Single-nucleus RNA and ATAC sequencing uncovers the molecular and cellular characteristics in the musk gland of Chinese forest musk deer (Moschus berezovskii).

The Chinese forest musk deer (FMD; Moschus berezovskii) is an endangered artiodactyl mammal. Musk secreted by the musk gland of male has extremely high economic and medicinal value. However, the molecular and cellular characteristics of the musk gland have not been studied. Here, we investigated the diversity and transcriptional composition of musk gland cell types and the effect of cell type-specific chromatin accessibility on gene expression using single-nucleus RNA sequencing (snRNA-seq) and single-nucleus ATAC sequencing (snATAC-seq) association analysis. Based on uniform manifold approximation and projection (UMAP) analysis, we identified 13 cell types from the musk gland, which included two different acinar cells (cluster 0 and cluster 10). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that many pathways related to musk secretion were enriched in acinar cells. Our analysis also revealed acinar cell core transcription factors and core target genes, and further constructed acinar cell-specific regulatory networks. In cluster 0, 11 core target genes (Nedd4l, Adcy9, Akr1c1, Vapb, Me1, Acsl1, Acss3, Srd5a1, Scnn1a, Acadm, and Nceh1) possibly related to musk secretion were regulated by 24 core transcription factors (SP3, NFIC, NR6A1, EHF, RUNX1, TFAP2A, RREB1, GRHL2, NFIB, ELF1, MAX, KLF5, REL, HES1, POU2F3, TFDP1, NR2C1, ATF7, MEIS1, NR4A2, NFIA, PBX1, ZNF652, and NFKB1). In cluster 10, four core target genes (Akr1c1, Pcca, Atp1b1, and Sgk1) possibly related to musk secretion were regulated by 10 core transcription factors (BARX2, EHF, PBX1, RUNX1, NFIB, FOXP1, KLF3, KLF6, ETV6, and NR3C2). Moreover, the credibility of snRNA-seq and snATAC-seq data was verified by fluorescence in situ hybridization and immunohistochemistry. Finally, cell communication analysis demonstrated that the two types of acinar cells mainly have communications in musk secretion-related processes. In conclusion, we provided important insights and invaluable resources for the molecular and cellular characteristics of the musk gland, which will lay a foundation for the study of musk secretion mechanism in the future.

Molecular detection of Cryptosporidium in Alpine musk deer (Moschus chrysogaster) in Gansu Province, Northwest China.

Cryptosporidium spp. are protozoa commonly found in domestic and wild animals. Limited information is available on Cryptosporidium in deer worldwide. In this study, 201 fecal samples were collected from Alpine musk deer on three farms in Gansu Province, China. Detection and subtyping of Cryptosporidium were performed by PCR and sequence analysis of the SSU rRNA and gp60 genes. The prevalence of Cryptosporidium infection in Alpine musk deer was 3.9% (8/201), with infection rates of 1.0% (1/100), 2.8% (1/36), and 9.2% (6/65) in three different farms. All positive samples for Cryptosporidium were from adult deer. Two Cryptosporidium species were identified, including C. parvum (n = 2) and C. xiaoi (n = 6). The C. parvum isolates were subtyped as IIdA15G1, while the C. xiaoi isolates were subtyped as XXIIIa (n = 2) and XXIIIg (n = 4). The IIdA15G1 subtype of C. parvum was found for the first time in deer. These results provide important insights into the identity and human infectious potential of Cryptosporidium in farmed Alpine musk deer.

Maslinic acid supplementation prevents di(2-ethylhexyl) phthalate-induced apoptosis via PRDX6 in peritubular myoid cells of Chinese forest musk deer.

