Preliminary Metabolomic Analysis: Serum Metabolomic Dynamics During Estrus Synchronization in Kazakh Mares
by
, ,
Jiahao Liu
1, 
Jintao Gan
1,
Xinkui Yao
1,2,
Jianwen Wang
1,2,
Wanlu Ren
1,2,
Jun Meng
1,2 and
Yaqi Zeng
1,2,* 1
College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China
2
Xinjiang Key Laboratory of Equine Breeding and Exercise Physiology, Urumqi 830052, China
*
Author to whom correspondence should be addressed.
Animals 2026, 16(14), 2222; https://doi.org/10.3390/ani16142222
Submission received: 31 May 2026
/
Revised: 10 July 2026
/
Accepted: 14 July 2026
/
Published: 17 July 2026
(This article belongs to the Section Animal Reproduction)
Simple Summary
This study aimed to systematically characterize dynamic serum metabolomic variations across four critical time points during estrus synchronization in Kazakh mares, screen stage-specific differential serum metabolites and core metabolic pathways, and identify hub metabolic markers closely associated with follicular development and reproductive hormone fluctuations. We adopted untargeted metabolomics to detect serum metabolites at four key stages of estrus synchronization: intravaginal device insertion stage (M1), device removal stage (M3), and 24 h after PG (prostaglandin F2α, PGF2α). All PG injections were combined with hCG administration as the estrus synchronization protocol described in Materials and Methods. We identified significant differential metabolites (DAMs) across stages, dominated by lipids and lipid-like molecules, organic acids and derivatives, and organic heterocyclic compounds. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that nicotinate and nicotinamide metabolism, amino acid biosynthesis, glycerophospholipid metabolism, and the TCA cycle were the most enriched pathways. Nicotinate and nicotinamide metabolism is associated with pathways related to energy metabolism, steroidogenesis, and redox balance via the generation of nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADPH), while amino acid biosynthesis provides substrates for follicular cell proliferation and microenvironment regulation. Temporal clustering identified hub metabolites including N-hexanoylsphingosine, palmitoyl sphingomyelin, L-leucyl-L-proline, uric acid, and glycerophosphocholine, which were closely correlated with reproductive hormone levels. These findings reveal the dynamic metabolic regulatory patterns during estrus synchronization in Kazakh horses fill the gap in metabolomic research on estrus synchronization in this breed, and may provide insights for refining estrus synchronization strategies and reproductive management in Kazakh horses.
Abstract
This study aimed to analyze the differences in serum metabolomes at different stages of estrus synchronization in Kazakh horses, to characterize dynamic changes in serum metabolites during estrus synchronization, and to screen potential metabolic markers and key regulatory pathways. Four sampling stages (M1: intravaginal device insertion; M3: device removal, labeled consistently with the pre-experiment timeline; P: 24 h post-PG injection; L: tertiary follicle emergence) were selected. The research results showed that 37 significantly different metabolites (DAMs) were identified in the M1-vs.-M3 positive ion mode, with 16 metabolites significantly upregulated and 21 significantly downregulated. In the M1-vs.-M3 negative ion mode, 47 significantly different metabolites were identified, including 17 upregulated and 30 downregulated. In the M3-vs.-P positive ion mode, 69 significantly different metabolites were identified, with 53 upregulated and 16 downregulated. In the M3-vs.-P negative ion mode, 63 significantly different metabolites were identified, including 39 upregulated and 24 downregulated. In the Pvs-L positive ion mode, 65 significantly different metabolites were identified, with 16 upregulated and 49 downregulated. In the P-vs.-L negative ion mode, 61 significantly different metabolites were identified, including 13 upregulated and 48 downregulated. The significantly different metabolites mainly included lipids and lipid-like molecules, organic heterocyclic compounds, and organic acids and their derivatives. KEGG enrichment analysis showed that the significantly different metabolites in Kazakh horses at different stages were mainly enriched in nicotinic acid and nicotinamide metabolism, amino acid synthesis, and related pathways. This study revealed significant differences in the serum metabolome, and nicotinic acid and nicotinamide metabolism, by generating NAD+ and NADPH, simultaneously supporting energy supply, steroid hormone synthesis, and antioxidant protection, which are the basic metabolic guarantees for the development of follicles from quiescence to growth; amino acid synthesis not only provides protein raw materials for follicular cell proliferation but also participates in the regulation of the follicular microenvironment by synthesizing signaling molecules and antioxidant substances. This study enriched the research on the metabolic regulation of estrus synchronization in equine species, filled the gap in the metabolomics research of estrus synchronization in Kazakh horses, and provided an important theoretical basis and practical reference for optimizing the estrus synchronization treatment plan for Kazakh horses, adjusting the focus of feeding and management at each stage and improving the conception rate.
Keywords:
Kazakh horse; synchronization of estrus; serum metabolomics; DAMs
