A Comparative Analysis of the Gene Expression Profiles in the Mammary Glands of Lactating and Nonlactating Mares at the Second Month of Gestation.

To investigate molecular regulation involved in lactation during pregnancy, this study focused on the transcriptomic profiles of mammary tissue from lactating and non-lactating Mongolian mares at the second month of gestation. A total of 4197 differentially expressed genes were identified by comparing mammary tissues from pregnant mares at two different states, including 1974 differentially expressed genes such as the milk protein-related genes a-s1-casein (CSN1S1), k-casein (CSN3), lactalbumin (LALBA), and lactoferrin (LTF), which were highly expressed in the lactating mares group, and overall, these differentially expressed genes were mainly associated with biological processes such as endoplasmic reticulum protein processing, the Toll-like receptor signaling pathway, steroid biosynthesis, cytokine-cytokine receptor interactions, and amino sugar and nucleotide glycolysis. These findings serve as a foundation for investigating the molecular underpinnings of lactation in pregnant equids.

Proteome and metabolomic profile of Mongolian horse follicular fluid during follicle development.

During follicular development, changes in the composition of the follicular fluid are synchronized with the development of oocytes. Our aim was to screen the key factors affecting oocyte maturation and optimize the in vitro culture protocol by understanding the changes of proteins and metabolites in follicular fluid. Follicles are divided into three groups according to their diameter (small follicle fluid (SFF): 10 mm < d < 20 mm; medium follicle fluid (MFF): 20 mm < d < 30 mm; large follicle fluid (LFF): 30 mm < d). Proteins and metabolites from the follicular fluid were analyzed by mass spectrometry. The results showed that: in LFF vs MFF, 20 differential abundant protein (DAP) and 88 differential abundant metabolites (DAM) were screened out; In SFF vs MFF, 3 DAPs and 65 DAMs were screened out; In MFF vs SFF, 24 DAPs and 35 DAMs were screened out. The analysis of differential proteins and metabolites showed that glycerophosphate hydrolysis decreased during follicular development, and proteins played a major role in metabolism and binding. In addition, DAMs and DAPs are co-enriched in the "linoleic acid metabolism" pathway. Combinatorial analysis reveals the dynamic profile of follicular fluid during follicular development and provides fundation for further exploring the function of follicular fluid in Mongolian horse.

Comparative Study on the Sperm Proteomes of Horses and Donkeys.

The reproductive performance of horse sperm and donkey sperm has been reported to differ. Sperm proteins play a crucial role in sperm viability and fertility. Although differences between species are known, no prior study has investigated disparities in the sperm proteome between horses and donkeys. Therefore, this study characterized and compared the sperm proteomes of horses and donkeys using 4D-DIA mass spectrometry technology. We identified 3436 proteins in horse sperm and 3404 proteins in donkey sperm. Of these, 3363 proteins were expressed in both horse and donkey sperm, with 73 proteins being specifically expressed in horse sperm, and 41 in donkey sperm. According to data analysis, donkeys exhibited a greater percentage of motility and progressive movement in straight-line sperm than horses, as well as lower percentages of static and slow sperm than horses. Joint analysis of the results from the horse and donkey sperm proteomes and their CEROS II-read parameters demonstrated a possible association between sperm proteins and their sperm viability patterns. These findings suggest that there are discrepancies in the expression levels and protein compositions of horse and donkey sperm and that certain specific proteins may be responsible for the differences in performance between these two species.

Optimizing the preparation of paraffin sections from stallion testes.

The preparation of paraffin sections is an important experimental technique in animal histological research, and key factors that determine the quality of a section include the dehydration time, waxing time, and drying temperature of the paraffin section. Paraffin sections obtained from testis tissue of adult horses exhibited higher quality with clear tissue structure and complete cell morphology after they underwent gradient dehydration for 6 hours, were immersed in wax for 60 minutes, and were dried in a 75-degree oven for 15 minutes. The detailed, optimized procedures that are developed in the current study may simplify histological experiments and research on equine testes.