Chinese forest musk deer (FMD), an endangered species, have exhibited low reproductive rates even in captivity due to stress conditions. Investigation revealed the presence of di(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, in the serum and skin of captive FMDs. Feeding FMDs with maslinic acid (MA) has been observed to alleviate the stress response and improve reproductive rates, although the precise molecular mechanisms remain unclear. Therefore, this study aims to investigate the molecular mechanisms underlying the alleviation of DEHP-induced oxidative stress and cell apoptosis in primary peritubular myoid cells (PMCs) through MA intake. Primary PMCs were isolated and exposed to DEHP in vitro. The results demonstrated that DEHP significantly suppressed antioxidant levels and promoted cell apoptosis in primary PMCs. Moreover, interfering with the expression of PRDX6 was found to induce excessive reactive oxygen species (ROS) production and cell apoptosis in primary PMCs. Supplementation with MA significantly upregulated the expression of PRDX6, thereby attenuating DEHP-induced oxidative stress and cell apoptosis in primary PMCs. These findings provide a theoretical foundation for mitigating stress levels and enhancing reproductive capacity of in captive FMDs.

Marked variations in gut microbial diversity, functions, and disease risk between wild and captive alpine musk deer.

Maintaining a healthy status is crucial for the successful captive breeding of endangered alpine musk deer (Moschus chrysogaster, AMD), and captive breeding programs are beneficial to the ex-situ conservation and wild population recovery of this species. Meanwhile, the gut microbiota is essential for host health, survival, and environmental adaptation. However, changes in feeding environment and food can affect the composition and function of gut microbiota in musk deer, ultimately impacting their health and adaptation. Therefore, regulating the health status of wild and captive AMD through a non-invasive method that targets gut microbiota is a promising approach. Here, 16S rRNA gene sequencing was employed to reveal the composition and functional variations between wild (N = 23) and captive (N = 25) AMD populations. The results indicated that the gut microbiota of wild AMD exhibited significantly higher alpha diversity (P < 0.001) and greater abundance of the phylum Firmicutes, as well as several dominant genera, including UCG-005, Christensenellaceae R7 group, Monoglobus, Ruminococcus, and Roseburia (P < 0.05), compared to captive AMD. These findings suggest that the wild AMD may possess more effective nutrient absorption and utilization, a more stable intestinal microecology, and better adaption to the complex natural environment. The captive individuals displayed higher metabolic functions with an increased abundance of the phylum Bacteroidetes and certain dominant genera, including Bacteroides, Rikenellaceae RC9 gut group, NK4A214 group, and Alistipes (P < 0.05), which contributed to the metabolic activities of various nutrients. Furthermore, captive AMD showed a higher level of 11 potential opportunistic pathogens and a greater enrichment of disease-related functions compared to wild AMD, indicating that wild musk deer have a lower risk of intestinal diseases and more stable intestinal structure in comparison to captive populations. These findings can serve as a valuable theoretical foundation for promoting the healthy breeding of musk deer and as a guide for evaluating the health of wild-released and reintroduced musk deer in the future. KEY POINTS: • Wild and captive AMD exhibit contrasting gut microbial diversity and certain functions. • With higher diversity, certain bacteria aid wild AMD's adaptation to complex habitats. • Higher potential pathogens and functions increase disease risk in captive AMD.

SSR Marker Acquisition and Application from Transcriptome of Captive Chinese Forest Musk Deer (Moschus berezovskii).

