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.

Horses and donkeys, as integral members of the equine family, exhibit distinct reproductive capabilities and characteristics. Seminal plasma, the fluid component of semen, contains a variety of proteins that play critical roles in sperm function and fertility. This study aimed to systematically compare the protein profiles in the seminal plasma of horses and donkeys, thereby elucidating the molecular differences between these two species. The study utilized 4D-DIA proteomics technology to analyze seminal plasma from horses and donkeys and further validated key proteins through Western blot. Our findings revealed significant variations in seminal plasma protein composition between horses and donkeys. We identified 2380 and 2385 proteins in the seminal plasma of horses and donkeys. Among these proteins, 59 are solely present in the seminal plasma of horses, and 64 uniquely exsit in that of donkeys, respectively. These insights enhance our understanding of the biological mechanisms underlying the reproductive distinctions between these equine species. Moreover, the identified species specific proteins may be essential for thier sperm quality and function, which holds practical value for breeding programs and investigations.

Introduction: Equine chorionic girdle cells can give rise to the endometrial cup, a structure that secretes equine chorionic gonadotropin (eCG). To date, the mechanisms underlying the proliferation, differentiation, invasion, and hormone secretion of equine chorionic girdle cells are not fully understood. During human pregnancy, interleukin-6 (IL-6) is maternally expressed to stimulate the invasion and migration of human syncytiotrophoblast cells and influence the synthesis of human chorionic gonadotropin. Although many previous studies reported the same upregulation of IL-6 during equine placentation, its effects on equine chorionic girdle cells have never been tested. Methods: In this study, we tested the effects of different concentrations of IL-6 on the morphology, differentiation, proliferation, hormone production, invasion, migration, and gene expression profiles of equine chorionic girdle cells. Results: We found that, with respect to morphology and proliferation, IL-6 had no significant effect; with respect to eCG production, 30 ng/mL IL-6 significantly increased the expression of genes related to eCG production, whereas treatment with 70 ng/mL IL-6 downregulated these genes. However, no significant increase in intracellular protein levels or eCG secretion was observed following treatment with any concentration of IL-6. Regarding cell migration and invasion, we found that IL-6 treatment had no significant effect on the migration capability of equine chorionic girdle cells but did enhance the invasion capability of equine chorionic girdle cells and upregulated the expression of the invasion-related genes, Mmp2 and Mmp9. Finally, our transcriptomic study revealed that in equine chorionic girdle cells, IL-6 treatment mainly affected the expression of genes related to the NOD-like receptor signaling pathway and the JAK–STAT signaling pathways, which are involved in immune and inflammatory responses. Conclusions: In summary, this study demonstrates the positive effects of IL-6 on the cytokine secretion and invasive ability of equine chorionic girdle cells.

Background: The reproductive ability of equine species is a critical component of equine breeding programs, with sperm quality serving as a primary determinant of reproductive success. In this study, we perform an integrative analysis of proteomics and metabolomics in seminal plasma to identify proteins and metabolites associated with sperm quality and reproductive ability in equine species. Methods: We utilized the CEROS instrument to assess the morphology and motility of sperm samples from three horses and three donkeys. Additionally, we statistically analyzed the mating frequency and pregnancy rates in both species. Meanwhile, the 4D-DIA high-throughput proteomic and metabolomic profiling of seminal plasma samples from horses and donkeys revealed a complex landscape of proteins and metabolites. Results: Our findings reveal a certain degree of correlation between seminal plasma proteins and metabolites and sperm quality, as well as overall fertility. Notably, we found that the proteins B3GAT3, XYLT2, CHST14, HS2ST1, GLCE, and HSPG2 in the glycosaminoglycan biosynthesis signaling pathway; the metabolites D-glucose, 4-phosphopantetheine, and 4-hydroxyphenylpyruvic acid in the tyrosine metabolism, starch, and source metabolisms; and pantothenate CoA biosynthesis metabolism present unique characteristics in the seminal plasma of equine species. Conclusions: This comprehensive approach provides new insights into the molecular mechanisms underlying sperm quality and has identified potential proteins and metabolites that could be used to indicate reproduction ability. The findings from this study could be instrumental in developing novel strategies to enhance equine breeding practices and reproductive management. Future research will focus on exploring their potential for clinical application in the equine industry.

Genomic imprinting is critical for mammalian development, but its regulation varies across species. The insulin-like growth factor 2 receptor (IGF2R), which is a maternally expressed imprinted gene critical for cell proliferation and differentiation, as well as embryonic and placental development, is classically regulated by differentially methylated regions (DMRs) and lncRNA-Airn in mice. However, studies on this in equus are scarce, especially in terms of mechanistic studies. In the present study, heart, liver, spleen, lung, kidney, brain, and muscle samples were obtained from horses, donkeys, and hybrids, and gene expression and imprinting state were tested to investigate the imprinting regulation of Igf2r in these animals. Bisulfite sequencing combined with an allele-specific expression analysis revealed a tissue-specific loss of imprinting in the mule liver and hybrid brain tissues. Strikingly, we found that the maternal-specific expression of equine Igf2r did not rely on the canonical DMRs or lncRNA-Airn. Surprisingly, DNA methylation of a specific region called CpG island 2 (CpGI2) in the Igf2r promoter showed cis-acting inheritance, meaning that the DNA methylation patterns of the parental alleles are retained in hybrid tissues. Notably, the DNA methylation of CpGI2 correlated negatively with Igf2r expression in the spleen (R² = 0.8797, p = 6.46 × 10⁻⁶), lung (R² = 0.8569, p = 1.57 × 10⁻⁵), and kidney (R² = 0.8650, p = 3.85 × 10⁻⁶). Our findings suggest that imprinting may work differently in other species. This study provides a framework for understanding imprinting diversity in hybrids and shows that equine hybrids can be used to study how epigenetic inheritance works.