Single-Cell Transcriptome Sequencing Reveals Molecular Expression Differences and Marker Genes in Testes during the Sexual Maturation of Mongolian Horses.

This study aimed to investigate differences in testicular tissue morphology, gene expression, and marker genes between sexually immature (1-year-old) and sexually mature (10-year-old) Mongolian horses. The purposes of our research were to provide insights into the reproductive physiology of male Mongolian horses and to identify potential markers for sexual maturity. The methods we applied included the transcriptomic profiling of testicular cells using single-cell sequencing techniques. Our results revealed significant differences in tissue morphology and gene expression patterns between the two age groups. Specifically, 25 cell clusters and 10 cell types were identified, including spermatogonial and somatic cells. Differential gene expression analysis highlighted distinct patterns related to cellular infrastructure in sexually immature horses and spermatogenesis in sexually mature horses. Marker genes specific to each stage were also identified, including APOA1, AMH, TAC3, INHA, SPARC, and SOX9 for the sexually immature stage, and PRM1, PRM2, LOC100051500, PRSS37, HMGB4, and H1-9 for the sexually mature stage. These findings contribute to a deeper understanding of testicular development and spermatogenesis in Mongolian horses and have potential applications in equine reproductive biology and breeding programs. In conclusion, this study provides valuable insights into the molecular mechanisms underlying sexual maturity in Mongolian horses.

Leucine promotes energy metabolism and stimulates slow-twitch muscle fibers expression through AMPK/mTOR signaling in equine skeletal muscle satellite cells.

Previous research has shown that leucine (Leu) can stimulate and enhance the proliferation of equine skeletal muscle satellite cells (SCs). The gene expression profile associated with Leu-induced proliferation of equine SCs has also been documented. However, the specific role of Leu in regulating the expression of slow-twitch muscle fibers (slow-MyHC) and mitochondrial function in equine SCs, as well as the underlying mechanism, remains unclear. During this investigation, equine SCs underwent culturing in differentiation medium and were subjected to varying concentrations of Leu (0 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM) over a span of 3 days. AMP-activated protein kinase (AMPK) inhibitor Compound C and mammalian target of rapamycin complex (mTOR) inhibitor Rapamycin were utilized to explore its underlying mechanism. Here we showed that the expression of slow-MyHC at 2 mM Leu level was significantly higher than the concentration levels of 0 mM,0.5 mM and 10 mM (P <0.01), and there was no significant difference compared to other groups (P > 0.05); the basal respiration, maximum respiration, standby respiration and the expression of slow-MyHC, PGC-1α, Cytc, ND1, TFAM, and COX1 were significantly increased with Leu supplementation (P < 0.01). We also found that Leu up-regulated the expression of key proteins on AMPK and mTOR signaling pathways, including LKB1, p-LKB1, AMPK, p-AMPK, S6, p-S6, 4EBP1, p-4EBP1, mTOR and p-mTOR (P < 0.05 or P < 0.01). Notably, when we treated the equine SCs with the AMPK inhibitor Compound C and the mTOR inhibitor Rapamycin, we observed a reduction in the beneficial effects of Leu on the expression of genes related to slow-MyHC and signaling pathway-related gene expressions. This study provides novel evidence that Leu promotes slow-MyHC expression and enhances mitochondrial function in equine SCs through the AMPK/mTOR signaling pathways, shedding light on the underlying mechanisms involved in these processes for the first time.

Identification of mulberry leaf flavonoids and evaluating their protective effects on H2O2-induced oxidative damage in equine skeletal muscle satellite cells.

