Putative proteomic signatures associated with post-thaw semen quality in Peranakan Etawah and Saanen goats: an exploratory study

Abstract

Objective: The objective of this study was to compare post-thaw semen quality and sperm kinematics between Peranakan Etawah (PE) and Saanen goats and to identify breed-associated proteomic signatures related to semen cryotolerance. Methods: Frozen semen straws were obtained from BIB Singosari, Indonesia, and grouped by breed. Post-thaw evaluations included viability, morphology/abnormality, plasma membrane integrity (HOST), and computer-assisted sperm analysis for motility and kinematic parameters. Sperm proteomes were profiled using UHPLC-Orbitrap LC-MS/MS. Data were processed in Proteome Discoverer and analyzed using multivariate models (PCA, PLS-DA), differential abundance testing (q-values), functional enrichment (g:Profiler), biomarker selection (VIP > 1; AUC ≥ 0.70), and correlation with semen traits (MetaboAnalyst). Results: PE and Saanen differed significantly in survival-related traits, including viability, abnormality, and plasma membrane integrity (p < 0.05), while total motility and all kinematic parameters were similar between breeds (p > 0.05). Proteomic patterns showed partial separation by PCA and clearer discrimination by validated PLS-DA. Multiple proteins were differentially abundant between breeds (q < 0.05) and clustered consistently by breed. Enrichment analysis highlighted mitochondrion-associated functions, particularly fatty acid β-oxidation. Candidate discriminative proteins included ACADVL, ETFB, GPD2, CYLC1, and LOC102188264, which were significantly associated with semen quality traits within breeds. Conclusion: Breed-related differences in frozen-thawed goat semen were mainly reflected in post-thaw survival traits and in proteomic signatures dominated by mitochondrial energy metabolism. Integrating proteomics with conventional semen assessment may help explain semen cryotolerance and support biomarker discovery.

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