Condition optimization for electroporation transfection in horse skeletal muscle satellite cells.

Satellite cells are an important cellular model for studying muscle growth and development and mammalian locomotion-related molecular mechanisms. In this study, we investigated the effects of voltage, pulse duration, and DNA dosage on horse skeletal muscle satellite cells' electroporation transfection efficiency using the eukaryotic expression plasmid Td Tomato-C1 (5.5 kb) encoding the red fluorescent protein gene mainly based on fluorescence-positive cell rate and cell survival rate. By comparison of different voltages, pulse durations, and DNA doses, horse skeletal muscle satellite cells have nearly 80% transfection efficiency under the condition of voltage 120 V, DNA dosage 7 µg/ml, and pulse duration 30 ms. This optimized electroporation condition would facilitate the application of horse skeletal muscle satellite cells in genetic studies of muscle function and related diseases.

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.

Transcriptome profiling of developing testes and spermatogenesis in the Mongolian horse.

BACKGROUND: Horse testis development and spermatogenesis are complex physiological processes. METHODS: To study these processes, three immature and three mature testes were collected from the Mongolian horse, and six libraries were established using high-throughput RNA sequencing technology (RNA-Seq) to screen for genes related to testis development and spermatogenesis. RESULTS: A total of 16,237 upregulated genes and 8,641 downregulated genes were detected in the testis of the Mongolian horse. These genes play important roles in different developmental stages of spermatogenesis and testicular development. Five genes with alternative splicing events that may influence spermatogenesis and development of the testis were detected. GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed for functional annotation of the differentially expressed genes. Pathways related to "spermatogenesis," male gamete generation," "spermatid development" and "oocyte meiosis" were significantly involved in different stages of testis development and spermatogenesis. CONCLUSION: Genes, pathways and alternative splicing events were identified with inferred functions in the process of spermatogenesis in the Mongolian horse. The identification of these differentially expressed genetic signatures improves our understanding of horse testis development and spermatogenesis.

Analysis of the miRNA transcriptome during testicular development and spermatogenesis of the Mongolian horse.

Numerous studies have shown that microRNAs (miRNAs) are essential for testicular development and spermatogenesis. In order to further characterise these physiological processes, three immature and three mature testes of the Mongolian horse were collected and six libraries were established. Using small RNA sequencing technology, 531 mature miRNAs were identified, including 46 novel miRNAs without previously ascribed functions. Among the 531 miRNAs, 421 were expressed in both immature and mature libraries, 65 miRNAs were found solely in immature testis libraries and 45 miRNAs were found solely in mature testis libraries. Furthermore, among the miRNAs that were identified in both immature and mature libraries, 107 were significantly differentially expressed (corrected P value (padj)<0.05). Among the miRNAs that were only expressed in immature testes, two miRNAs were differentially expressed, whereas among the miRNAs that were only expressed in mature testes, nine miRNAs were differentially expressed. Comprehensive analysis of miRNA and mRNA expression profiles predicted 107 miRNA-mRNA interaction sites. Gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis of the predicted target genes suggested roles of the differentially expressed miRNAs in testicular development and spermatogenesis. These findings identify miRNAs as key factors in the development of the testes and spermatogenesis in the Mongolian horse, which may also help us to understand the mechanisms of fertility in related mammalian species.

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.

Regulatory pathway analysis of coat color genes in Mongolian horses.

BACKGROUND: Studies on the molecular genetics of horse skin pigmentation have typically focused on very few genes and proteins. In this study, we used Illumina sequencing to determine the global gene expression profiles in horses with white-colored coats and those with black-colored coats, with the goal of identifying novel genes that could regulate horse coat color. RESULTS: Genes encoding ribosomal-associated proteins were highly expressed in horse skin. We found a total of 231 unigenes that were differentially expressed between horses with white coats and horses with black coats; 119 were down-regulated, and 112 were up-regulated. Many of the up-regulated genes in black horses, such as genes related to tyrosine metabolism, may directly regulate dark coat color. Keratin genes, MIA family genes, fatty acid-related genes, and melanoma-associated genes were also differentially regulated, which suggests that they may play important roles in coat color formation. CONCLUSIONS: These findings show that the transcription profiles from white and black horse skin provide useful information to understand the genetics underlying the control of skin melanin synthesis in horses, which may enhance our knowledge of human skin diseases, such as melanoma and albinism.

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.

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.

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.

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.

Proteomic Differences Between the Ovulatory and Anovulatory Sides of the Mare's Follicular and Oviduct Fluid.

The follicular fluid and oviduct fluid play major roles in oocyte maturation, sperm activation, and fertilization. To better understand the physiological environments for equine oocyte maturation and fertilization, here we conducted the proteome analysis and comparison on follicular fluids and oviduct fluids from the ovulatory side and the anovulatory side. The results showed that there is no significant difference between two side oviduct fluids, but a total of 71 differential abundance proteins (DAPs) were identified between two side follicular fluids, of which 9 are up-regulated and 62 are down-regulated in ovulatory side follicle fluid versus anovulatory side follicle fluid. As we expected, the function classification and enrichment results indicate that up- and down-regulated proteins are largely related to oocyte meiosis, maturation and ovulation. Noticeably, among 9 up-regulated DAPs in ovulatory side follicle fluid, as the DAP with the greatest fold change, PLA2G1B may be a newly discovered component that influences the efficacy of horse IVM/IVF. The current findings add to our knowledge of the in vivo conditions and regulation of equine reproduction, as well as the regulatory mechanism underpinning alternative ovulation.

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.

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.

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.

MSTN Regulatory Network in Mongolian Horse Muscle Satellite Cells Revealed with miRNA Interference Technologies.

Myostatin (MSTN), a member of the transforming growth factor-ß superfamily, inhibits the activation of muscle satellite cells. However, the role and regulatory network of MSTN in equine muscle cells are not well understood yet. We discovered that MSTN knockdown significantly reduces the proliferation rate of equine muscle satellite cells. In addition, after the RNA sequencing of equine satellite cells transfected with MSTN-interference plasmid and control plasmid, an analysis of the differentially expressed genes was carried out. It was revealed that MSTN regulatory networks mainly involve genes related to muscle function and cell-cycle regulation, and signaling pathways, such as Notch, MAPK, and WNT. Subsequent real-time PCR in equine satellite cells and immunohistochemistry on newborn and adult muscle also verified the MSTN regulatory network found in RNA sequencing analysis. The results of this study provide new insight into the regulatory mechanism of equine MSTN.

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.

[Effects of Kit gene on coat depigmentation in white horses].

Coat color of horse is an important basis for both species identification and individual recognition and is also one of the important references traits for breeding. Therefore, the research on the mechanism of coat fading has become an important part of horses' coat color study. It has been found that the white phenotype is closely related to the mutation of kit gene, which is located on chromosome 3. Investigated results showed that the formation of the epidermal melanoblast and melanin relies on the expression of kit gene, which determines the presence of white phenotype. Nevertheless, studies results have shown that the mutation of kit gene in the white horse exhibited significant differences among species. Horses that the coat color completely faded are very rare and are found occasionally in a few species. However, a larger number of horses that coat color completely faded, called Mongolian white horse, are found in West Ujimqin , Xilin Gol League, Inner Mongolia. Therefore, genetic mechanism of color fading in Mongolian white horses is still not clear. No typical mutations have been observed in 21 exons of kit gene in Mongolian white horse. This paper summarized recent international studies on molecular mechanism of color fading and tried to lay the foundation for the study of formation mechanism of Mongolian white horse. The aim of this review is to provide some valuable references to horses coat color research and breeding.

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.

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.