Introduction: Horses are susceptible to oxidative stress during strenuous endurance exercise, leading to muscle fatigue and damage. Mulberry leaf flavonoids (MLFs) possess significant antioxidant properties. However, the antioxidant efficacy of MLFs can be influenced by the extraction process, and their impact on H2O2-induced oxidative stress in equine skeletal muscle satellite cells (ESMCs) remains unexplored. Methods: Our study employed three extraction methods to obtain MLFs: ultrasound-assisted extraction (CEP), purification with AB-8 macroporous resin (RP), and n-butanol extraction (NB-EP). We assessed the protective effects of these MLFs on H2O2-induced oxidative stress in ESMCs and analyzed the MLF components using metabolomics. Results: The results revealed that pre-treatment with MLFs dose-dependently protected ESMCs against H2O2-induced oxidative stress. The most effective concentrations were 0.8 mg/mL of CEP, 0.6 mg/mL of RP, and 0.6 mg/mL of NB-EP, significantly enhancing EMSC viability (p < 0.05). These optimized MLF concentrations promoted the GSH-Px, SOD and T-AOC activities (p < 0.05), while reducing MDA production (p < 0.05) in H2O2-induced ESMCs. Furthermore, these MLFs enhanced the gene expression, including Nrf2 and its downstream regulatory genes (TrxR1, GPX1, GPX3, SOD1, and SOD2) (p < 0.05). In terms of mitochondrial function, ESMCs pre-treated with MLFs exhibited higher basal respiration, spare respiratory capacity, maximal respiration, ATP-linked respiration compared to H2O2-induced ESMCs (p < 0.05). Additionally, MLFs enhanced cellular basal glycolysis, glycolytic reserve, and maximal glycolytic capacity (p < 0.05). Metabolomics analysis results revealed significant differences in mulberrin, kaempferol 3-O-glucoside [X-Mal], neohesperidin, dihydrokaempferol, and isobavachalcone among the three extraction processes (p < 0.05). Discussion: Our study revealed that MLFs enhance antioxidant enzyme activity, alleviate oxidative damage in ESMCs through the activation of the Nrf2 pathway, and improve mitochondrial respiration and cell energy metabolism. Additionally, we identified five potential antioxidant flavonoid compounds, suggesting their potential incorporation into the equine diet as a strategy to alleviate exercise-induced oxidative stress.

Comprehensive analysis of the whole-transcriptome landscape of the ovarian cortex from Mongolian horses that reproduce seasonally.

The reproductive cycle of equines tends to be seasonal and is influenced by factors such as light and temperature. The process and methods of regulating the mare oestrous cycle in the anestrus period are still immature. The effects of noncoding RNAs and mRNAs on the oestrous cycle have aroused much interest, but corresponding analyses of seasonal mare ovaries have not been reported. Here, we report a whole transcriptome analysis of the Mongolian horse ovarian cortex collected in anestrus and diestrus periods. In total, 1081 mRNAs, 205 lncRNAs, 54 circRNAs, and 13 miRNAs were upregulated in winter anestrus ovarian cortex (WAO), and 1261 mRNAs, 90 lncRNAs, 29 circRNAs, and 40 miRNAs were upregulated in summer diestrus ovarian cortex (SDO). The GO and KEGG enrichment analysis of differentially expressed mRNAs and target genes of differentially expressed lncRNAs, circRNAs, and miRNAs revealed some key functions and pathways that may be related to follicle and oocyte development. We found that estrogen-related pathways were enriched in different RNAs. Our data were used to generate miRNA, circRNA, lncRNA, and mRNA databases from the Mongolian horse ovary and differential expression profiles between WAO and SDO; these results provide clues for exploring methods of estrus regulation in mares during the anestrus period.

Diversity and functional prediction of fungal communities in different segments of mongolian horse gastrointestinal tracts.

