The sperm freezability during cryopreservation varies widely in chicken breeds and individuals, however, the underlying mechanisms are especially less examined. Comparative 4D label-free quantitative proteomic analysis of four high-freezability (HF) and four low-freezability (LF) Beijing-You chickens identified 42 differentially expressed proteins (DEPs) among 2309 proteins. The DEPs were mainly associated with metabolism and cellular processes. Voltage-dependent anion-selective channel protein 1 (VDAC1) was mainly related to mitochondrial function and ATP metabolism. The DEPs analyzed by quantitative proteomic analysis were validated by parallel reaction monitoring (PRM), and identified changes in VDAC1 that were specially linked to chicken sperm freezability. Furthermore, we confirmed that the freezing caused damage to the sperm mitochondrial ultrastructure and the mitochondrial membrane potential (MMP) in the LF group decreased extensively after freezing. The molecular docking showed that VDAC1 was binding highly stable with the mitochondrial-targeted antioxidant. The addition of mitoquinone (MitoQ) to the diluent increased the fertility of post-thawed sperm, from 33.95 % to 48.34 %, might be sperm mitochondria antioxidant potential enhanced. In conclusion, the difference abundance of sperm mitochondria VDAC1 protein might be one of the factors leading to different freezability of chicken sperm.
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