Forest musk deer (Moschus berezovskii) is one of the most endangered medicinally important wild animals in the world. Forest musk deer farming is the main way of production of musk. However, the single provenance and lack of genetic information lead to reduced genetic diversity of forest musk deer. Therefore, more SSR markers need to be developed to identify forest musk deer germplasm. In this study, bone marrow derived mesenchymal cells were used to construct cDNA library for transcriptome sequencing. The datasets were de novo assembled and annotated. 9 polymorphic simple sequence repeat (SSR) markers were finally identified and used to detect population genetic diversity. 6.07 Gb clean data were generated using Illumina sequencing technology, and de novo assembled into 138,591 transcripts and 81,553 unigenes. 5,777 simple sequence repeats (SSRs) were identified, in which there were 578 repeating motif types, with mono-nucleotide and tri-nucleotides comprising 55.88% and 25.60%, respectively. 100 primer pairs were designed to validate amplification and polymorphism using DNA from fecal samples. 9 polymorphic SSRs were developed and used to detect population genetic diversity of 122 forest musk deer in 2 farms. The average number of alleles per locus varied from 4 to 15 (average = 8.3). The observed heterozygosity (HO) per locus ranged from 0.102 to 0.941, while the expected heterozygosity (HE) per locus was from 0.111 to 0.651. All loci deviated significantly from the Hardy-Weinberg equilibrium (p < 0.001). The polymorphism information content (PIC) of these loci varied from 0.108 to 0.619. 9 polymorphic SSR markers were developed in this research. These sites can be used for breeding planning and conservation of germplasm resources.

Population genomics reveals moderate genetic differentiation between populations of endangered Forest Musk Deer located in Shaanxi and Sichuan.

BACKGROUND: Many endangered species exist in small, genetically depauperate, or inbred populations, hence promoting genetic differentiation and reducing long-term population viability. Forest Musk Deer (Moschus berezovskii) has been subject to illegal hunting for hundreds of years due to the medical and commercial values of musk, resulting in a significant decline in population size. However, it is still unclear to what extent the genetic exchange and inbreeding levels are between geographically isolated populations. By using whole-genome data, we reconstructed the demographic history, evaluated genetic diversity, and characterized the population genetic structure of Forest Musk Deer from one wild population in Sichuan Province and two captive populations from two ex-situ centers in Shaanxi Province. RESULTS: SNP calling by GATK resulted in a total of 44,008,662 SNPs. Principal component analysis (PCA), phylogenetic tree (NJ tree), ancestral component analysis (ADMIXTURE) and the ABBA-BABA test separated Sichuan and Shaanxi Forest Musk Deer as two genetic clusters, but no obvious genetic differentiation was observed between the two captive populations. The average pairwise FST value between the populations in Sichuan and Shaanxi ranged from 0.05-0.07, suggesting a low to moderate genetic differentiation. The mean heterozygous SNPs rate was 0.14% (0.11%-0.15%) for Forest Musk Deer at the genomic scale, and varied significantly among three populations (Chi-square = 1.22, p < 0.05, Kruskal-Wallis Test), with the Sichuan population having the lowest (0.11%). The nucleotide diversity of three populations varied significantly (p < 0.05, Kruskal-Wallis Test), with the Sichuan population having the lowest genetic θπ (1.69 × 10-3). CONCLUSIONS: Genetic diversity of Forest Musk Deer was moderate at the genomic scale compared with other endangered species. Genetic differentiation between populations in Sichuan and Shaanxi may not only result from historical biogeographical factors but also be associated with contemporary human disturbances. Our findings provide scientific aid for the conservation and management of Forest Musk Deer. They can extend the proposed measures at the genomic level to apply to other musk deer species worldwide.

Transcriptome profiling of lung immune responses potentially related to acute respiratory distress syndrome in forest musk deer.

BACKGROUND: Forest musk deer is an endangered species globally. The death of captive forest musk deer can be caused by certain respiratory system diseases. Acute respiratory distress syndrome (ARDS) is a huge threat to the life of forest muck deer that breed in our department. METHODS: Lung histopathologic analysis was conducted by hematoxylin and eosin (HE) staining. The lung gene changes triggered by ARDS were examined by RNA sequencing and related bioinformatics analysis in forest musk deer. The potential functions of unigenes were investigated by NR, SwissProt KOG, GO, and KEGG annotation analyses. Vital biological processes or pathways in ARDS were examined by GO and KEGG enrichment analyses. RESULTS: A total of 3265 unigenes were differentially expressed (|log2fold-change|> 2 and adjusted P value < 0.01) in lung tissues of 3 forest musk deer with ARDS compared with normal lung tissues of the non-ARDS group. These differentially expressed unigenes (DEGs) played crucial roles in immunity and defense responses to pathogens. Moreover, we identified the DEGs related to one or more of the following biological processes: lung development, immunity, and bacterial/viral/fungal infection. And six DEGs that might be involved in lung injury caused by immune dysregulation or viral/fungal infection were identified. CONCLUSION: ARDS-mediated lung gene alterations were identified in forest musk deer. Moreover, multiple genes involved in lung development and lung defense responses to bacteria/viruses/fungi in ARDS were filtered out in forest musk deer.

