ABSTRACT High‐altitude environments expose mammals and their gut symbionts to multifaceted stressors—hypoxia, cold, and intense UV radiation. Whether gut microbial communities undergo compositional restructuring in response to these stressors, and whether such restructuring carries translational value for captive conservation, remain unresolved questions. Here, we integrated deep shotgun metagenomics (≥ 15 Gb per sample), untargeted fecal metabolomics, and culturomics in 75 captive forest musk deer (Moschus berezovskii Flerov, 1929) housed at high altitude (~3900 m) and low altitude (~1450 m) facilities under uniform husbandry. Neutral community modeling showed a greater contribution of deterministic processes at high altitude (only 34.3% of species conformed to neutral expectations vs. 89.3% at low altitude), consistent with stronger environmental filtering. At high altitude, we observed enrichment of a functionally coherent guild of short‐chain fatty acid (SCFA)‐producing bacteria—centered on Flavonifractor plautii, Intestinimonas butyriciproducens, and Enterococcus faecium—that formed antagonistic co‐occurrence networks with opportunistic pathogens including Clostridioides difficile and Campylobacter species, mirroring SCFA enrichment in phylogenetically diverse high‐altitude mammals. Fecal metabolomics revealed coordinated shifts in urolithin biosynthesis, branch‐specific regulation of the tryptophan–kynurenine pathway, and energy metabolism remodeling, all robustly predicted by microbiome composition via neural network modeling. Culturomics yielded seven safety‐validated isolates with confirmed gastrointestinal stress tolerance and broad‐spectrum pathogen‐antagonistic activity in vitro. These findings provide an actionable framework for altitude‐informed facility siting, fecal microbiota transplantation (FMT) donor selection, host‐derived probiotic development, and non‐invasive health surveillance in captive endangered species, and are broadly transferable to other taxa facing microbiome‐associated disease pressure in captivity.

Age-related changes throughout the lifespan are known to influence gut microbiota composition, microbial functional potential, and host-associated metabolic processes. Understanding these age-related variations is important for elucidating their potential physiological implications at different life stages. However, information regarding the gut microbiome and metabolomic characteristics of super-geriatric captive giant pandas (Ailuropoda melanoleuca) remains limited. In this study, fecal samples were collected from adult and super-geriatric captive giant pandas and analyzed using metagenomic sequencing combined with untargeted metabolomics. The gut microbiota of super-geriatric individuals exhibited a marked decrease in Bacillota and an enrichment of Pseudomonadota compared with adult individuals. Functional profiling revealed age-associated shifts in microbial metabolic potential, with a transition from biosynthesis-dominated pathways toward pathways related to substrate degradation and energy utilization. Metabolomic analyses further revealed pronounced metabolic alterations in super-geriatric giant pandas, including elevated levels of unsaturated fatty acids and changes in bile acid-related metabolites. Alterations in gut microbiota composition, particularly the relative enrichment of Pseudomonadota-associated taxa, were associated with inflammation-related metabolic features. Collectively, these findings indicate coordinated changes in gut microbial composition and metabolic profiles during aging. Overall, this study characterizes age-associated alterations in gut microbiota structure and fecal metabolic signatures in super-geriatric captive giant pandas, providing a scientific basis for future studies on microbiota-metabolism interactions and for improving nutritional management and health monitoring strategies in aged individuals of this endangered species.

Adaptation to arboreal environments requires overcoming gravitational constraints, driving repeated morphological innovations across snake lineages. Among these, elongated tails represent a key adaptation that enhances branch-gripping ability, yet the genomic changes underlying this trait remain poorly understood. Here, ancestral state reconstruction revealed that arboreality evolved independently in multiple snake clades, with tail elongation as a recurrent morphological adaptation. To investigate its genetic underpinnings, we generated a high-quality, chromosome-level genome assembly for the green cat snake (Boiga cyanea) and performed comparative analyses with the Asian vine snake (Ahaetulla prasina). We identified accelerated evolution in genes associated with somite specification, a critical process for axial elongation, and detected positive selection in key somitogenesis regulators, including HES7 and TBX18. Notably, LOXL3, which contributes to somite boundary formation, exhibited a conserved amino acid substitution in both arboreal lineages. In addition, convergent divergence of conserved nonexonic elements (CNEs) was observed in genomic regions linked to the GDF11-LIN28-HOX13 pathway, which governs the axial-to-tail transition. Functional assays confirmed that divergence in these CNEs alters regulatory activity, potentially modulating gene expression within critical developmental pathways. Collectively, our findings establish a genomic framework for snake axial elongation, highlighting how arboreal specialization shaped tail length evolution.