BACKGROUND: Anaerobic fungi are effective fibre-degrading microorganisms in the digestive tract of horses. However, our understanding of their diversity and community structure is limited, especially in different parts of the gastrointestinal tract. RESULTS: For the first time, high-throughput sequencing technology was used to analyse and predict fungal microbial diversity in different parts of the gastrointestinal tract of Mongolian horses. The results revealed that the richness and diversity of fungi in the hindgut of Mongolian horses were much higher than those in the foregut. The foregut was dominated by Basidiomycota and Ascomycota, whereas the hindgut was dominated by Neocallimastigomycota and Basidiomycota. At the genus level, the relative abundance of many pathogenic fungi (Cryptococcus, Cladosporium, Alternaria, and Sarocladium) in the foregut was significantly higher than that in the posterior gut, indicating that Mongolian horses have strong disease resistance. The prediction of fungal function also showed significant differences in the fungal flora between the foregut and the hindgut. The fungi in Mongolian horses' foreguts were mainly pathologically nutritive and contained many animal and plant pathogens, particularly in the small intestine (jejunum and ileum). This indicates that the foregut may be the most important immune site in the digestive system of Mongolian horses, which explains the high disease resistance of Mongolian horses. The number of unassigned functional groups in the posterior gut was significantly higher than that in the anterior gut, indicating that the functions of fungal groups in the posterior gut have not been fully explored, and further studies are required in the future. CONCLUSIONS: Analysis of high-throughput sequencing results revealed that the fungal composition varied greatly among different gastrointestinal tract segments in Mongolian horses, whose hindgut contains many anaerobic fungi involved in plant cellulose degradation. This provides important basic data for studying fungal diversity in the digestive system of healthy horses, which can be used for the health assessment of horses and provides clues for further research on the disease resistance and digestive capacity of horses, as well as a reference for the early diagnosis of intestinal diseases and innovative treatment methods.

Integrated analysis of transcriptome and proteome for exploring mechanism of promoting proliferation of equine satellite cells associated with leucine.

The proliferation and differentiation of skeletal muscle satellite cells (SCs) are necessary for the development of mature skeletal muscle. Leucine (Leu) is both an essential amino acid (EAA) and a branched-chain amino acid (BCAA), which has attracted worldwide attention due to its ability to repair and become new fibers. We separated the equine SCs into the control group (CON) and the Leu-supplemented group (LEU), which the cells were cultured in Leu-deprived and Leu-supplemented media respectively. We combined the transcriptome (RNA-Seq) and quantitative proteome (TMT) profiling analyses on proliferation of equine SCs associated with Leu. 1839 up-regulated and 631 down-regulated genes made up the 2470 differentially expressed genes (DEGs), and the 253 differentially abundant proteins (DEPs) included 118 up-regulated and 135 down-regulated proteins. 110 overlapping genes were verified based on the mRNA and protein translation relationship. Moreover, by comparing overlapped pathways through enrichment analysis, we found 13 genes not only appeared among 110 key DEGs/DEPs but also enriched in the KEGG overlapping signaling pathway, including CCL26, STAT2, PCK2, ASNS, GPT2, SHMT2, PHGDH, PGAM2, PSAT1, FTL, HMOX1, STEAP1 and STEAP2. To our knowledge, this is the first report in the world to systematically show how Leu regulated the growth of equine SCs. Leu deficiency inhibits the proliferation of equine SCs and development of fresh muscle fibers was proved in this paper. The main genes in charge of the Leu-induced proliferation of horse SCs have been found. These genes will make it easier to understand the mechanism at work and offer new information for enhancing the performance of sport horses and alleviating the equine muscle damage during exercise in the future.

A genome-wide landscape of mRNAs, miRNAs, lncRNAs, and circRNAs of skeletal muscles during dietary restriction in Mongolian horses.

The proportion of different muscle fibers is essential for the horse breed's aptitude for athletic activities. Adaptation of locomotor muscle is correlated with altered physiologic conditions. To investigate the adaptive changes of muscle fiber phenotype and transcriptome in horse skeletal muscle during dietary restriction (DR). The muscle fiber type distribution and deep RNA-seq analysis of detecting differentially expressed mRNAs (DEGs), miRNA (DEMIRs), lncRNAs (DELs), circRNAs (DECs), and their function analysis were investigated in gluteus medius muscle of Mongolian horses during DR. A total of 1433 DEGs, 5 DEMIRs, 329 DELs, and 53 DECs were identified. Differing from non-uniform muscle fiber type changing, functional enrichment analysis showed that most downregulated DEGs were associated in muscle contraction, fuel energy metabolism, and protein balance. Linkages between non-coding RNA and mRNA landscape were detected from their functional changes. Our study provides new insights into the expressional changes of mRNA and non-coding RNA in horse skeletal muscles during DR, which might improve our understanding of the molecular mechanisms regulating muscle adaption during DR for racing horses.

