR]

The giant panda, a strict herbivore that feeds on bamboo, still retains a typical carnivorous digestive system. Reference catalogs of microbial genes and genomes are lacking, largely limiting the antibiotic resistome and functional exploration of the giant panda gut microbiome. Here, we integrated 177 fecal metagenomes of captive and wild giant pandas to construct a giant panda integrated gene catalog (GPIGC) comprised of approximately 4.5 million non-redundant genes and reconstruct 393 metagenome-assembled genomes (MAGs). Taxonomic and functional characterization of genes revealed that the captivity of the giant panda significantly changed the core microbial composition and the distribution of microbial genes. Higher abundance and prevalence of antibiotic resistance genes (ARGs) were detected in the guts of captive giant pandas, and ARG distribution was influenced by geography, for both captive and wild individuals. Escherichia, as the prevalent genus in the guts of captive giant pandas, was the main carrier of ARGs, meaning there is a high risk of ARG transmission by Escherichia. We also found that multiple mcr gene variants, conferring plasmid-mediated mobile colistin resistance, were widespread in the guts of captive and wild giant pandas. There were low proportions of carbohydrate-active enzyme (CAZyme) genes in GPIGC and MAGs compared with several omnivorous and herbivorous mammals. Many members of Clostridium MAGs were significantly enriched in the guts of adult, old and wild giant pandas. The genomes of isolates and MAGs of Clostridiaceae harbored key genes or enzymes in complete pathways for degrading lignocellulose and producing short-chain fatty acids (SCFAs), indicating the potential of these bacteria to utilize the low-nutrient bamboo diet. Overall, our data presented an exhaustive reference gene catalog and MAGs in giant panda gut and provided a comprehensive understanding of the antibiotic resistome and microbial adaptability for a high-lignocellulose diet. [Display omitted] • Captivity of giant panda changed the composition and diversity of gut microbiome. • Lifestyle and geography influenced on ARGs abundance and distribution, respectively. • Escherichia was the main carrier of ARGs in the guts of captive giant pandas. • Clostridium harbored key genes in metabolisms of lignocellulose. [ABSTRACT FROM AUTHOR]

[Display omitted] Extant giant pandas are divided into Sichuan and Qinling subspecies. The giant panda has many species-specific characteristics, including comparatively small organs for body size, small genitalia of male individuals, and low reproduction. Here, we report the most contiguous, high-quality chromosome-level genomes of two extant giant panda subspecies to date, with the first genome assembly of the Qinling subspecies. Compared with the previously assembled giant panda genomes based on short reads, our two assembled genomes increased contiguity over 200-fold at the contig level. Additional sequencing of 25 individuals dated the divergence of the Sichuan and Qinling subspecies into two distinct clusters from 10,000 to 12,000 years ago. Comparative genomic analyses identified the loss of regulatory elements in the dachshund family transcription factor 2 (DACH2) gene and specific changes in the synaptotagmin 6 (SYT6) gene, which may be responsible for the reduced fertility of the giant panda. Positive selection analysis between the two subspecies indicated that the reproduction-associated IQ motif containing D (IQCD) gene may at least partly explain the different reproduction rates of the two subspecies. Furthermore, several genes in the Hippo pathway exhibited signs of rapid evolution with giant panda-specific variants and divergent regulatory elements, which may contribute to the reduced inner organ sizes of the giant panda. [ABSTRACT FROM AUTHOR]

A recent study has described the normal vaginal bacterial community in giant pandas, but there is a lack of knowledge of the fungal community residing in the vagina of giant pandas. In order to comprehensively understand the vaginal fungal microbial diversity and abundance in giant pandas, high throughput sequencing was used to analyse the ITS1 region, based on thirteen samples taken from the pandas' vaginas, which were grouped by sampling points and age. The results showed that the most abundant phyla were Basidiomycota (73.37%), followed by Ascomycota (20.04%), Zygomycota (5.23%), Glomeromycota (0.014%) and Chytridiomycota (0.006%). At the genus level, Guehomyces (37.92%) was the most abundant, followed by Cladosporium (9.072%), Trichosporon (6.2%) and Mucor (4.97%). Furthermore, Candida only accounted for a low percentage of the vaginal fungal community. With the saturation of rarefaction curves and fungal diversity indices, the samples from Dujiangyan and Chungking Safari Park (DC group) showed a higher fungal species richness and diversity than other living environments. Shannon diversity indices showed significant difference between group WL (Wolong nature reserve) and DC ( P  < .05). Additionally, a higher diversity was found in ten to fifteen years old (Group 2) than other groups. Group 2 and Group 3 displayed significant differences in the diversities of their vaginal fungal communities ( P  < .05). These data that has been collected from this research will be helpful for further study to improve the reproductive status of giant pandas. [ABSTRACT FROM AUTHOR]

Data on numbers and distribution of free-ranging giant panda are essential to the formulation of effective conservation strategies. There is still no ideal method to identify individuals and sex this species. The traditional bite-size method using bamboo fragments in their feces lacks accuracy. The modern DNA-based estimation is expensive and demands fresh samples. The lack of identifiable individual features on panda pelage and no apparent sexual dimorphism impede reliable estimation from camera trap images. Here, we propose an innovative and non-invasive technique to identify and sex this species using a footprint identification technique (FIT). It is based on a pairwise comparison of trails (unbroken series of footprints) using discriminant analysis, with a Ward's clustering method. We collected footprints from 30 captive animals to train our algorithm and used another 11 animals for model validation. The accuracy for individual identification was > 90% for individuals with more than six footprints and 89% with fewer footprints per trail. The accuracy for sex discrimination was about 84% using a single footprint and 91% using trails. This cost-effective method provides a promising future for monitoring wild panda populations and understanding their dynamics and especially useful for monitoring reintroduced animals after the detachment of GPS collars. The data collection protocol is straightforward and accessible to citizen scientists and conservation professionals alike. [ABSTRACT FROM AUTHOR]