Microbiome-mediated longevity mechanisms in conservation wildlife remain largely uncharacterized, particularly in species with specialized diets where host-microbe coevolution plays essential compensatory roles. We conducted cross-sectional metagenomic profiling of gut microbiota in 82 captive giant pandas (Ailuropoda melanoleuca) spanning juvenile, adult, and geriatric cohorts. The cohort included 14 long-lived individuals (≥ 25 years). Our approach integrated enterotype analysis, functional pathway characterization, and lactic acid bacteria (LAB) isolation. Age-specific compositional shifts occurred throughout the lifespan, characterized by reciprocal dominance between Bacillota (declining from 84.77% to 10.26%) and Pseudomonadota (increasing from 12.74% to 81.86%). Streptococcus dominated adolescent microbiomes, followed by a transition to cellulose-degrading taxa in young adults. Enterotype analysis revealed four distinct community types with age-dependent succession patterns. Long-lived pandas exhibited elevated abundance of Akkermansia muciniphila, Bifidobacterium longum, Lactobacillus intestinalis, and Ruminococcus gnavus. Functional profiling revealed age-dependent shifts from nucleotide and amino acid biosynthesis in young animals to lipid metabolism and amino acid degradation in aged individuals. Carbohydrate-active enzyme analysis demonstrated enrichment of cellulolytic enzymes (such as GH3, GH5, GH9) in long-lived adults. Biosynthetic gene cluster profiling revealed progressive age-associated accumulation of genes encoding NRPS, RiPP-like, arylpolyene, and terpene biosynthetic pathways. Culture-based isolation from fecal samples of long-lived giant pandas yielded 98 LAB strains representing eight species (Lactobacillus johnsonii, Limosilactobacillus reuteri, L. intestinalis, among others), including 89 non-hemolytic strains that were sensitive to six common antibiotics. These findings suggest potential cross-species conservation of microbiome-longevity mechanisms, with implications for understanding physiological adaptations in specialist herbivores and applications in conservation medicine.

While diet is known to regulate the composition, function, and diversity of the human gut microbiome, its effects on wildlife remain understudied. Here, noninvasive sampling methods were first used to conduct metagenomic analyses of the gut microbiomes of 10 protected wild animals in the Wolong Nature Reserve. There were significant differences in microbiota composition and function between herbivores and carnivores. Herbivores exhibited higher microbial diversity and evenness (Shannon and Pielou indices), with Bacillota and Acinetobacter predominating, whereas carnivores were enriched in Pseudomonadota and Escherichia. Cellulose-degrading bacterium Ruminococcus champanellensis was abundant in herbivores, while Rhodococcus and Pediococcus, which were associated with toxin degradation and pathogen inhibition, were more prevalent in carnivores. Carnivores showed higher lipid metabolism and protein degradation, as evidenced by the enrichment of leucyl aminopeptidase and oligopeptidase B, while herbivores demonstrated superior cellulose and starch digestion, characterized by the enrichment of cellulose 1,4-beta-cellobiosidase. Stochastic processes shaped gut microbiome assembly, especially in herbivores. Potential health risks from pathogens such as Escherichia and Listeria were identified, and Escherichia abundance was positively correlated with niche width. Furthermore, the findings suggest that high-altitude environments may promote the persistence and spread of pathogens. Overall, our findings underscore the intricate linkages between diet, gut microbiota composition, assembly processes, and host ecology in protected wildlife, address a key knowledge gap, and provide important theoretical and practical insights for ecological conservation, species restoration, and environmental management.

The giant panda is a vulnerable species endemic to China and serves as a flagship species for biodiversity conservation. Accurately assessing population size in small populations has always been the foundation of conservation efforts. Owing to higher dropout rate and instability, the microsatellite marker has always caused the overestimation of the population size. The microhaplotype genetic marker, which is made from several adjacent SNPs, is as stable as the SNP marker and as polymorphic as the microsatellite marker. This study screened a panel of 32 candidate microhaplotype markers with an average polymorphic information content of 0.628 from a whole‐genome sequencing dataset of 195 giant pandas. Afterwards, we successfully demonstrated the feasibility and reliability of the microhaplotype markers by genotyping half of them via the Illumina paired‐end sequencing, with the markers amplified in four quadruplex PCR reactions, ranging from 142 and 234 bp in length. The cumulative non‐exclusion probabilities for the full and half panel of markers are 6.477 × 10 −27 and 1.444 × 10 −13 , respectively. These markers offer a potential tool for individual identification, sex determination, the management and conservation of giant pandas.

