Effects of HMG on Superovulation Efficacy in Kazakh Sheep and Screening of Plasma Reproductive Hormones and Metabolic Markers

Superovulation and embryo transfer techniques are effective strategies for improving reproductive efficiency in sheep flocks. To characterize changes in plasma metabolites and reproductive hormones following HMG or FSH superovulatory treatment, and to screen for biomarkers closely associated with embryo quality, we employed liquid chromatography-based untargeted metabolomics and enzyme-linked immunosorbent assay (ELISA) to analyze plasma samples from donor ewes on day 6 after hormone administration. This study aimed to identify small-molecule biomarkers affecting embryo quality under different hormonal regimens. A total of 46 Kazakh ewes were allocated into two groups: the HMG group received HMG treatment, while the FSH group received FSH treatment; both groups were superovulated using a tapering dose regimen administered via intramuscular injection. Our results show that the average number of viable embryos in the HMG group (5.36 ± 3.29, n = 11) was significantly higher than that in the FSH group (2.91 ± 2.60, n = 35) (p < 0.05). Plasma progesterone concentrations differed significantly between the two groups (p = 0.01). Metabolomic analysis identified three key differential metabolites: 5-fluorouridine, ADP, and hexadecanoic acid. Functional enrichment analysis revealed 45 significantly enriched metabolic pathways. In conclusion, HMG effectively improves sheep embryo quality by modulating the levels of small-molecule metabolites associated with embryonic development.