Abstract: The object of this study is to investigate the changes of serum metabolites during multiple ovulation in sheep and look for metabolites related to follicular maturation. In this study, the untargeted metabolomics analysis was processed through LC-MS/MS to analyze the metabolites in the serum of donor sheep after simultaneous oestrus, before FSH injection (1st) and before artificial insemination (AI) (2nd). The metabolomics analysis showed that 1 158 metabolites (721 in positive iron mode and 437 in negative iron mode) were detected and 617 were annotated. The diferential metabolites were screened based on the following criteria: VIP≥1; FC>1.2 or＜0.83; P≤0.05. A total of 295 metabolites were differentially expressed between 2nd and 1st (186 up-regulated and 109 down-regulated). KEGG pathway enrichment showed that 88 functional pathways were enriched in IE groups, including steroid hormone biosynthesis, arachidonic acid metabolism and fatty acid biosynthesis. This study showed that high content of deoxycorticosterone, phosphatidylcholine and arachidonic acid in serum would be helpful for follicular developing in multiple ovulation. The results would help to explain the relation between blood material and follicular development and provide a theoretical basis for the nutrition regulation and donor selection in multiple ovulation.

Key words: superovulation, non-targeted metabonomics, sheep, follicular development