Specialized herbivores like giant pandas (Ailuropoda melanoleuca), red pandas (Ailurus fulgens), and bamboo rats, which primarily consume bamboo, are at risk of nutrient deficiencies, particularly vitamin B12 (VB12), potentially leading to cardiovascular diseases. This study explored the effects of VB12 supplementation on cardiovascular health in silver star bamboo rats (Rhizomys pruinosus). We first conducted a comprehensive genome annotation of R. pruinosus, laying the foundation for in-depth evolutionary studies. Comparative transcriptomic analysis revealed that genes related to cardiovascular disease (e.g., Sgcb, Adcy2, Itga1, Itgb8, Ifng, and Gpc1) were upregulated in the livers of R. pruinosus compared to carnivorous and omnivorous rodents, indicating a higher cardiovascular disease risk. After 60 days of VB12 supplementation, liver transcriptome analysis revealed significant improvements in cardiovascular health markers, including reduced cholesterol synthesis and enhanced fatty acid metabolism. Serum biochemical assays indicated that VB12 supplementation led to reduced homocysteine levels, decreased low-density lipoprotein (LDL)-to-high-density lipoprotein (HDL) ratios, and increased the apolipoprotein A-to-apolipoprotein B ratio. These findings suggest that VB12 may mitigate cardiovascular disease risk and could be considered in the dietary management of specialized bamboo-eating species. Our study provides valuable insights into disease prevention strategies for these species with similar dietary habits.

The hypoxic, cold, and high-ultraviolet radiation environments at high altitude pose severe challenges to mammalian immune and metabolic systems. However, little is known about how captive forest musk deer adapt to high-altitude environments and their seasonal variations. This study analyzed peripheral blood transcriptomes of 33 captive forest musk deer (Moschus berezovskii) at high altitude (~3900 m) and low altitude (~1450 m) during autumn-winter and spring-summer seasons. Results revealed comprehensive immune suppression in the high-altitude group during autumn-winter (downregulation of complement system CFB/C2/C3, interferon pathway genes including FLT3, with only natural killer (NK) cell PRKCQ upregulated), coupled with energy-conserving metabolic reprogramming (altered carbohydrate metabolism, inhibited lipid synthesis, fat mobilization, suppressed protein degradation). During spring-summer, neutrophil antimicrobial responses (SLPI/NCF1/ELANE) and humoral immunity (B cell differentiation genes PAX5/RUNX1; class-switch enzyme AICDA) partially recovered while cellular immunity (IL15/B2M) remained suppressed, accompanied by enhanced anabolic metabolism and adipocyte differentiation. Notably, NK cell-mediated cytotoxicity showed selective enhancement despite comprehensive immune suppression, representing an energy-efficient innate defense strategy. This study provides the first characterization of seasonal immune dynamics in a high-altitude cervid species. These findings reveal persistent immune constraints in high-altitude populations and provide theoretical foundations for disease prevention and health management in captive forest musk deer at high altitudes.

Addressing the escalating global burdens of inflammatory bowel disease and antimicrobial resistance demanded innovative food-based approaches to fortify gut health and suppress pathogens. We introduced a novel edible probiotic, Enterococcus faecalis HY0110, isolated from marine Thunnus thynnus. Through comprehensive in vitro, in vivo, and metabolomic analyses, we demonstrated its superior antibacterial effects compared to Lactobacillus rhamnosus GG, along with significantly enhanced antioxidant and free-radical scavenging capacities. Notably, elevated acetic acid production strongly correlated with its antimicrobial efficacy (R ≥ 0.999). HY0110 also exerted antiproliferative effects on HT-29 colorectal cancer cells by attenuating β-catenin and BCL-2 expression while upregulating pro-apoptotic markers P62 and c-PARP. In a DSS-induced colitis model, HY0110 alleviated inflammation, restored gut microbial homeostasis, and enhanced deterministic processes in community assembly dynamics. Furthermore, fermenting Periplaneta americana powder with HY0110 triggered extensive metabolic remodeling, notably a 668.73-fold rise in astragaloside A, plus increases in L-Leucyl-L-Alanine, S-lactoylglutathione, and 16,16-dimethyl prostaglandin A1. These shifts diminished harmful components and amplified essential amino acids and peptides to bolster immune modulation, redox balance, and anti-inflammatory responses. This work established a transformative paradigm for utilizing marine probiotics and novel entomological substrates in functional foods, presenting strategic pathways for precision nutrition and inflammatory disease management.

