Proteomic Profiling of Follicular Fluid Reflects Follicle Size and Development in Bali Cattle (Bos javanicus domesticus)

ABSTRACT Follicular fluid supports oocyte development by providing a dynamic biochemical microenvironment during folliculogenesis. This study aimed to characterize the follicular-fluid proteomic profile in Bali cattle ( Bos javanicus domesticus ) and explore discriminating candidate proteins between small (SF) (< 6 mm) and large follicles (LS) (≥ 6 mm). Ovaries were collected from 20 Bali cattle at a local slaughterhouse, and follicular fluid was aspirated and classified according to follicle diameter. Proteomic analysis was performed using Liquid Chromatography–High-Resolution Mass Spectrometry (LC-HRMS), followed by multivariate statistical, functional enrichment, and exploratory receiver operating characteristic (ROC) analyses. Proteins (98) with both shared and group-specific distributions between follicle sizes were identified. Multivariate (principal component (PCA) and partial least squares discriminant (PLS-DA)) analyses revealed clear separation between groups, indicating distinct proteomic signatures. Differential expression analysis identified several significantly altered proteins, whereas variable importance in projection (VIP) analysis highlighted key discriminative proteins. Functional enrichment analysis revealed that these proteins were predominantly associated with extracellular localization, complement activation, and protease inhibition pathways. During exploratory ROC analysis, ADIPOQ and ITIH1 were identified as promising candidate discriminatory performance proteins, with the highest discriminatory performance (area under the curve (AUC) ≥ 0.80). ADIPOQ (AUC = 0.86) and ITIH1 (AUC = 0.81) were more abundant in LS and SF, respectively. This exploratory study suggests that follicular-fluid proteomics may help distinguish follicle size and identify candidate proteins associated with follicular development in Bali cattle. These findings provide preliminary molecular insights and identify candidate proteins that warrant further investigation as potential follicular-status biomarkers in indigenous cattle.