Serum metabolomics reveals stage-dependent metabolic transitions during CIDR-PGF₂α-induced estrus in Kazak ewes

Serum metabolomics is a powerful approach for characterizing the systemic biochemical changes accompanying reproductive transitions in livestock. A hormone-based protocol, CIDR-PGF₂α, is used to drive Kazak ewes through a defined sequence of reproductive stages: the LP (luteal phase, early stage), LDP (luteolysis, middle stage), and OES (estrus, late stage when the ewe exhibits estrus). However, the serum metabolic dynamics during these transitions remain uncharacterized.

Serum samples were collected from 18 Kazak ewes at the three stages LP, LDP, and OES (n = 6 per group) after they received CIDR-PGF₂α synchronization treatment. Untargeted serum metabolomics was performed using a technique called UHPLC-MS/MS (Vanquish-Orbitrap Exploris 120). Differential metabolites were identified between stages using two statistical methods: OPLS-DA (VIP > 1.0) combined with Student's t-test (p < 0.05), and pathway enrichment analysis was conducted using the KEGG database for sheep (Ovis aries).

A total of 1,861 metabolites were identified. Of these, 213, 119, and 203 were significantly different in the LP vs. LDP, LP vs. OES, and LDP vs. OES. All OPLS-DA models demonstrated strong ability to distinguish between stages (R ² Y = 0.994, 0.989, and 0.995; Q ² = 0.580, 0.296, and 0.423), and 200-permutation testing confirmed the absence of overfitting. KEGG enrichment analysis identified 15 significantly enriched pathways in the LP vs. LDP, 11 in LP vs. OES, and 15 significantly enriched pathways in LDP vs. OES (p < 0.05). Notably, these pathways centered on four key areas of metabolism: (1) choline-mediated one-carbon metabolism, (2) mitochondrial fatty acid β-oxidation, (3) sphingolipid signaling, and (4) glycerophospholipid biosynthesis. Choline and betaine aldehyde dropped markedly during the middle stage LDP and partially recovered at the late stage OES. Two short-chain acylcarnitines, 3-hydroxybutyrylcarnitine and acetyl carnitine accumulated progressively from the early stage LP through to the late stage OES, reflecting increasing fatty acid β-oxidation activity. Sphingosine was the only metabolite that was consistently downregulated across all three comparisons (FC = 0.604, 0.336, and 0.556), a pattern consistent with sustained pro-survival sphingosine-1-phosphate signaling throughout the transition. Pregnanediol 3-O-glucuronide showed the largest fold change of any metabolite at OES (FC = 4.6), which verifies efficient hepatic progesterone clearance following luteolysis.

In summary, this study provides the first serum metabolomics characterization of Kazak ewes undergoing CIDR-PGF₂α-induced estrus. It identified five metabolites, choline, betaine aldehyde, 3-hydroxybutyrylcarnitine, acetyl carnitine, and sphingosine, as physiologically meaningful, stage-sensitive biomarkers of the reproductive transition. These results provide a quantitative metabolic baseline for Kazak ewe reproductive physiology and set the stage for future comparative metabolomics studies in naturally cycling ewes. The ultimate aim is to develop metabolite-targeted nutritional strategies to induce estrus during the non-breeding season.