Selection signatures for local and regional adaptation in Chinese Mongolian horse breeds reveal candidate genes for hoof health.

BACKGROUND: Thousands of years of natural and artificial selection since the domestication of the horse has shaped the distinctive genomes of Chinese Mongolian horse populations. Consequently, genomic signatures of selection can provide insights into the human-mediated selection history of specific traits and evolutionary adaptation to diverse environments. Here, we used genome-wide SNPs from five distinct Chinese Mongolian horse populations to identify genomic regions under selection for the population-specific traits, gait, black coat colour, and hoof quality. Other global breeds were used to identify regional-specific signatures of selection. RESULTS: We first identified the most significant selection peak for the Wushen horse in the region on ECA23 harbouring DMRT3, the major gene for gait. We detected selection signatures encompassing several genes in the Baicha Iron Hoof horse that represent good biological candidates for hoof health, including the CSPG4, PEAK1, EXPH5, WWP2 and HAS3 genes. In addition, an analysis of regional subgroups (Asian compared to European) identified a single locus on ECA3 containing the ZFPM1 gene that is a marker of selection for the major domestication event leading to the DOM2 horse clade. CONCLUSIONS: Genomic variation at these loci in the Baicha Iron Hoof may be leveraged in other horse populations to identify animals with superior hoof health or those at risk of hoof-related pathologies. The overlap between the selection signature in Asian horses with the DOM2 selection peak raises questions about the nature of horse domestication events, which may have involved a prehistoric clade other than DOM2 that has not yet been identified.

A Transcriptomic Regulatory Network among miRNAs, lncRNAs, circRNAs, and mRNAs Associated with L-leucine-induced Proliferation of Equine Satellite Cells.

In response to muscle injury, muscle stem cells are stimulated by environmental signals to integrate into damaged tissue to mediate regeneration. L-leucine (L-leu), a branched-chain amino acid (BCAA) that belongs to the essential amino acids (AAs) of the animal, has gained global interest on account of its muscle-building and regenerating effects. The present study was designed to investigate the impact of L-leu exposure to promote the proliferation of equine skeletal muscle satellite cells (SCs) on the regulation of RNA networks, including mRNA, long non-coding RNA (lncRNA), covalently closed circular RNA (circRNA), and microRNA (miRNA) in skeletal muscles. Equine SCs were used as a cell model and cultured in different concentrations of L-leu medium. The cell proliferation assay found that the optimal concentration of L-leu was 2 mM, so we selected cells cultured with L-leu concentrations of 0 mM and 2 mM for whole-transcriptiome sequencing, respectively. By high-throughput sequencing analysis, 2470 differentially expressed mRNAs (dif-mRNAs), 363 differentially expressed lncRNAs (dif-lncRNAs), 634 differentially expressed circRNAs (dif-circRNAs), and 49 differentially expressed miRNAs (dif-miRNAs) were significantly altered in equine SCs treated with L-leu. To identify the function of autoimmunity and anti-inflammatory responses after L-leu exposure, enrichment analysis was conducted on those differentially expressed genes (DEGs) related to lncRNA, circRNA, and miRNA. The hub genes were selected from PPI Network, including ACACB, HMGCR, IDI1, HAO1, SHMT2, PSPH, PSAT1, ASS1, PHGDH, MTHFD2, and DPYD, and were further identified as candidate biomarkers to regulate the L-leu-induced proliferation of equine SCs. The up-regulated novel 699_star, down-regulated novel 170_star, and novel 360_mature were significantly involved in the competing endogenous RNA (ceRNA) complex network. The hub genes involved in cell metabolism and dif-miRNAs may play fundamental roles in the L-leu-induced proliferation of equine SCs. Our findings suggested that the potential network regulation of miRNAs, circ-RNAs, lncRNAs, and mRNAs plays an important role in the proliferation of equine SCs, so as to build up new perspectives on improving equine performance and treatment strategies for the muscle injuries of horses.