DNA methylation plays a crucial role in species development and environmental adaptation. In mammals, there are significant dietary changes from infancy to adulthood. Notably, the red panda transitions from milk consumption as juveniles to a bamboo-based diet as adults, with significant alterations in food characteristics and nutritional content. However, the regulatory role of DNA methylation in this process remains unclear. In this study, we investigate the regulatory role of DNA methylation on the expression of digestive and metabolic genes in the liver and pancreas during the red panda’s dietary transition from suckling stage to adulthood. Our findings reveal significant differences in DNA methylation patterns before and after dietary transition, highlighting the specific alterations in the methylation profiles of genes involved in lipid, carbohydrate, and amino acid metabolism. We found that perilipin-4 (PLIN4) is hypomethylated and highly expressed in the liver of adult red pandas, facilitating lipid droplet formation and storage, crucial for adapting to the low-fat content in bamboo. In contrast, genes like lipoprotein lipase (LPL), crucial for lipid breakdown, exhibited hypermethylated with low-expression patterns, reflecting a reduced lipid metabolism capacity in adults. Carbohydrate metabolism-related genes like ADH4 and FAM3C are hypomethylated and highly expressed in adults, enhancing glycogen production and glucose utilization. Genes involved in protein metabolism like CTSZ and GLDC, exhibit hypomethylated with high-expression and hypermethylated with low-expression patterns in the pancreas of adults, respectively, contributing to protein metabolism balance post-weaning. This study reveals the regulatory role of DNA methylation in the dietary transition of red pandas from milk to bamboo and provides methylation evidence for the molecular regulation of adaptive expression of digestive and metabolic genes in red pandas with specialized diets.

The prevalence of abscess disease significantly limits the population expansion of captive forest musk deer, which is an endangered species protected by the legislation of China. Our prior work had demonstrated that Trueperella pyogenes and Pseudomonas aeruginosa are two important microorganisms in causing the abscess disease of forest musk deer, and furazolidone could inhibit the growth and virulence of the pathogens in vitro. In this study, the in vivo protection activity of furazolidone was evaluated by using mouse models chronically infected with T. pyogenes and P. aeruginosa. The results showed that furazolidone treatment significantly increased the survival rates of mice in the co-infection group, all the mice survived at 14 days post-infection. The damage degree of the lung tissues caused by bacterial infection was ameliorated by the treatment of furazolidone from 7 to 14 days post-infection, which also reduced the residual bacterial burden in the lungs. Compared to the untreated control group, the expression levels of genes activated by the quorum-sensing system of P. aeruginosa and the core virulence regulatory genes of T. pyogenes were significantly suppressed by furazolidone. In addition, the results of transcriptomic analyses showed that 270 DEGs were identified in the co-infection group. This finding further revealed that the immune responses of mice could be enhanced by the treatment of furazolidone, and this might also contribute to the clearance of bacteria from the lungs. Therefore, this study clearly reveals the protection activity of furazolidone against P. aeruginosa and T. pyogenes infection, and thus provides a promising candidate in the treatment of abscess disease.

Epigenetic regulation plays an important role in the evolution of species adaptations, yet little information is available on the epigenetic mechanisms underlying the adaptive evolution of bamboo-eating in both giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens). To investigate the potential contribution of epigenetic to the adaptive evolution of bamboo-eating in giant and red pandas, we performed hepatic comparative transcriptome and methylome analyses between bamboo-eating pandas and carnivorous polar bears (Ursus maritimus). We found that genes involved in carbohydrate, lipid, amino acid, and protein metabolism showed significant differences in methylation and expression levels between the two panda species and polar bears. Clustering analysis of gene expression revealed that giant pandas did not form a sister group with the more closely related polar bears, suggesting that the expression pattern of genes in livers of giant pandas and red pandas have evolved convergently driven by their similar diets. Compared to polar bears, some key genes involved in carbohydrate metabolism and biological oxidation and cholesterol synthesis showed hypomethylation and higher expression in giant and red pandas, while genes involved in fat digestion and absorption, fatty acid metabolism, lysine degradation, resistance to lipid peroxidation and detoxification showed hypermethylation and low expression. Our study elucidates the special nutrient utilization mechanism of giant pandas and red pandas and provides some insights into the molecular mechanism of their adaptive evolution of bamboo feeding. This has important implications for the breeding and conservation of giant pandas and red pandas.