Genetic diversity of Blastocystis subtypes in the Alpine musk deer (Moschus chrysogaster) in Gansu province, northwestern China.

Blastocystis sp. is a gastrointestinal pathogen that is frequently found in humans and animals worldwide. In this study, 201 fecal samples were collected from captive Alpine musk deer (Moschus chrysogaster) at three farms in Gansu province. Blastocystis was detected and subtyped by amplifying and sequencing the small subunit ribosomal DNA gene. The overall prevalence of Blastocystis was 39.8% (80/201). Five known Blastocystis subtypes (STs), including ST1 (n = 1), ST4 (n = 12), ST10 (n = 50), ST14 (n = 6), and ST24 (n = 11) were identified using subtyping and evolutionary analysis. ST10 was the most common ST observed in each farm. This study showed the infection status and genetic characteristics of Blastocystis in M. chrysogaster. Based on the surveyed data, because various potentially zoonotic STs, such as ST1, ST4, ST10, ST14, and ST24, were detected, it is believed that the zoonotic risk of Blastocystis from the Alpine musk deer in this area cannot be ignored.

Comparative study of the function and structure of the gut microbiota in Siberian musk deer and Forest musk deer.

Musk deer are famous for their secretion of musk; however, the scale of artificial breeding of musk deer is limited. Considering the lack of a comprehensive understanding of the gut microbiota, there is a need to study the gut microbiota of Siberian musk deer (SMD). Quantitative PCR analysis and high-throughput sequencing were used to show the differences in gut microbial communities and functions between SMD and forest musk deer (FMD). The relative abundance of Firmicutes was significantly higher in SMD than in FMD, with a corresponding decrease in Bacteroidetes, and showed significant seasonal variation. The gut microbiome of FMD has enriched activity for carbohydrate metabolism, while in SMD, amino acids and energy metabolism was higher. The key enzymatic reactions were related to pyruvate metabolism in SMD; however, in FMD, enzymes that digest cellulose (EC:3.2.1.21, EC:3.2.1.4.) were more abundant, and these were related to the living environment and feeding habits. This indicates that FMD and SMD have significant differences in their microbial communities and functions. Furthermore, antibiotic resistances were identified and significantly different in gut microbes of SMD and FMD. For SMD, seasonal variations alter microbial communities and function. The key enzymes of the short-chain fatty acids (EC:1.3.1.44, EC:6.4.1.2) were significantly different, with higher relative abundance in winter-a mechanism of natural selection and environmental adaptation. This study is the first to analyze the composition of the gut microbiota of SMD and can be used to develop or modify conservation and husbandry strategies for musk deer, to improve their productivity. KEY POINTS: • Significant differences in microbial communities and their function between FMD and SMD. • The energy metabolism and the relative abundance of Firmicutes were significantly higher in SMD. • Seasonal variations alter microbial function in SMD, carbohydrate metabolism was higher in summer.

Comparative analysis of gut microbial composition and potential functions in captive forest and alpine musk deer.