Common protein-coding variants influence the racing phenotype in galloping racehorse breeds.

Selection for system-wide morphological, physiological, and metabolic adaptations has led to extreme athletic phenotypes among geographically diverse horse breeds. Here, we identify genes contributing to exercise adaptation in racehorses by applying genomics approaches for racing performance, an end-point athletic phenotype. Using an integrative genomics strategy to first combine population genomics results with skeletal muscle exercise and training transcriptomic data, followed by whole-genome resequencing of Asian horses, we identify protein-coding variants in genes of interest in galloping racehorse breeds (Arabian, Mongolian and Thoroughbred). A core set of genes, G6PC2, HDAC9, KTN1, MYLK2, NTM, SLC16A1 and SYNDIG1, with central roles in muscle, metabolism, and neurobiology, are key drivers of the racing phenotype. Although racing potential is a multifactorial trait, the genomic architecture shaping the common athletic phenotype in horse populations bred for racing provides evidence for the influence of protein-coding variants in fundamental exercise-relevant genes. Variation in these genes may therefore be exploited for genetic improvement of horse populations towards specific types of racing.

Analysis of the Whole-Genome Sequences from an Equus Parent-Offspring Trio Provides Insight into the Genomic Incompatibilities in the Hybrid Mule.

Interspecific hybridization often shows negative effects on hybrids. However, only a few multicellular species, limited to a handful of plants and animals, have shown partial genetic mechanisms by which hybridization leads to low fitness in hybrids. Here, to explore the outcome of combining the two genomes of a horse and donkey, we analyzed the whole-genome sequences from an Equus parent-offspring trio using Illumina platforms. We generated 41.39× and 46.21× coverage sequences for the horse and mule, respectively. For the donkey, a 40.38× coverage sequence was generated and stored in our laboratory. Approximately 24.86 million alleles were discovered that varied from the reference genome. Single nucleotide polymorphisms were used as polymorphic markers for assigning alleles to their parental genomic inheritance. We identified 25,703 Mendelian inheritance error single nucleotide polymorphisms in the mule genome that were not inherited from the parents through Mendelian inheritance. A total of 555 de novo single nucleotide polymorphisms were also identified. The rate of de novo single nucleotide polymorphisms was 2.21 × 10-7 in the mule from the Equus parent-offspring trio. This rate is obviously higher than the natural mutation rate for Equus, which is also consistent with the previous hypothesis that interracial crosses may have a high mutation rate. The genes associated with these single nucleotide polymorphisms are mainly involved in immune processes, DNA repair, and cancer processes. The results of the analysis of three genomes from an Equus parent-offspring trio improved our knowledge of the consequences of the integration of parental genomes in mules.

Fast and slow myofiber-specific expression profiles are affected by noncoding RNAs in Mongolian horses.

