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

## TL;DR

This study shows how adding glycerol to the diet of sheep can activate their reproductive systems during the non-breeding season through changes in metabolism and hormone signaling.

## Contribution

The study introduces a novel theoretical framework of 'metabolic reprogramming for estrus' and identifies key metabolic pathways involved in seasonal reproduction in ruminants.

## Key findings

- Glycerol supplementation significantly activated the bile acid metabolic pathway and increased estrus rates in Kazakh ewes.
- L-carnitine and kisspeptin/GPR54 signaling were found to synergistically regulate follicular development and reproductive hormone cycles.
- Metabolic reprogramming through glycerol improved mitochondrial β-oxidation and re-established HPO axis rhythms in non-breeding ewes.

## Abstract

This study focuses on seasonal reproduction in sheep, the core problem of efficient production, and innovatively proposes a theoretical framework of “metabolic reprogramming for estrus”. Because the key metabolic nodes and interactions across tissues in nutrition-induced estrus in non-breeding ewes have not been elucidated, this project uses glycerol as a metabolic intervention carrier and reveals three major scientific points through the strategy of metabolomics integration: (1) How energy signalling regulates the activation of Kisspeptin/GPR54 signalling in the hypothalamus through metabolite-hormone axes; (2) The lipolytic metabolite (L-carnitine) cooperates with the microenvironment of ovarian steroid production to promote follicular development; (3) The contribution of the bile acid–metabolism axis to seasonal reproductive regulation. The research results will be the first to map out the “metabolite–neuroendocrine–reproductive phenotype” panoramic regulatory network, providing an innovative theoretical paradigm for the precise nutritional control of the reproduction cycle in ruminants.

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.