Comparison of Muscle Metabolomics Between Two Chinese Horse Breeds

The unique athletic capabilities and locomotion of horses are intrinsically linked to their skeletal muscle metabolism. Understanding the metabolic mechanisms underlying muscle development is crucial for optimizing training, nutrition, and overall performance. This study delves into the breed-specific metabolic differences between Guanzhong (GZ) horses, an athletic breed with a larger stature, and Ningqiang (NQ) ponies, a smaller breed typically used for ornamental purposes, both native to the same region in China.

Introduction

Horses, with their impressive athletic abilities, serve as excellent models for studying muscle metabolism. While previous research has explored exercise-related metabolites and identified biomarkers for certain equine conditions, a comprehensive understanding of the metabolic regulation governing muscle development across different breeds remains an area requiring further investigation. Factors such as exercise, climate, and diet significantly influence muscle metabolism. This study focuses on two distinct Chinese horse breeds, Guanzhong (GZ) and Ningqiang (NQ) horses, which, despite similar environmental conditions and management, exhibit clear phenotypic differences. GZ horses are known for their adaptability, taller stature, and rapid development, while NQ ponies are characterized by their smaller size, adapted for mountainous terrains. These size and performance disparities likely stem from variations in muscle composition and metabolism. Untargeted metabolomics, particularly using liquid chromatography-mass spectrometry (LC-MS), offers a powerful approach to comprehensively analyze the metabolite profiles of tissues and uncover differences under various conditions. By employing both MS1 and MS2 ion data, this study aims to identify key metabolites and pathways associated with muscle development in these two breeds, providing insights into their distinct physiological characteristics.

Materials and Methods

Animal Ethics and Sample Collection

All animal procedures were approved by the Animal Care Committee of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences. Muscle biopsies were collected from the gluteus medius of 12 male horses (6 GZ, 6 NQ) under local anesthesia. Samples were immediately flash-frozen in liquid nitrogen and stored at -80°C for subsequent analysis. Post-biopsy wound care involved disinfection and application of Yunnan Baiyao powder for hemostasis, a traditional Chinese herbal medicine.

Biochemical Analysis

Muscle glycogen concentration, along with the activities of citrate synthase (CS) and hexokinase (HK) – key enzymes in the tricarboxylic acid (TCA) cycle – were measured using ELISA kits.

LC-MS Measurement and Data Processing

Metabolomic profiling was conducted using high-resolution tandem mass spectrometry (LC-MS/MS). Muscle tissue was extracted, and metabolites were analyzed using an ultra-high-performance liquid chromatography (UPLC) system coupled with a TripleTOF 5600+ mass spectrometer. Data processing involved peak detection, alignment, and metabolite identification using software packages like XCMS, msConvert, and CAMERA, referencing databases such as HMDB and KEGG. Quality control samples were utilized to ensure data stability and reproducibility. Principal component analysis (PCA) was performed to assess sample variability and identify potential outliers.

Statistical Analysis

Statistical analyses, including Student’s t-tests and Partial Least Squares Discriminant Analysis (PLS-DA), were employed to identify significantly differential metabolites between the GZ and NQ horse groups. Metabolites were considered significant if they met criteria for fold change (FC ≥ 2), variable importance in projection (VIP ≥ 1), and adjusted p-value (q-value ≤ 0.05). Correlation analyses and pathway enrichment analyses (MetPA) were conducted to elucidate the functional roles of identified metabolites.

Results

Biochemical Differences in Muscle Tissue

Guanzhong (GZ) horses exhibited significantly higher muscle glycogen concentrations compared to Ningqiang (NQ) ponies. Furthermore, the activities of citrate synthase and hexokinase, crucial enzymes for energy metabolism, were also significantly elevated in GZ horses, suggesting a greater capacity for aerobic energy production and utilization.

Metabolite Profiling and Identification

LC-MS/MS analysis identified a substantial number of metabolites in the muscle tissue. Principal component analysis (PCA) clearly separated the GZ and NQ horse groups, indicating distinct metabolic profiles. Further analysis using PLS-DA confirmed this separation. A total of 13 significant differential metabolites were identified between the two breeds. These metabolites were predominantly categorized under lipids and lipid-like molecules.