The heterogeneity and plasticity of muscle fibers are essential for the athletic performance of horses, mainly at the adaption of exercises and the effect on muscle diseases. Skeletal muscle fibers can be generally distinguished by their characteristics of contraction as slow and fast type myofibers. The diversity of contractile properties and metabolism enable skeletal muscles to respond to the variable functional requirements. We investigated the muscle fiber composition and metabolic enzyme activities of splenius muscle and gluteus medius muscle from Mongolian horses. The deep RNA-seq analysis of detecting differentially expressed mRNAs, lncRNAs, circRNAs and their correlation analysis from two muscles were performed. Splenius muscle and gluteus medius muscle from Mongolian horses showed a high divergence of myofiber compositions and metabolic enzyme activities. Corresponding to their phenotypic characteristics, 57 differentially expressed long noncoding RNAs and 12 differentially expressed circle RNAs were found between two muscles. The analysis results indicate multiple binding sites were detected in lncRNAs and circRNAs with myofiber-specific expressed miRNAs. Among which we found significant correlations between the above noncoding RNAs, miRNAs, their target genes, myofiber-specific developmental transcript factors, and sarcomere genes. We suggest that the ceRNA mechanism of differentially expressed noncoding RNAs by acting as miRNA sponges could be fine tuners in regulating skeletal muscle fiber composition and transition in horses, which will operate new protective measures of muscle disease and locomotor adaption for racehorses.

A Fast PCR Test for the Simultaneous Identification of Species and Gender in Horses, Donkeys, Mules and Hinnies.

Having considered that the current methods are costly and time-consuming, we designed an only 3 pairs primer-based PCR test to accurately identify the species and gender in horses, donkeys, mules and hinnies. Through a thorough sequence comparison between horse and donkey's highly similar genomes, and a vast amount of preliminary confirmation, we found that three fragments, CNGB3 gene on an autosome, displacement loop region on mitochondrial DNA and SRY genes on chromosome Y, within these equine DNA, are enough to enable us achieving our goal. The PCR test described here would be an economical, fast and accurate alternative for the most commonly-used methods, polymerase chain reaction-restriction fragment length polymorphism, microsatellite assay, and sequencing.

The Composition and Predictive Function of the Fecal Microbiota Differ Between Young and Adult Donkeys.

The community of microorganisms inhabiting the gastrointestinal tract of monogastric herbivores played critical roles in the absorption of nutrients and keeping the host healthy. However, its establishment at different age groups has not been quantitatively and functionally examined. The knowledge of microbial colonization and its function in the intestinal tract of different-age donkeys is still limited. By applying the V3-V4 region of the bacterial 16S rRNA gene and functional prediction on fecal samples from different-age donkeys, we characterized the gut microbiota during the different age groups. In contrast to the adult donkeys, the gut microbiota diversity and richness of the young donkeys showed significantly less resemblance. The microbial data showed that diversity and richness increased with age, but a highly individual variation of microbial composition was observed at month 1. Principal coordinate analysis (PCoA) revealed a significant difference across five time points in the feces. The abundance of Bacteroides, Lactobacillus, and Odoribacter tended to decrease, while the proportion of Streptococcus was significantly increased with age. For functional prediction, the relative abundance of pathways had a significant difference in the feces across different age groups, for example, Terpenoids and Polyketides and Folding, Sorting, and Degradation (P < 0.05 or P < 0.01). The analysis of beta diversity (PCoA and LEfSe) and microbial functions predicted with PICRUSt (NSTIs) clearly divided the donkeys into foals (≤3 months old) and adults (≥7 months old). Microbial community composition and structure had distinctive features at each age group, in accordance with functional stability of the microbiota. Our findings established a framework for understanding the composition and function of the fecal microbiota to differ between young and adult donkeys.

Hair follicle regional specificity in different parts of bay Mongolian horse by histology and transcriptional profiling.

BACKGROUND: Different morphological structures of hairs having properties like defense and camouflage help animals survive in the wild environment. Horse is one of the rare kinds of animals with complex hair phenotypes in one individual; however, knowledge of horse hair follicle is limited in literature and their molecular basis remains unclear. Therefore, the investigation of horse hair follicle morphogenesis and pigmentogenesis attracts considerable interest. RESULT: Histological studies revealed the morphology and pigment synthesis of hair follicles are different in between four different parts (mane, dorsal part, tail, and fetlock) of the bay Mongolian horse. Hair follicle size, density, and cycle are strongly associated with the activity of alkaline phosphatase (ALP). We observed a great difference in gene expression between the mane, tail, and fetlock, which had a greater different gene expression pattern compared with the dorsal part through transcriptomics. The development of the hair follicle in all four parts was related to angiogenesis, stem cells, Wnt, and IGF signaling pathways. Pigmentogenesis-related pathways were involved in their hair follicle pigment synthesis. CONCLUSIONS: Hair follicle morphology and the activity of ALP differ among four body parts in bay Mongolian horse. Hair follicles of the different body parts of the are not synchronized in their cycle stages. GO terms show a regional specificity pattern between different skin parts of the bay Mongolian horse. These results provide an insight into the understanding of the biological mechanism of the hair follicle in other mammals.