Comparative Analysis of mRNA and lncRNA Expression Profiles in Testicular Tissue of Sexually Immature and Sexually Mature Mongolian Horses.

Testicular development and spermatogenesis are tightly regulated by both coding and non-coding genes, with mRNA and lncRNA playing crucial roles in post-transcriptional gene expression regulation. However, there are significant differences in regulatory mechanisms before and after sexual maturity. Nevertheless, the mRNAs and lncRNAs in the testes of Mongolian horses have not been systematically identified. In this study, we first identified the testicular tissues of sexually immature and sexually mature Mongolian horses at the tissue and protein levels, and comprehensively analyzed the expression profiles of mRNA and lncRNA in the testes of 1-year-old (12 months, n = 3) and 10-year-old (n = 3) Mongolian horses using RNA sequencing technology. Through gene expression analysis, we identified 16,582 mRNAs and 2128 unknown lncRNAs that are commonly expressed in both sexually immature and sexually mature Mongolian horses. Meanwhile, 9217 mRNAs (p < 0.05) and 2191 unknown lncRNAs (p < 0.05) were identified as differentially expressed between the two stages, which were further validated by real-time fluorescent quantitative PCR and analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The analysis results showed that genes in the sexually immature stage were mainly enriched in terms related to cellular infrastructure, while genes in the sexually mature stage were enriched in terms associated with hormones, metabolism, and spermatogenesis. In summary, the findings of this study provide valuable resources for a deeper understanding of the molecular mechanisms underlying testicular development and spermatogenesis in Mongolian horses and offer new perspectives for future related research.

Different expression patterns of DNA methyltransferases during horse testis development.

DNA methyltransferases (DNMTs) are important epigenetic modification during spermatogenesis. To further evaluate the pattern of DNMTs in horse testes during development, we investigated the expression and localization of DNMT1, DNMT3a and DNMT3b at different time points. The qRT-PCR results showed that DNMT1 expression was maintained in testes tissue from 6-month-old (0.5y) to 2-year-old (2y) of age and decreased after 3-year-old (3y) (P < 0.01). The expression levels of DNMT3a and DNMT3b peaked in testes tissue at 3y (P < 0.01). At 4-year-old (4y), the expression of DNMT3a and DNMT3b was decreased and became similar to that at 0.5y. Immunofluorescence of DNMT1, DNMT3a and DNMT3b on testis samples confirmed the differential expression and localization of these three DNA methylation transferases during horse development. Further molecular biological studies are needed to understand the implications of the expression patterns of these DNMTs in horse testes.

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.

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.

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.

Lower expression of the equine maternally imprinted gene IGF2R is related to the slow proliferation of hinny embryonic fibroblast in vitro.

BACKGROUND: Proliferation of embryonic fibroblasts under the same cell culture conditions, hinny embryonic fibroblasts (HiEFs) was slower than horse embryonic fibroblast (HEFs), donkey embryonic fibroblasts (DEFs) and mule embryonic fibroblasts (MuEFs). The imprinted genes IGF2 and IGF2R are important for cell proliferation. Therefore, we investigated whether the slower proliferation of HiEFs is related to an aberrant gene expression of IGF2 or its receptors or genes influencing the expression of the IGF2 system. METHODS AND RESULTS: Real-time polymerase chain reaction, immunofluorescence and cell starving experiment in HEFs, DEFs, MuEFs and HiEFs revealed that the slower proliferation of HiEF in vitro was related to its lower expression of IGF2R (P < 0.001). Moreover, quantification of allele-specific expression and bisulfate assay confirmed that in both MuEFs and HiEFs, IGF2R had normal maternal imprinting, implying that the imprint aberrant was not involved in the lower IGF2R expression in HiEFs. CONCLUSIONS: The reduction of IGF2R expression in HiEFs is associated with its slower proliferation in vitro.

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.

OCT4 expression transactivated by GATA protein is essential for non-rodent trophectoderm early development.