The total arsenic concentration and the arsenic speciation in urine and feces samples of the two Giant Pandas living at Vienna zoo and of their feed, bamboo, were determined with ICPMS and HPLC-ICPMS. Urine was the main excretion route and accounted for around 90% of the ingested arsenic. The urinary arsenic concentrations were very high, namely up to 179 μg/L. Dimethylarsinic acid (DMA) was the dominating arsenic compound in the urine samples and ranged from 73 to 92% of the total arsenic, which is unusually high for a terrestrial mammal. The feces samples contained around 70% inorganic arsenic and 30% DMA. The arsenic concentrations in the bamboo samples were between 16 and 920 μg/kg dry mass. The main arsenic species in the bamboo extracts was inorganic arsenic. This indicates that the Giant Panda possesses a unique way of very efficiently methylating and excreting the provided inorganic arsenic. This could be essential for the survival of the animal in its natural habitat, because parts of this area are contaminated with arsenic. [ABSTRACT FROM AUTHOR]

While the health effects of the colonization of the reproductive tracts of mammals by bacterial communities are widely known, there is a dearth of knowledge specifically in relation to giant panda microbiomes. In order to investigate the vaginal and uterine bacterial diversity of healthy giant pandas, we used high-throughput sequence analysis of portions of the 16S rRNA gene, based on samples taken from the vaginas (GPV group) and uteri (GPU group) of these animals. Results showed that the four most abundant phyla, which contained in excess of 98% of the total sequences, were Proteobacteria (59.2% for GPV and 51.4% for GPU), Firmicutes (34.4% for GPV and 23.3% for GPU), Actinobacteria (5.2% for GPV and 14.0% for GPU) and Bacteroidetes (0.3% for GPV and 10.3% for GPU). At the genus level, Escherichia was most abundant (11.0%) in the GPV, followed by Leuconostoc (8.7%), Pseudomonas (8.0%), Acinetobacter (7.3%), Streptococcus (6.3%) and Lactococcus (6.0%). In relation to the uterine samples, Janthinobacterium had the highest prevalence rate (20.2%), followed by Corynebacterium (13.2%), Streptococcus (19.6%), Psychrobacter (9.3%), Escherichia (7.5%) and Bacteroides (6.2%). Moreover, both Chao1 and abundance-based coverage estimator (ACE) species richness indices, which were operating at the same sequencing depth for each sample, demonstrated that GPV had more species richness than GPU, while Simpson and Shannon indices of diversity indicated that GPV had the higher bacterial diversity. These findings contribute to our understanding of the potential influence abnormal reproductive tract microbial communities have on negative pregnancy outcomes in giant pandas. [ABSTRACT FROM AUTHOR]

Abstract: 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 4.60×3.99μm (n=50). The isolate was genetically analyzed using the partial 18S rRNA, 70kDa 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. [Copyright &y& Elsevier]

• Reported miRNA profiles of giant panda's heart, liver, spleen, lung, and kidney. • Differential expression analysis identified tissue-specific DEmiRs of five tissues. • Identified tissue-specific enrichment GO terms and KEGG pathways. • Providing unique genetic resources for giant panda. microRNA (miRNA) is a small endogenous noncoding RNA molecule that plays multiple roles in regulating most biological processes. However, for China's national treasure giant panda, a world-famous rare and protected species, reports of its miRNA have been found only in blood and breast milk. To explore the miRNA expression differences between different giant panda tissues, here, we generated the miRNA profiles of five tissues (heart, liver, spleen, lung, and kidney) from four giant pandas with Illumina Hiseq 2500 platform, and filtered the differentially expressed miRNAs (DEmiRs) in each tissue, predicted the target genes of miRNA from each tissue based on the DEmiRs. Then, the GO and KEGG enrichment analysis were conducted using the target genes predicted from DEmiRs in each tissue. The RNA-seq generated an average of 0.718 GB base per sample. A total of 1,256 known miRNAs and 12 novel miRNAs were identified, and there were 215, 131, 185, 83, and 126 tissue-specific DEmiRs filtered in the heart, liver, spleen, lung, and kidney, respectively, including miR-1b-5p, miR-122-5p, miR-143, miR-126-5p, and miR-10b-5p, respectively. The predicted target genes, including MYL2, LRP5, MIF, CFD, and PEBP1 in the heart, liver, spleen, lung, and kidney, respectively, were closely associated with tissue-specific biological functions. The enrichment analysis results of target genes showed tissue-specific characteristics, such as the significantly enriched GO terms extracellular matrix in the heart and insulin-like growth factor binding in the liver. The miRNA profiles of the heart, liver, spleen, lung, and kidney of giant panda have been reported in this study, it reveals the miRNA expression differences between different tissues of the giant panda, and provides valuable genetic resources for the further related molecular genetic research of the rare and protected species giant panda and other mammals. [ABSTRACT FROM AUTHOR]