Gut microbiota forms a unique microecosystem and performs various irreplaceable metabolic functions for ruminants. The gut microbiota is important for host health and provides new insight into endangered species conservation. Forest musk deer (FMD) and alpine musk deer (AMD) are typical small ruminants, globally endangered due to excessive hunting and habitat loss. Although nearly 60 years of captive musk deer breeding has reduced the hunting pressure in the wild, fatal gastrointestinal diseases restrict the growth of captive populations. In this study, 16S rRNA high-throughput sequencing revealed the differences in gut microbiota between FMD and AMD based on 166 fecal samples. The alpha diversity was higher in FMD than in AMD, probably helping FMD adapt to different and wider habitats. The ß-diversity was higher between adult FMD and AMD than juveniles and in winter than late spring. The phylum Firmicutes and the genera Christensenellaceae R7 group, Ruminococcus, Prevotellaceae UCG-004, and Monoglobus were significantly higher in abundance in FMD than in AMD. However, the phylum Bacteroidetes and genera Bacteroides, UCG-005, Rikenellaceae RC9 gut group, and Alistipes were significantly higher in AMD than FMD. The expression of metabolic functions was higher in AMD than in FMD, a beneficial pattern for AMD to maintain higher energy and substance metabolism. Captive AMD may be at higher risk of intestinal diseases than FMD, with higher relative abundances of most opportunistic pathogens and the expression of disease-related functions. These results provide valuable data for breeding healthy captive musk deer and assessing their adaptability in the wild. KEY POINTS: • Alpha diversity of gut microbiota was higher in FMD than that in AMD • Expression of metabolic and disease-related functions was higher in AMD than in FMD.

Antibacterial and anti-virulence effects of furazolidone on Trueperella pyogenes and Pseudomonas aeruginosa.

BACKGROUND: Trueperella pyogenes and Pseudomonas aeruginosa are two important bacterial pathogens closely relating to the occurrence and development of forest musk deer respiratory purulent disease. Although T. pyogenes is the causative agent of the disease, the subsequently invaded P. aeruginosa will predominate the infection by producing a substantial amount of quorum-sensing (QS)-controlled virulence factors, and co-infection of them usually creates serious difficulties for veterinary treatment. In order to find a potential compound that targets both T. pyogenes and P. aeruginosa, the antibacterial and anti-virulence capacities of 55 compounds, which have similar core structure to the signal molecules of P. aeruginosa QS system, were tested in this study by performing a series of in vitro screening experiments. RESULTS: We identified that furazolidone could significantly reduce the cell densities of T. pyogenes in mono-culture or in the co-culture with P. aeruginosa. Although the growth of P. aeruginosa could also be moderately inhibited by furazolidone, the results of phenotypic identification and transcriptomic analysis further revealed that sub-inhibitory furazolidone had remarkable inhibitory effect on the biofilm production, motility, and QS system of P. aeruginosa. Moreover, furazolidone could efficiently protect Caenorhabditis elegans models from P. aeruginosa infection under both fast-killing and slow-killing conditions. CONCLUSIONS: This study reports the antibacterial and anti-virulence abilities of furazolidone on T. pyogenes and P. aeruginosa, and provides a promising strategy and molecular basis for the development of novel anti-infectious drugs to dealing with forest musk deer purulent disease, or other diseases caused by T. pyogenes and P. aeruginosa co-infection.

Whole-genome sequencing of wild Siberian musk deer (Moschus moschiferus) provides insights into its genetic features.

BACKGROUND: Siberian musk deer, one of the seven species, is distributed in coniferous forests of Asia. Worldwide, the population size of Siberian musk deer is threatened by severe illegal poaching for commercially valuable musk and meat, habitat losses, and forest fire. At present, this species is categorized as Vulnerable on the IUCN Red List. However, the genetic information of Siberian musk deer is largely unexplored. RESULTS: Here, we produced 3.10 Gb draft assembly of wild Siberian musk deer with a contig N50 of 29,145 bp and a scaffold N50 of 7,955,248 bp. We annotated 19,363 protein-coding genes and estimated 44.44% of the genome to be repetitive. Our phylogenetic analysis reveals that wild Siberian musk deer is closer to Bovidae than to Cervidae. Comparative analyses showed that the genetic features of Siberian musk deer adapted in cold and high-altitude environments. We sequenced two additional genomes of Siberian musk deer constructed demographic history indicated that changes in effective population size corresponded with recent glacial epochs. Finally, we identified several candidate genes that may play a role in the musk secretion based on transcriptome analysis. CONCLUSIONS: Here, we present a high-quality draft genome of wild Siberian musk deer, which will provide a valuable genetic resource for further investigations of this economically important musk deer.