Characterization and comparison of the bacterial microbiota in different gastrointestinal tract compartments of Mongolian horses.

The intestinal microbiota plays an important role in the health and metabolism of the host. Next-generation sequencing technology has enabled the characterization of the gut microbiota of several animal species. We analyzed the intestinal microbiota in six different parts of the gastrointestinal tracts (GITs) of five Mongolian horses by sequencing the 16S rRNA gene V3-V4 hypervariable region. All horses were kept in the natural habitat of the Inner Mongolia grassland. Significant differences were observed among the microbiota compositions of the distinct GIT regions. In addition, while the microbial community structures of the small and large intestine were significantly different, those of the cecum and colon were similar. In the foregut, Firmicutes (65%) and Proteobacteria (23%) were the most abundant, while Firmicutes (45%) and Bacteroidetes (42%) were the most common in the hindgut. At the level of family, Ruminococcaceae (p = .203), Lachnospiraceae (p = .157), Rikenellaceae (p = .122), and Prevotellaceae (p = .068) were predominant in the hindgut, while the relative abundance of the Akkermansia genus (5.7%, p = .039) was higher in the ventral colon. In terms of the putative functions, the ratio of microbial abundance in the different parts of the GIT was similar, the result can help characterize the gut microbial structure of different animals.

The distinct transcriptomes of fast-twitch and slow-twitch muscles in Mongolian horses.

Skeletal muscle is the largest organ system in the mammalian body and plays a key role in locomotion of horses. Fast and slow muscle fibers have different abilities and functions to adapt to exercises. To investigate the RNA and miRNA expression profiles in the muscles with different muscle fiber compositions on Mongolian horses. We examined the muscle fiber type population and produced deep RNA sequencing for different parts of skeletal muscles. And chose two of them with the highest difference in fast and slow muscle fiber population (splenius and gluteus medius) for comparing the gene expression profile of slow and fast muscle fiber types. We identified a total of 275 differentially expressed genes (DEGs), and 11 differentially expressed miRNAs (DEmiRs). In addition, target gene prediction and alternative splicing analysis were also performed. Significant correlations were found between the differentially expressed gene, miRNAs, and alternative splicing events. The result indicated that differentially expressed muscle-specific genes and target genes of miRNAs might co-regulating the performance of slow and fast muscle fiber types in Mongolian horses.

Microbial diversity within the digestive tract contents of Dezhou donkeys.

Gastrointestinal microbiota has significant impact on the nutrition and health of monogastric herbivores animals including donkey. However, so far the microbiota in different gastrointestinal compartments of healthy donkey has not been described. Therefore, we investigated the abundance and function of microbiota at different sites of the gastrointestinal tract (GIT) (foregut: stomach, duodenum, jejunum and ileum; hindgut: cecum, ventral colon, dorsal colon, and rectum) of healthy adult donkeys mainly based on 16S rRNA gene sequencing and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis. Collectively, our results showed that donkey has a rich, diverse and multi-functional microbiota along the GIT. In general, the richness and diversity of the microbiota are much higher in the hindgut relative to that in the foregut; at phylum level, the Firmicutes is dominant in the foregut while both Firmicutes and Bacteroides are abundant in the hindgut; at the genus level, Lactobacillus was dominant in the foregut while Streptococcus was more dominant in the hindgut. Our further PICRUSt analysis showed that varying microbiota along the GIT is functionally compatible with the corresponding physiological function of different GIT sites. For example, the microbes in the foregut are more active at carbohydrate metabolism, and in the hindgut are more active at amino acid metabolism. This work at the first time characterized the donkey digestive system from the aspects of microbial composition and function, provided an important basic data about donkey healthy gastrointestinal microbiota, which may be utilized to evaluate donkey health and also offer clues to further investigate donkey digestive system, nutrition, even to develop the microbial supplements.