Oct4 is exclusively expressed in rodent inner cell mass (ICM) but silenced in its trophectoderm (TE). However, for many non-rodent animals, including pig, cattle, rabbit, goat, and human, OCT4 has a remarkable expression in early TE. This study, applying pig as the main research model, proves that OCT4 expression in TE is supported by a unique GATA motif in the OCT4 upstream conserved regulatory region, and GATA4 is responsible for its activation. Moreover, OCT4 acts as a specific regulator of a narrow range of genes (including BCL2A1 and HNRNP2AB1) that are essential for the first wave of rapid proliferation in early TE. This study describes the regulatory mechanism to direct the OCT4 expression and its significance in TE of porcine preimplantation embryo.

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.

The transcriptome landscapes of allantochorion and vitelline-chorion in equine day 30 conceptus.

During equine early gestation, trophectoderm forms chorion tissue, which is composed of two parts that one is covering allantoin, called allantochorion (AC) and another is covering yolk sac, which here we call vitelline-chorion (VC). Given that little is known about the equine trophoblast-derived chorion differentiation at an early stage, we first compared the transcriptome of AC and VC of day 30 equine conceptus based on RNA-sequencing. As a result, we found that compared to VC, there are 484 DEGs, including 305 up- and 179 down-regulated genes in AC. GO and KEGG analysis indicated that up-regulated genes in AC are mainly cell proliferation and cell adhesion-related genes, participating in allantois expansion and allantochorionic-placenta formation; dominant genes in VC are extracellular exosome and other cell adhesion-related genes implicated in direct and indirect conceptus-maternal communication. Additionally, as for the progenitor chorion tissue of equine chorionic gonadotropin secreting endometrium cup-the chorionic girdle (CG), which locates at the junction of the dilating AC and regressing VC, we revealed its unique gene expression pattern and the gene regulation during its further differentiation in vitro. Collectively, this study sheds light on the molecular events regarding the trophoblast differentiation and function at an early stage of the equine preimplantation conceptus.

Effect of NANOG overexpression on porcine embryonic development and pluripotent embryonic stem cell formation in vitro.

The efficiency of establishing pig pluripotent embryonic stem cell clones from blastocysts is still low. The transcription factor Nanog plays an important role in maintaining the pluripotency of mouse and human embryonic stem cells. Adequate activation of Nanog has been reported to increase the efficiency of establishing mouse embryonic stem cells from 3.5 day embryos. In mouse, Nanog starts to be strongly expressed as early as the morula stage, whereas in porcine NANOG starts to be strongly expressed by the late blastocyst stage. Therefore, here we investigated both the effect of expressing NANOG on porcine embryos early from the morula stage and the efficiency of porcine pluripotent embryonic stem cell clone formation. Compared with intact porcine embryos, NANOG overexpression induced a lower blastocyst rate, and did not show any advantages for embryo development and pluripotent embryonic stem cell line formation. These results indicated that, although NANOG is important pluripotent factor, NANOG overexpression is unnecessary for the initial formation of porcine pluripotent embryonic stem cell clones in vitro.

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.

Interspecies cell fusion between mouse embryonic stem cell and porcine pluripotent cell.

In the area of stem cell research, fusion of somatic cells into pluripotent cells such as mouse embryonic stem (ES) cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell on the pluripotent state of somatic nucleus. As many other groups, we previously established a porcine pluripotent cell line at a low potential. Therefore, here, we performed experiments to investigate if the fusion with mouse ES cell could improve the pluripotent state of porcine pluripotent cell. Our data showed that resultant mouse-porcine interspecies fused cells are AP positive, and could be passaged up to 20 passages. Different degrees of increases in expression of porcine pluripotent genes proved that pig-origin gene network can be programmed by mouse ES. Further differentiation study also confirmed these fused cells' potential to form three germ layers. However, unexpectedly, we found that chromosome loss and aberrant (especially in porcine chromosomes) is severe after the cell fusion, implying that interspecies cell fusion may be not suitable to study porcine pluripotency without additional supportive conditions for genome stabilization.

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.