Key Metabolic Pathways Identified

Pathway enrichment analysis revealed that the most significantly altered metabolic pathways between GZ and NQ horses were glutathione metabolism and taurine and hypotaurine metabolism. These pathways are strongly associated with antioxidant defense and cellular protection against oxidative stress, which are critical during muscle development and function.

Muscle Metabolic Network

A metabolic network analysis indicated complex interactions between identified metabolites. Notably, metabolites related to glutathione metabolism and taurine/hypotaurine metabolism were found to interact with pathways involved in energy production, such as the TCA cycle. Several key metabolites, including those involved in valine, leucine, and isoleucine degradation, as well as intermediates in carnitine synthesis, were found in higher abundance in NQ horses, suggesting potential differences in fatty acid oxidation and energy storage mechanisms.

Discussion

The findings of this study highlight significant metabolic differences between the athletic Guanzhong (GZ) horses and the Ningqiang (NQ) ponies. The higher muscle glycogen content and elevated activities of key glycolytic and TCA cycle enzymes (HK and CS) in GZ horses are consistent with their greater athletic potential and capacity for energy storage and utilization during strenuous activity. The abundance of lipids and lipid-like molecules, further categorized into glycerophospholipids, organic acids, and fatty acyls, underscores the importance of lipid metabolism in providing energy for muscle function in horses.

The identification of significantly altered glutathione and taurine metabolism pathways points to differences in antioxidant capacity between the breeds. Glutathione is a crucial endogenous antioxidant, protecting cells from oxidative damage, while taurine plays vital roles in osmoregulation, membrane stabilization, and antioxidant defense. The observed alterations in these pathways suggest that GZ horses may have a more robust system for managing oxidative stress associated with high levels of physical activity.

The metabolites found to be more abundant in NQ horses, such as those involved in carnitine synthesis and fatty acid oxidation, suggest potential differences in how these breeds utilize and store energy. Carnitine is essential for transporting fatty acids into mitochondria for energy production. These metabolic variations likely contribute to the distinct athletic phenotypes observed between the two breeds.

Conclusion

This study successfully differentiated the muscle metabolic profiles of Guanzhong (GZ) and Ningqiang (NQ) horses using untargeted metabolomics. GZ horses, the more athletic breed, demonstrated higher muscle glycogen content and enzymatic activity associated with energy metabolism. Conversely, differences in lipid metabolism, along with the glutathione and taurine/hypotaurine pathways, were prominent between the breeds, suggesting distinct mechanisms for energy utilization and antioxidant defense. These findings provide a foundational understanding of the metabolic basis for athletic performance in different horse breeds and could inform future breeding strategies and nutritional management aimed at enhancing equine performance. Further in vivo and in vitro studies are warranted to fully elucidate the biological mechanisms underlying these observed metabolic differences.

Data Availability Statement

The original contributions presented in the study are included in the article and its supplementary material. Further inquiries can be directed to the corresponding author.

Ethics Statement

The animal study was reviewed and approved by the Animal Care Committee of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (approval number: IAS2019-24).

Author Contributions

SM conceptualized the study, performed sample preparation and statistical analyses, and drafted the manuscript. YZ collected samples and contributed to sample preparation and manuscript review. SL and ZZ were involved in sample collection. XL and LJ supervised the project, conceived the study, and reviewed the manuscript. All authors have read and approved the final manuscript.

Funding

This research was supported by grants from the National Natural Science Foundation of China (nos. 32002144, 31560620, 31972530, and 31772553). XL also received support from the International Postdoctoral Exchange Fellowship Program (20190102) and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (No. 101027750).

Acknowledgments

The authors acknowledge the financial support from the National Natural Science Foundation of China. They are grateful to the horse owners for their collaboration and to Lianchuan Biotechnology Co., Ltd. for their assistance with liquid chromatography–mass spectrometry analysis.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2023.1162953/full#supplementary-material

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