Refinement of Global Domestic Horse Biogeography Using Historic Landrace Chinese Mongolian Populations.

The Mongolian horse is one of the oldest extant horse populations and although domesticated, most animals are free-ranging and experience minimal human intervention. As an ancient population originating in one of the key domestication centers, the Mongolian horse may play a key role in understanding the origins and recent evolutionary history of horses. Here we describe an analysis of high-density genome-wide single-nucleotide polymorphism (SNP) data in 40 globally dispersed horse populations (n = 895). In particular, we have focused on new results from Chinese Mongolian horses (n = 100) that represent 5 distinct populations. These animals were genotyped for 670K SNPs and the data were analyzed in conjunction with 35K SNP data for 35 distinct breeds. Analyses of these integrated SNP data sets demonstrated that the Chinese Mongolian populations were genetically distinct from other modern horse populations. In addition, compared to other domestic horse breeds, the Chinese Mongolian horse populations exhibited relatively high genomic diversity. These results suggest that, in genetic terms, extant Chinese Mongolian horses may be the most similar modern populations to the animals originally domesticated in this region of Asia. Chinese Mongolian horse populations may therefore retain ancestral genetic variants from the earliest domesticates. Further genomic characterization of these populations in conjunction with archaeogenetic sequence data should be prioritized for understanding recent horse evolution and the domestication process that has led to the wealth of diversity observed in modern global horse breeds.

Comparison of Fecal Microbiota of Mongolian and Thoroughbred Horses by High-throughput Sequencing of the V4 Region of the 16S rRNA Gene.

The hindgut of horses is an anaerobic fermentative chamber for a complex and dynamic microbial population, which plays a critical role in health and energy requirements. Research on the gut microbiota of Mongolian horses has not been reported until now as far as we know. Mongolian horse is a major local breed in China. We performed high-throughput sequencing of the 16S rRNA genes V4 hypervariable regions from gut fecal material to characterize the gut microbiota of Mongolian horses and compare them to the microbiota in Thoroughbred horses. Fourteen Mongolian and 19 Thoroughbred horses were used in the study. A total of 593,678 sequence reads were obtained from 33 samples analyzed, which were found to belong to 16 phyla and 75 genera. The bacterial community compositions were similar for the two breeds. Firmicutes (56% in Mongolian horses and 53% in Thoroughbred horses) and Bacteroidetes (33% and 32% respectively) were the most abundant and predominant phyla followed by Spirochaete, Verrucomicrobia, Proteobacteria, and Fibrobacteres. Of these 16 phyla, five (Synergistetes, Planctomycetes, Proteobacteria, TM7, and Chloroflexi) were significantly different (p<0.05) between the two breeds. At the genus level, Treponema was the most abundant genus (43% in Mongolian horses vs 29% in Thoroughbred horses), followed by Ruminococcus, Roseburia, Pseudobutyrivibrio, and Anaeroplasma, which were detected in higher distribution proportion in Mongolian horses than in Thoroughbred horses. In contrast, Oscillibacter, Fibrobacter, Methanocorpusculum, and Succinivibrio levels were lower in Mongolian horses. Among 75 genera, 30 genera were significantly different (p<0.05) between the two breeds. We found that the environment was one of very important factors that influenced horse gut microbiota. These findings provide novel information about the gut microbiota of Mongolian horses and a foundation for future investigations of gut bacterial factors that may influence the development and progression of gastrointestinal disease in horses.