IntroductionIn an era increasingly defined by the challenge of antibiotic resistance, this study offers groundbreaking insights into the antibacterial properties of two distinct Lactiplantibacillus plantarum strains, TE0907 and TE1809, hailing from the unique ecosystem of Bufo gargarizans. It uniquely focuses on elucidating the intricate components and mechanisms that empower these strains with their notable antibacterial capabilities.MethodsThe research employs a multi-omics approach, including agar diffusion tests to assess antibacterial efficacy and adhesion assays with HT-29 cells to understand the preliminary mechanisms. Additionally, gas chromatography-mass spectrometry (GC-MS) is employed to analyze the production of organic acids, notably acetic acid, and whole-genome sequencing is utilized to identify genes linked to the biosynthesis of antibiotics and bacteriocin-coding domains.ResultsThe comparative analysis highlighted the exceptional antibacterial efficacy of strains TE0907 and TE1809, with mean inhibitory zones measured at 14.97 and 15.98 mm, respectively. A pivotal discovery was the significant synthesis of acetic acid in both strains, demonstrated by a robust correlation coefficient (cor ≥ 0.943), linking its abundance to their antimicrobial efficiency. Genomic exploration uncovered a diverse range of elements involved in the biosynthesis of antibiotics similar to tetracycline and vancomycin and potential regions encoding bacteriocins, including Enterolysin and Plantaricin.ConclusionThis research illuminates the remarkable antibacterial efficacy and mechanisms intrinsic to L. plantarum strains TE0907 and TE1809, sourced from B. gargarizans. The findings underscore the strains' extensive biochemical and enzymatic armamentarium, offering valuable insights into their role in antagonizing enteric pathogens. These results lay down a comprehensive analytical foundation for the potential clinical deployment of these strains in safeguarding animal gut health, thereby enriching our understanding of the role of probiotic bacteria in the realm of antimicrobial interventions.

With the context of increasing global concern about antibiotic resistance, the study presented the antibacterial properties of two novel edible bacterial strains, Pediococcus pentosaceus TE0302 and Lactococcus lactis TE0801, derived from the unique ecosystem of Bufo gargarizans. . Significant antibacterial efficacy of these strains, with inhibitory zones of 14.77 mm and 15.26 mm, were confirmed through a multi-omics approach involving agar diffusion assays, mouse models, metabolomics, and genomics. These strains also mitigate pathogen-induced colitis and improve gut microbiota by increasing beneficial bacteria and reducing harmful ones. A crucial investigation is that the bacteria's substantial production of N-butyric acid is linked to antimicrobial effectiveness with a correlation coefficient >= 0.972. Genomic analysis shown that these strains feature diverse biosynthetic pathways similar to established antibiotics like Tetracycline and Vancomycin and contain genes for powerful bacteriocin production, especially Enterolysin_A. Pan-genome analysis indicated the unique gene counts of 124 and 179, greatly enhanced the genomic diversity compared to typical reference strains to emphasize the novelty. This study can facilitate a clear understanding of probiotics' genetic and biochemical properties. It highlighted the pivotal role in providing sustainable solutions to combat antibiotic resistance and improve gastrointestinal health, foreshadowing a new era of food science biotechnology.

