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. 2025 Aug 5;15(15):2291.
doi: 10.3390/ani15152291.

Metabolomic and Molecular Mechanisms of Glycerol Supplementation in Regulating the Reproductive Function of Kazakh Ewes in the Non-Breeding Season

Ying Nan  1 , Baihui Jiang  1 , Xingdong Qi  1 , Cuifang Ye  1 , Mengting Xie  1 , Zongsheng Zhao  1
Affiliations

Metabolomic and Molecular Mechanisms of Glycerol Supplementation in Regulating the Reproductive Function of Kazakh Ewes in the Non-Breeding Season

Ying Nan et al. Animals (Basel). .

Abstract

The activation mechanism of the reproductive axis in Kazakh ewes during the non-breeding season was explored by supplementation with glycerol complex (7% glycerol + tyrosine + vitamin B9). The experiment divided 50 ewes into five groups (n = 10). After 90 days of intervention, it was found that significant changes in serum DL-carnitine, N-methyl-lysine and other differential metabolites were observed in the GLY-Tyr-B9 group (p < 0.05, "p < 0.05" means significant difference, "p < 0.01" means "highly significant difference"). The bile acid metabolic pathway was specifically activated (p < 0.01). The group had a 50% estrus rate, ovaries contained 3-5 immature follicles, and HE staining showed intact granulosa cell structure. Serum E2/P4 fluctuated cyclically (p < 0.01), FSH/LH pulse frequency increased (p < 0.01), peak Glu/INS appeared on day 60 (p < 0.05), and LEP was negatively correlated with body fat percentage (p < 0.01). Molecular mechanisms revealed: upregulation of hypothalamic kiss-1/GPR54 expression (p < 0.01) drove GnRH pulses; ovarian CYP11A1/LHR/VEGF synergistically promoted follicular development (p < 0.05); the HSL of subcutaneous fat was significantly increased (p < 0.05), suggesting involvement of lipolytic supply. Glycerol activates the reproductive axis through a dual pathway-L-carnitine-mediated elevation of mitochondrial β-oxidation efficacy synergizes with kisspeptin/GPR54 signalling enhancement to re-establish HPO axis rhythms. This study reveals the central role of metabolic reprogramming in regulating seasonal reproduction in ruminants.

Keywords: estrus induction; glycerol; kisspeptin/GnRH pulse; lipid metabolism reprogramming; metabolic–neural axis regulation.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Vulva of a spent ewe (A), vulva of an estrous ewe (B).
Figure 2
Figure 2
Ovary of an estrous ewe (A) and HE-stained section (B).
Figure 3
Figure 3
Hormone levels in the estrous cycle of ewes. (A) P4 hormone level, (B) E2 hormone level, (C) LH hormone level, and (D) FSH hormone level. Note: “p < 0.05” indicates a significant difference, which is labeled with “*” in the figure, and “p < 0.01” indicates “highly significant difference, which is labeled with “**” in the figure.
Figure 4
Figure 4
Hormonal changes in lipid metabolism. (A) Glu levels. (B) INS levels. (C) LEP levels. “p < 0.05” indicates a significant difference, which is labeled with “*” in the figure.
Figure 5
Figure 5
Gene expression quantity. (A) Oestrus gene expression in hypothalamic tissue; (B) Oestrus gene expression in ovarian tissue; (C) Oestrus gene expression in subcutaneous adipose tissue. “p < 0.05” indicates a significant difference, which is labeled with “*” in the figure, and “p < 0.01” indicates “highly significant difference, which is labeled with “**” in the figure.
Figure 6
Figure 6
(A) Metabolite classification. (B) QC sample correlation analysis. (C) Total sample PCA analysis.
Figure 7
Figure 7
(A) PLS-DA Score Scatter Plot. (B) Ranked Validation Plot.
Figure 8
Figure 8
(A) Volcano diagram. (B) Matchstick diagram.
Figure 8
Figure 8
(A) Volcano diagram. (B) Matchstick diagram.
Figure 9
Figure 9
Clustering heat map of differential metabolites.
Figure 10
Figure 10
Correlation diagram of differential metabolites.
Figure 11
Figure 11
KEGG enrichment bubble plot.
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