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Journal of Integrative Agriculture
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Single-nucleus RNA sequencing and metabolomics reveal evolutionary divergence in muscle fiber programming between Equus caballus and Equus asinus

Cong Li1, 2, Xuyong Qin2, Jiale Han2Na Li2, Zhaofei Wang2, Qiwen Yang2, Halima Jafari2, Chuzhao Lei2, Ruihua Dang1, 2#

1 College of Animal Science and Technology, Tarim University, Alar 843300, China

2 Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.

 Highlights 

Ø Single-nucleus RNA-seq atlas of equine skeletal muscle reveals donkey and horse distinct cell types, with species-specific age-related shifts in muscle fiber composition.

Ø The conserved upregulation pattern of LDHA reveals the critical role of propanoate metabolism in energy supply for equine locomotion.

Ø Cross-species transcriptomic comparison delineates a highly conserved atlas of skeletal muscle cell types.

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摘要  

骨骼肌细胞异质性及其分子调控机制是理解马科动物运动生理学的基础。然而,这些机制在驴(Equus asinus)和马(Equus caballus)中仍未得到充分解析。本研究整合了单核转录组学与代谢组学技术,系统比较了两种物种背最长肌在不同发育阶段的特征。我们分别鉴定了驴和马的9种和12种不同的骨骼肌细胞类型。肌纤维组成呈现物种特异性的年龄相关变化:成年马中,I型和II型肌纤维比例均有所增加;而成年驴则相反,I型肌纤维比例下降,II型肌纤维比例上升。马体内II型肌纤维的主导地位可能反映了其适应高强度运动需求的物种特异性适应策略。伪时间分析描绘了肌纤维发育轨迹,并揭示了这些路径上动态变化的基因表达谱。免疫细胞亚群分析显示,成年组中促炎信号通路(TNFNOD样受体通路)被激活,同时树突状细胞的抗炎能力减弱,共同表明存在与年龄相关的促炎状态转变。细胞间通讯分析进一步揭示了关键信号通路(包括BMP(成脂分化)、Notch(免疫调节)和IGF(组织修复)通路)中与年龄相关的失调,这可能共同导致肌肉代谢与再生能力受损。跨物种比较显示,驴和马的骨骼肌转录组具有高度保守性(皮尔逊相关系数为0.87),但也观察到如内皮细胞中SLC29A1等物种特异性标记基因的表达。代谢组学分析识别了整体代谢物类别组成的显著差异,并揭示了两物种间明显不同的基因-代谢物网络。总之,这些发现全面阐明了马科动物骨骼肌发育的细胞动态变化、代谢重塑及进化保守性,为理解哺乳动物肌肉适应机制提供了重要见解,并为提升马科动物运动能力或治疗肌肉疾病提供了潜在靶点。



Abstract  

Skeletal muscle cellular heterogeneity and molecular regulation are fundamental to understanding exercise physiology in Equus species. However, these mechanisms remain incompletely characterized in donkeys (Equus asinus) and horses (Equus caballus). Here, we integrated single-nucleus transcriptomics and metabolomics to systematically compare the longissimus dorsi muscle across developmental stages in both species. We identified nine and twelve distinct skeletal muscle cell types in donkeys and horses, respectively. Muscle fiber composition exhibited species-specific age-related changes: in adult horses, the proportion of both type I and II fibers increased; in adult donkeys, by contrast, the proportion of type I fibers decreased while that of type II fibers increased. The predominance of type II fibers in horses likely reflects a species-specific adaptation to high-intensity locomotor demands. Pseudotime analysis delineated muscle fiber trajectories and revealed dynamic gene expression profiles along these paths. Subpopulation analysis of immune cells revealed the activation of pro-inflammatory signaling pathways (TNF and NOD-like receptor pathways) in adult groups, coupled with diminished anti-inflammatory capacity in dendritic cells, collectively indicating an age-associated pro-inflammatory shift. Intercellular communication analysis further indicated age-related dysregulation in key signaling pathways, including BMP (adipogenic differentiation), Notch (immune regulation), and IGF (tissue repair), which may contribute to impaired muscle metabolism and regenerative capacity. Cross-species comparison revealed that skeletal muscle transcriptomes of donkeys and horses are highly conserved (Pearson correlation coefficient 0.87), although species-specific marker gene expression, such as SLC29A1 in endothelial cells, was observed. Metabolomic profiling identified distinct differences in overall metabolite category composition and revealed significantly divergent gene-metabolite networks between the two species. Together, these findings comprehensively illuminate the cellular dynamics, metabolic remodeling, and evolutionary conservation of skeletal muscle development in Equus species, providing valuable insights into mammalian muscle adaptation and identifying potential targets for enhancing locomotor performance or managing myopathies in equids.

Keywords:  Equus genus        skeletal muscle development        muscle fiber        single-nucleus RNA sequencing        metabolomics  
Online: 10 February 2026  
Fund: 

This study was supported by the Accurate Identification of Lactation and Meat Quality Traits in Jiami Donkeys (K4050425388), High-Level Talent Introduction Program of Tarim University (2025)The Special Program for Introducing Talents to Southern Xinjiang (2025), National Key R&D Program of China (2024YFD1300500), Key Research and Development Program of Shaanxi Province (2025NC-YBXM-110, 2025NC-YBXM-113).

About author:  Cong Li, E-mail: lc863749946@163.com; #Correspondence Ruihua Dang, dangruihua@nwsuaf.edu.cn

Cite this article: 

Cong Li , Xuyong Qin, Jiale Han, Na Li, Zhaofei Wang, Qiwen Yang, Halima Jafari, Chuzhao Lei, Ruihua Dang. 2026. Single-nucleus RNA sequencing and metabolomics reveal evolutionary divergence in muscle fiber programming between Equus caballus and Equus asinus. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.02.014

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