Red pandas evolved from carnivores to herbivores and are unique within Carnivora. Red pandas and carnivorous mammals consume milk during the suckling period, while they consume bamboo and meat during the adult period, respectively. Red pandas and carnivorous mammal ferrets have a close phylogenetic relationship. To further investigate the molecular mechanisms of dietary changes and nutrient utilization in red pandas from suckling to adult, comparative analysis of the whole transcriptome was performed on stomach tissues from red pandas and ferrets during the suckling and adult periods. The main results are as follows: (1) we identified ncRNAs for the first time in stomach tissues of both species, and found significant expression changes of 109 lncRNAs and 106 miRNAs in red pandas and 756 lncRNAs and 109 miRNAs in ferrets between the two periods; (2) up-regulated genes related to amino acid transport regulated by lncRNA-miRNA-mRNA networks may efficiently utilize limited bamboo amino acids in adult red pandas, while up-regulated genes related to amino acid degradation regulated by lncRNAs may maintain the balance of amino acid metabolism due to larger daily intakes in adult ferrets; and (3) some up-regulated genes related to lipid digestion may contribute to the utilization of rich nutrients in milk for the rapid growth and development of suckling red pandas, while up-regulated genes associated with linoleic acid metabolism regulated by lncRNA-miRNA-mRNA networks may promote cholesterol decomposition to reduce health risks for carnivorous adult ferrets. Collectively, our study offers evidence of gene expression adaptation and ncRNA regulation in response to specific dietary changes and nutrient utilization in red pandas during suckling and adult periods.

MicroRNAs can regulate various biological functions including cell proliferation, differentiation, embryo formation, and implantation. The giant panda exhibits embryonic diapause, with embryo development resuming in late pregnancy. However, the changes in microRNAs during late pregnancy remain poorly understand. After mating, plasma samples were collected on day 40 of early pregnancy (EP; n = 3) and 30 days before delivery of late pregnancy (LP; n = 3). Following microRNAs screening, a total of 120 microRNAs were detected in the plasma exosomes of pregnant pandas. Nine differentially expressed microRNAs (DEmicroRNAs) were identified in LP compared to EP, including three that were upregulated and six that were downregulated. Notably, miR-25b and miR-47 were significantly downregulated in LP group. All DEmicroRNAs were predicted to target a total of 2,675 genes. Pathway enrichment analysis of these target genes revealed significant enrichment in the MAPK and Rap1 signaling pathways, which are closely related to cell proliferation, differentiation, and cell-cell and cell-matrix interactions. Analysis of protein-protein interaction networks showed that most of the hub genes (five out of eight), including Fgfr1, Fgf2, Fgf18, Erbb4, and Kras within the MAPK and Rap1 pathways are associated with the cell proliferation and differentiation. Significantly, Erbb4 was regulated by significantly differentially expressed miRNA-47. We suggest that plasma exosomal microRNAs are involved in cell proliferation and differentiation during embryonic development by regulating key hub genes within MAPK and Rap1 pathways. These findings provided new insights into the development of giant panda embryos.

A non-invasive, fast, and reliable method for sex identification of the giant panda ( Ailuropoda melanoleuca ) was developed by genotyping five SNPs located at the sex chromosome amelogenin ( AMEL ) paralogs. The giant panda’s sex can be accurately distinguished by the homozygous or heterozygous SNP genotypes. Mass spectrometry assay and Sanger sequencing both successfully genotyped tissue and fresh fecal samples collected from known-sex giant pandas. Additionally, we simulated the conditions of wild fecal samples, which were naturally degraded for up to two weeks, and successfully genotyped these samples by mass spectrometry assay in parallel. These results suggest that this is a powerful method for sex determination in highly degraded and lower concentrated DNA samples.

The changes in the expression of genes related to digestion and metabolism may be various in different dietary mammals from juvenile to adult, especially, the giant panda ( Ailuropoda melanoleuca ) and red panda ( Ailurus fulgens ), which were once carnivores but have shifted to being specialized bamboo eaters, are unique features of their changes are more unclear. To elucidate the changing patterns of gene expression related to digestion and metabolism from juvenile to adult in different dietary mammals, we performed transcriptome analysis of the liver or pancreas in giant and red pandas, herbivorous rabbits ( Oryctolagus cuniculus ) and macaques ( Macaca mulatta ), carnivorous ferrets ( Mustela putorius furo ), and omnivorous mice ( Mus musculus ) from juvenile to adult. During the transition from juvenile to adulthood, giant and red pandas, as well as rabbits and macaques, show significant upregulation of key genes for carbohydrate metabolism, such as starch hydrolysis and sucrose metabolism, and unsaturated fatty acid metabolism, such as linoleic acid, while there is no significant difference in the expression of key genes for fatty acid β-oxidation. A large number of amino acid metabolism related genes were upregulated in adult rabbits and macaques compared to juveniles. While adult giant and red pandas mainly showed upregulation of key genes for arginine synthesis and downregulation of key genes for arginine and lysine degradation. In adult stages, mouse had significantly higher expression patterns in key genes for starch hydrolysis and sucrose metabolism, as well as lipid and protein metabolism. In contrast to general expectations, genes related to lipid, amino acid and protein metabolism were significantly higher expressed in adult group of ferrets, which may be related to their high metabolic levels. Our study elucidates the pattern of changes in the expression of genes related to digestion and metabolism from juvenile to adult in different dietary mammals, with giant and red pandas showing adaptations associated with specific nutritional limitations of bamboo.

