Proteomic signatures of cervical mucus associated with fertility in Bali heifers (Bos javanicus): Implications for biomarker-based selection in artificial insemination programs

Abstract

Background and Aim: ) remains suboptimal, with low conception rates following artificial insemination (AI). Cervical mucus (CM) is a critical factor in sperm transport and fertilization; however, its molecular basis in relation to fertility has not been elucidated in this indigenous breed. This study aimed to characterize the proteomic profile of CM in Bali heifers and to identify protein biomarkers associated with fertility-related mucus quality. Materials and Methods: The study was conducted between February and August 2024 in South Sulawesi, Indonesia. Forty clinically healthy Bali heifers (2-3 years old) were sampled during natural oestrus and divided into good CM (GCM; n = 20) and poor CM (PCM; n = 20) groups using a validated five-parameter biophysical scoring system. CM proteins were extracted and analyzed using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. High-confidence protein identification was achieved at <1% false discovery rate, and differential abundance was evaluated using Benjamini-Hochberg correction (p < 0.05). Functional enrichment, correlation analysis with mucus traits, and receiver-operating-characteristic (ROC) analyses with cross-validation were performed. Results: Significant differences (p < 0.05) were observed between GCM and PCM groups for appearance, viscosity, spinnbarkeit, and ferning pattern, while pH did not differ. A total of 52 proteins were identified after quality control, of which 13 showed significant differential abundance. GCM was characterized by higher levels of NT5E, lactoferrin, SCGB1D, and lactotransferrin, whereas PCM showed enrichment of complement factor I (CFI), haptoglobin (HP), MUC5AC, FAIM2, TIMP2, PEBP4, SAA3, GRP, and IGL. Functional enrichment analysis indicated anti-inflammatory and epithelial-protective pathways in GCM, in contrast to complement activation, proteolysis, and oxidative remodeling in PCM. ROC analysis demonstrated excellent discriminative performance for NT5E (GCM) and CFI and haptoglobin (PCM), each achieving an area under the curve of 1.00 in this cohort. Conclusion: This study offers the first proteomic evidence connecting CM composition to fertility-related traits in Bali heifers. NT5E, CFI, and HP stand out as promising biomarkers for fertility screening, providing a molecular framework to improve AI efficiency and selection strategies in indigenous cattle.

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