Viral metagenomics revealed diverse CRESS-DNA virus genomes in faeces of forest musk deer.

BACKGROUND: Musk deer can produce musk which has high medicinal value and is closely related to human health. Viruses in forest musk deer both threaten the health of forest musk deer and human beings. METHODS: Using viral metagenomics we investigated the virome in 85 faeces samples collected from forest musk deer. RESULTS: In this article, eight novel CRESS-DNA viruses were characterized, whole genomes were 2148 nt-3852 nt in length. Phylogenetic analysis indicated that some viral genomes were part of four different groups of CRESS-DNA virus belonging in the unclassified CRESS-DNA virus, Smacoviridae, pCPa-like virus and pPAPh2-like virus. UJSL001 (MN621482), UJSL003 (MN621469) and UJSL017 (MN621476) fall into the branch of unclassified CRESS-DNA virus (CRESSV1-2), UJSL002 (MN621468), UJSL004 (MN621481) and UJSL007 (MN621470) belong to the cluster of Smacoviridae, UJSL005 (MN604398) showing close relationship with pCPa-like (pCRESS4-8) clusters and UJSL006 (MN621480) clustered into the branch of pPAPh2-like (pCRESS9) virus, respectively. CONCLUSION: The virome in faeces samples of forest musk deer from Chengdu, Sichuan province, China was revealed, which further characterized the diversity of viruses in forest musk deer intestinal tract.

The olfactory subgenome and specific odor recognition in forest musk deer.

Olfactory receptors (ORs) are encoded by OR genes. The OR genes in forest musk deer (Moschus berezovskii), which rely on olfaction for reproductive and social communication, are poorly understood. In this study, we analyzed the genome sequence of the forest musk deer to obtain its olfactory subgenome and compared it to other species. A total of 1378 OR-related sequences were detected in the forest musk deer genome including 864 functional genes, 366 pseudogenes and 148 partial genes. These OR genes were classified into Class I and Class II and were further classified into 18 families and 244 subfamilies through sequence identity. Comparative analyses of the OR genes' protein sequences in species from different orders (forest musk deer, human, mouse and dog) showed that 12 clusters were specific to forest musk deer. However, when compared to other Artiodactyl species (i.e. cattle, yak and pig) only two clusters were specific to forest musk deer. The odor identification potential of the OR genes in the forest musk deer was focused mainly on floral, woody, lemon, sweet and fatty odors. We also found that OR genes specific to forest musk deer were involved in the identification of spearmint and caraway. Our work is the first genome-wide analysis of OR genes in forest musk deer. These findings will assist with better understanding the relationship between behavior and olfaction in the forest musk deer and the characteristics of the olfactory subgenome in Artiodactyl mammals.

Species dilemma of musk deer (Moschus spp) in India: molecular data on cytochrome c oxidase I suggests distinct genetic lineage in Uttarakhand compared to other Moschus species.

Musk deer are of high conservation priority owing to poaching pressure because of its musk pod. Representation of musk deer status using genetics is poorly documented in India, and it is not confirmed as to how many species of musk deer are present. We characterize for the first time, the genetic diversity of musk deer from Uttarakhand using Cytochrome Oxidase sub-unit (COI) gene (486 bp) and compared with the data available for other species. Results revealed the presence of six haplotypes in the Uttarakhand population amongst 17 sequences. Of these, 12 sequences shared the single haplotype. The intra-species sequences divergence was 0.003-0.017, whereas divergence with other species of musk deer was 0.071-0.081. Bayesian phylogenetic tree revealed that samples from Uttarakhand formed a separate clade with respect to other species of musk deer, whereas three species distributed in China clustered in the same clade and showed low sequences divergence, i.e., 0.002-0.061. Because of different ecomorph reported, we suggest using the barcoding based approach for inter and intra-species distinction and delineating species boundaries across the range for effective conservation. Besides, systematic classification, DNA barcoding would also help in dealing wildlife offence cases for disposal of the legal report in court.

