Identification and evaluation of candidate sperm proteins associated with cryopreservation-induced changes in semen quality in Thai native roosters.

Semen cryopreservation is essential for the long-term conservation of valuable poultry genetic resources; however, freezing and thawing can compromise sperm function through structural, metabolic, and oxidative alterations. This study evaluated cryopreservation-associated changes in five candidate proteins and their relationships with semen quality in Thai native roosters. Semen was collected from 50 roosters on five separate occasions, and qualified ejaculates were pooled to generate five paired pooled experimental units. Fresh and frozen-thawed semen were evaluated for sperm quality, lipid peroxidation, and the relative abundance and subcellular localization of TUBB, SPAG6, TEKT5, ATP5A1, and DPYS using immunoblotting and immunofluorescence. Exploratory correlation analysis, principal component analysis (PCA), and receiver operating characteristic (ROC) analysis were used to characterize relationships between candidate protein abundance and cryopreservation-associated changes in semen quality. Cryopreservation significantly reduced sperm motility, viability, mitochondrial membrane potential, acrosome integrity, and chromatin integrity and increased malondialdehyde concentration (FDR-adjusted P < 0.001). The relative abundance of TUBB, SPAG6, ATP5A1, and DPYS decreased, whereas TEKT5 increased after cryopreservation. Characteristic subcellular localization patterns were largely preserved after freezing and thawing. Exploratory correlation and PCA analyses indicated coordinated patterns of cryopreservation-associated changes in candidate protein abundance and semen quality, while ROC analysis indicated that SPAG6 and TEKT5 showed the highest discriminatory performance for distinguishing fresh from frozen-thawed semen within the present dataset. These findings identify protein-specific molecular changes associated with cryopreservation-induced sperm injury and highlight SPAG6 and TEKT5 as candidate molecular indicators for further investigation. Validation in individual males and larger independent populations is needed to determine whether these proteins predict semen freezability, cryotolerance, or reproductive performance.