Transcriptome-Based Investigation of Muscle Tissues in Three Anatomical Regions of Kazakh Horses: A Comparative Study Across Sexes

The Kazakh horse is a local breed known for its dual purpose of providing both milk and meat, characterized by stable genetic properties and a tolerance for coarse feeding. The differences in skeletal muscle fiber types and metabolic characteristics between sexes are crucial factors influencing athletic performance; however, the regulatory mechanisms through which sex affects skeletal muscle development in horses remain unclear. In this study, we examined the longest back muscles, abdominal muscles, and diaphragm muscles of three-year-old Kazakh horses (4 males and 4 females) using histochemical staining combined with transcriptome sequencing to systematically analyze the effects of sex on the characteristics of skeletal muscle fibers and their underlying molecular mechanisms. Our results revealed 361 differentially expressed genes (DEGs) in the longest back muscles of different genders, with 230 DEGs and 236 DEGs identified in the respective muscle types. Notably, TPM1, MYL1, and MYH3 exhibited significant differences between male and female muscles. Gene Ontology (GO) functional annotation indicated that the DEGs were predominantly enriched in categories such as contractile fiber and cytoskeletal framework. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis demonstrated significant enrichment of DEGs in muscle cell cytoskeleton and other signaling pathways associated with muscle fiber contraction and energy metabolism. RT-qPCR validation confirmed the reliability of the transcriptome data. Our findings suggest that sex influences the characteristics of muscle fibers in Kazakh horses by regulating the expression of genes related to the skeletal muscle cell framework. TPM1, MYL1, and MYH3 may serve as core candidate genes for sex-specific regulation of skeletal muscle development. This study may provide a molecular foundation for understanding gender differences in equine athletic performance and holds significant reference value for optimizing horse breeding and training strategies.哈萨克马（Kazakh horse）是一种地方良种，以兼具乳用与肉用双重价值而闻名，具备稳定的遗传特性与耐粗饲能力。性别间骨骼肌纤维类型及代谢特征的差异是影响运动性能的关键因素，但性别调控马骨骼肌发育的分子机制仍未明确。本研究选取3岁龄哈萨克马（公马、母马各4匹）的背最长肌、腹肌与膈肌，采用组织化学染色联合转录组测序技术，系统分析性别对骨骼肌纤维特性的影响及其潜在分子机制。研究结果显示，不同性别哈萨克马的背最长肌中鉴定出361个差异表达基因（DEGs），腹肌与膈肌这两类肌肉中分别鉴定出230个与236个DEGs；其中TPM1、MYL1与MYH3在公马与母马的肌肉组织中呈现显著表达差异。基因本体（Gene Ontology, GO）功能注释结果显示，差异表达基因主要富集于收缩纤维、细胞骨架结构等功能类别。进一步的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析表明，差异表达基因显著富集于肌细胞细胞骨架及其他与肌纤维收缩、能量代谢相关的信号通路。逆转录实时定量PCR（RT-qPCR）验证证实了转录组测序数据的可靠性。本研究结果表明，性别可通过调控骨骼肌细胞骨架相关基因的表达，影响哈萨克马的肌纤维特性；TPM1、MYL1与MYH3或可作为性别特异性调控骨骼肌发育的核心候选基因。本研究为解析马属动物运动性能的性别差异提供了分子基础，同时对优化马匹育种与训练策略具有重要参考价值。