[Microbial diversity of musk across its maturation process in forest musk deer].

Musk,with unique and intense perfume,was a kind of deep brown precious medicinal material in traditional Chinese medicine. However,the immature musk in musk pot was white and stench. Given the fact that bacterial diversity generated odorous metabolites in animal hosts,in this study,musk samples at three different mature stages,including MJ( the end of June),MA( the end of August) and MO( the end of October) were harvested from three male forest musk deer,and then next-generation sequencing was used to intensively survey the bacterial communities in musk harvested at different mature stages. RESULTS: indicated that the average OTUs per sample at the end of June,August and October were 47 116. 00 ± 1 567. 24( SE),52 009. 00 ± 8 958. 75( SE) and50 004. 67±4 135. 57( SE),respectively. Feature of the musk 16 S rRNA gene showed a total of 418 genera belonging to 52 phyla were observed in all samples. The main microbiota was bacteria,which accounted for 98. 82%,99. 95% and 99. 58% in MJ,MA and MO,respectively. At phylum level,Firmicutes was the most abundant bacterial of MA( 32. 75%) and MO( 39. 19%). While,the major bacterial in MJ was Proteobacteria( 49. 14%). PICRUSt analysis revealed the functions of bacterial in MJ were mainly involved in secretion,while bacterial functions of MA and MO were mainly involved in amino acid or other substance metabolism,which was in accord with the musk secretion physiological process of forest musk deer. This is the first study involved in the bacterial diversity in musk of forest musk deer across the maturation process,while may provide a new insight into the musk generation mechanism.

Sex hormones play roles in determining musk composition during the early stages of musk secretion by musk deer (Moschus berezovskii).

Musk is a secreted external hormone or information compound that is stored in musk scent glands of the males of species within the family Moschidae, such as Moschus berezovskii. The secretion of musk changes periodically during the courtship and reproduction periods, with the early stage of secretion occurring from May to July, and the maturation stage occurring from August to April of the following year. In this study, we analyzed the dynamic changes in musk components from June to April of the following year. The result showed that musk morphological character, water content, total ion chromatographic pattern, and composition undergo seasonal change. Luminescence immunoassay and radioimmunoassay analyses were performed to determine corresponding fecal hormone levels. The results showed that testosterone, estrogen, and cortisol levels in feces change on a seasonal basis, and are significantly higher in June than in other months (p < 0.01). Correlation analysis showed that the contents of four examined musk components (muscone, cyclopentadecanone, cholesterol, and cholestenol) from June to August were significantly highly negatively correlated with fecal testosterone and estradiol levels (p < 0.01). In contrast, the correlation coefficients were low or not significant from August to April of the following year. These results indicate that testosterone and estradiol may play a major role in determining musk composition during the early stage of musk secretion but not during the course of musk maturation, which suggests that musk secretion may be promoted by increases in sex hormones in June.

[Research progress on musk and artificial propagation technique of forest musk deer].

Forest musk deer(Moschus berezovskii) is mainly distribute in Shaanxi and Sichuan provinces of China. The musk is a dry exudates in the mature male sachet with complex chemical composition, abundant modern identification methods, various pharmacological action, extensive clinical application. The population quantity and quality of wild and captive forest musk deer failed to improve because of lacking scientific management, excessive hunting and habitat destruction, etc. Carrying out the captive forest musk deer is an effective way to protect them, fast breeding of forest musk deer is the key to increase the yield of musk. Therefore, we should develop new technologies such as natural estrus and artificial insemination in order to speed up the breeding of musk deer and increase the yield of musk. This article reviews the research progress of new artificial propagation technique and musk, which teases some feasible research basis for optimization of forest musk deer species and the research of musk.

