Xinyu Li’s research while affiliated with Inner Mongolia Agricultural University and other places

Cryopreservation of semen has advanced significantly with the development of artificial insemination techniques, but post-thawed sperm often exhibit reduced viability, membrane integrity, and acrosome integrity compared to fresh sperm, leading to decreased fertilization capacity. Oxidative stress is a major concern during cryopreservation. This study investigated the use of resveratrol (RSV), a potent antioxidant, in the cryopreservation of Mongolian horse semen. Different concentrations of RSV were incorporated into semen cryopreservation extenders, and the morphological and antioxidant indices of post-thawed sperm were assessed to determine the optimal RSV concentration. The study also employed tandem mass tag (TMT) quantitative proteomics technology to explore differential proteins and their pathways. The results showed that sperm quality parameters were positively correlated with RSV concentration within a certain range (10–40 µmol L−1) and were significantly higher than the control group. RSV also enhanced the antioxidant capacity of sperm, with the optimal effect observed at 40 µmol L−1. Proteomics analysis identified 10 differential proteins between the control and optimal RSV concentration groups, with 7 upregulated proteins primarily involved in antioxidant activity and maintaining intracellular redox balance. These findings were further validated through real-time fluorescent quantitative PCR and protein immunoblotting, suggesting that RSV has potential as an effective antioxidant for improving the cryopreservation of Mongolian horse semen.

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.

Fermented mare’s milk (koumiss), a traditional Central Asian dairy product derived from fermented mare’s milk, is renowned for its unique sour taste and texture. It has long been consumed by nomadic tribes for its nutritional and medicinal benefits. This study aimed to comprehensively analyze the protective effects of koumiss against alcohol-induced harm across behavioral, hematological, gastrointestinal, hepatic, and reproductive dimensions using a mouse model. Optimal intoxicating doses of alcohol and koumiss doses were determined, and their effects were explored through sleep tests and blood indicator measurements. Pretreatment with koumiss delayed inebriation, accelerated sobering, and reduced mortality in mice, mitigating alcohol’s impact on blood ethanol levels and various physiological parameters. Histopathological and molecular analyses further confirmed koumiss’s protective role against alcohol-induced damage in the liver, stomach, small intestine, and reproductive system. Transcriptomic studies on reproductive damage indicated that koumiss exerts its benefits by influencing mitochondrial and ribosomal functions and also shows promise in mitigating alcohol’s effects on the reproductive system. In summary, koumiss emerges as a potential natural agent for protection against alcohol-induced harm, opening avenues for future research in this field.

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.

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.

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.

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.