The giant panda (Ailuropoda melanoleuca) is the epitome of a flagship species for wildlife conservation and also an ideal model of adaptive evolution. As an obligate bamboo feeder, the giant panda relies on the olfaction for food recognition. The number of olfactory receptor (OR) genes and the rate of pseudogenes are the main factors affecting the olfactory ability of animals. In this study, we used the chromosome-level genome of the giant panda to identify OR genes and compared the genome sequences of OR genes with five other Ursidae species (spectacled bear (Tremarctos ornatus), American black bear (Ursus americanus), brown bear (Ursus arctos), polar bear (Ursus maritimus) and Asian black bear (Ursus thibetanus)). The giant panda had 639 OR genes, including 408 functional genes, 94 partial OR genes and 137 pseudogenes. Among them, 222 OR genes were detected and distributed on 18 chromosomes, and chromosome 8 had the most OR genes. A total of 448, 617, 582, 521 and 792 OR genes were identified in the spectacled bear, American black bear, brown bear, polar bear and Asian black bear, respectively. Clustering analysis based on the OR protein sequences of the six species showed that the OR genes distributed in 69 families and 438 subfamilies based on sequence similarity, and the six mammals shared 72 OR gene subfamilies, while the giant panda had 31 unique OR gene subfamilies (containing 35 genes). Among the 35 genes, there are 10 genes clustered into 8 clusters with 10 known human OR genes (OR8J3, OR51I1, OR10AC1, OR1S2, OR1S1, OR51S1, OR4M1, OR4M2, OR51T1 and OR5W2). However, the kind of odor molecules can be recognized by the 10 known human OR genes separately, which needs further research. The phylogenetic tree showed that 345 (about 84.56%) functional OR genes were clustered as Class-II, while only 63 (about 15.44%) functional OR genes were clustered as Class-I, which required further and more in-depth research. The potential odor specificity of some giant panda OR genes was identified through the similarity to human protein sequences. Sequences similar to OR2B1, OR10G3, OR11H6 and OR11H7P were giant panda-specific lacking, which may be related to the transformation and specialization from carnivore to herbivore of the giant panda. Since our reference to flavoring agents comes from human research, the possible flavoring agents from giant panda-specific OR genes need further investigation. Moreover, the conserved motifs of OR genes were highly conserved in Ursidae species. This systematic study of OR genes in the giant panda will provide a solid foundation for further research on the olfactory function and variation of the giant panda.

To investigate a safe and effective approach for enhancing the in vivo expression of recombinant genes and improving the systemic immunity of animals against infectious diseases, we employed the interleukin-7 (IL-7) gene from Tibetan pigs to construct a recombinant eukaryotic plasmid (VRTPIL-7). We first examined VRTPIL-7's bioactivity on porcine lymphocytes in vitro and then encapsulated it with polyethylenimine (PEI), chitosan copolymer (CS), PEG-modified galactosylated chitosan (CS-PEG-GAL) and methoxy poly (ethylene glycol) (PEG) and PEI-modified CS (CS-PEG-PEI) nanoparticles using the ionotropic gelation technique. Next, we intramuscularly or intraperitoneally injected mice with various nanoparticles containing VRTPIL-7 to evaluate their immunoregulatory effects in vivo. We observed a significant increase in neutralizing antibodies and specific IgG levels in response to the rabies vaccine in the treated mice compared to the controls. Treated mice also exhibited increased leukocytes, CD8+ and CD4+ T lymphocytes, and elevated mRNA levels of toll-like receptors (TLR1/4/6/9), IL-1, IL-2, IL-4, IL-6, IL-7, IL-23, and transforming growth factor-beta (TGF-β). Notably, the recombinant IL-7 gene encapsulated in CS-PEG-PEI induced the highest levels of immunoglobulins, CD4+ and CD8+ T cells, TLRs, and cytokines in the mice's blood, suggesting that chitosan-PEG-PEI may be a promising carrier for in vivo IL-7 gene expression and enhanced innate and adaptive immunity for the prevention of animal diseases.