CHARACTERIZATION OF VIRULENCE GENES AND ANTIMICROBIAL RESISTANCE OF LUNG PATHOGENIC ESCHERICHIA COLI ISOLATES IN FOREST MUSK DEER (MOSCHUS BEREZOVSKII).

This study investigated genotypic diversity, 26 virulence genes, and antimicrobial susceptibility of lung pathogenic Escherichia coli (LPEC) isolated from forest musk deer. Associations between virulence factors (VFs) and phylogenetic group, between antimicrobial resistance (AMR) and phylogenetic group, and between AMR and VFs were subsequently assessed. The results showed 30 LPEC isolated were grouped into seven different clusters (A, B, C, D, E, F, and G). The detection rates of crl (90%), kpsMT II (76.67%), mat (76.67%), and ompA (80%) were over 75%. The most frequent types of resistance were to amoxicillin (100%), sulfafurazole (100%), ampicillin (96.67%), and tetracycline (96.67%), with 93.33% (n = 28) of isolates resistant to more than eight types of drugs. There were significant relationships between resistance to cefalotin and the presence of iucD(a) (P < 0.001), papC (P = 0.032), and kpsMT II (P = 0.028); between resistance to chloromycetin and the presence of irp2 (P = 0.004) and vat (P = 0.047); between resistance to nalidixic acid and the presence of crl (P = 0.002) and iucD(a) (P = 0.004); and between resistance to ampicillin/sulbactam and the presence of vat (P = 0.013). These results indicated there could be some association between resistance and VFs, and there is a great need for the prudent use of antimicrobial agents in LPEC.

Akkermansia muciniphila isolated from forest musk deer ameliorates diarrhea in mice via modification of gut microbiota.

BACKGROUND: The forest musk deer, a rare fauna species found in China, is famous for its musk secretion which is used in selected Traditional Chinese medicines. However, over-hunting has led to musk deer becoming an endangered species, and their survival is also greatly challenged by various high incidence and high mortality respiratory and intestinal diseases such as septic pneumonia and enteritis. Accumulating evidence has demonstrated that Akkermannia muciniphila (AKK) is a promising probiotic, and we wondered whether AKK could be used as a food additive in animal breeding programmes to help prevent intestinal diseases. METHODS: We isolated one AKK strain from musk deer feces (AKK-D) using an improved enrichment medium combined with real-time PCR. After confirmation by 16S rRNA gene sequencing, a series of in vitro tests was conducted to evaluate the probiotic effects of AKK-D by assessing its reproductive capability, simulated gastrointestinal fluid tolerance, acid and bile salt resistance, self-aggregation ability, hydrophobicity, antibiotic sensitivity, hemolysis, harmful metabolite production, biofilm formation ability, and bacterial adhesion to gastrointestinal mucosa. RESULTS: The AKK-D strain has a probiotic function similar to that of the standard strain in humans (AKK-H). An in vivo study found that AKK-D significantly ameliorated symptoms in the enterotoxigenic Escherichia coli (ETEC)-induced murine diarrhea model. AKK-D improved organ damage, inhibited inflammatory responses, and improved intestinal barrier permeability. Additionally, AKK-D promoted the reconstitution and maintenance of the homeostasis of gut microflora, as indicated by the fact that AKK-D-treated mice showed a decrease in Bacteroidetes and an increase in the proportion of other beneficial bacteria like Muribaculaceae, Muribaculum, and unclassified f_Lachnospiaceae compared with the diarrhea model mice. CONCLUSION: Taken together, our data show that this novel AKK-D strain might be a potential probiotic for use in musk deer breeding, although further extensive systematic research is still needed.