Biochemical Profile of the Follicular Environment in Cattle Following Intrafollicular Transfer of Immature Oocytes

Intrafollicular transfer of immature oocytes (IFIOT) is a promising embryo production strategy, but outcomes remain poor and inconsistent. We investigated the effects of the injection procedure and the number of injected cumulus-oocyte complexes (COCs) on the biochemical and molecular environment of follicles. First, dominant follicles from hormonally synchronized heifers were either not injected (NIFIOT), injected with medium only (IFIOTM), or injected with medium containing COCs (IFIOT-COCs). Follicular fluid (FF) was analyzed for cell-free DNA (cfDNA), oxidative stress markers (GPx activity and FRAP), steroid hormones, and metabolomic profiles using UPLC-HRMS. Injection procedure increased cfDNA levels and altered steroidogenesis, with higher progesterone levels, lower estradiol levels, and lower E2/P4 ratios in the injected follicles. Metabolomic analysis revealed changes in amino acid and anti-inflammatory pathways on the injected follicle compared to the non injected. In addition, follicles were injected with 25 or 50 COCs (IFIOT25 and IFIOT50) to assess the impact of oocyte number on the follicular environment. IFIOT25 follicles showed higher GPx activity and estradiol concentrations compared to IFIOT50, whereas IFIOT50 follicles exhibited the downregulation of redox- and stress-related genes (SOD1, GPX4, NFE2L2, HSP70, and CASP3) and lower embryo yield compared to IFIOT25 (42 vs 52%, respectively). Metabolomic analysis indicated alterations in lipid and energy metabolism, with key metabolites (creatine, l-acetylcarnitine, dodecanoic acid, and panthenol) reduced in the IFIOT50 group, suggesting impaired metabolic homeostasis at higher oocyte density. Overall, both the injection process and oocyte number influenced follicular physiology, potentially affecting oocyte quality, highlighting the need to refine IFIOT protocols.