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Background: Cryoprotective agent (CPA) addition during sperm cryopreservation causes detrimental effects on sperm function and quality. We previously reported that CPA addition adversely affects bull sperm physiological functions and shows differentially expressed proteins., Objectives: To study functional and proteomic alterations between high CPA-tolerant spermatozoa (HCS) and low CPA-tolerant spermatozoa (LCS) in bull., Materials and Methods: Bull semen was collected from the cauda epididymides of Korean bull (Hanwoo) and suspended in Tris-egg yolk buffer (TYB). The collected fresh semen was diluted down to a final concentration of 6% glycerol TYB solution. After CPA exposure to the sperm cells from individual bulls, the percentage of sperm motility was examined by utilizing a computer-assisted sperm analysis system. According to sperm motility value, the HCS (motility above 80%) and LCS (motility below 60%) groups were evaluated for sperm function parameters (swimming speed, capacitation, viability, and mitochondrial function) and protein expression., Results: The HCS group had good sperm function parameters following CPA addition, whereas sperm functions in the LCS group were significantly reduced. There were differentially expressed proteins between the HCS and LCS groups. Cytosolic 5-nucleotidase 1B and fumarate hydratase were abundantly expressed in the HCS group, while F-actin-capping protein subunit beta, voltage-dependent anion-selective channel protein 2, and cytochrome b-c1 complex subunit 1 had a lower expression in the HCS group than in the LCS group., Discussion and Conclusion: Identified proteins implicate potential markers to predict CPA-tolerable spermatozoa, which could provide a method of selecting animals and breeds with cryoprotectant resistance., (© 2019 American Society of Andrology and European Academy of Andrology.)

Sperm cryopreservation is an important tool for storing genetic traits and assisted reproduction techniques. Several studies have developed semen cryopreservation protocols. However, the sperm proteome is different between ejaculated and epididymal spermatozoa and little is known about cryopreservation effects on epididymal spermatozoa. Therefore, our study aimed to (i) investigate the differences of sperm parameters based on the freezing tolerance of spermatozoa and (ii) identify potential markers to predict the freezability of bull epididymal spermatozoa. Our preliminary study demonstrated that spermatozoa from individual bulls differ in cryopreservation freezability. We categorized spermatozoa into high freezing-tolerant spermatozoa and low freezing-tolerant spermatozoa group based on sperm motility after freezing/thawing. We evaluated several sperm functional parameters, including sperm motility/motion kinematics, sperm speed parameters, viability, mitochondrial activity, and capacitation status. Our results demonstrated that motility, sperm speed parameters, viability, and mitochondrial membrane potential had significant differences between the two groups but motion kinematics and capacitation status did not. In addition, the concentration of three proteins - glutathione s-transferase mu 5, voltage-dependent anion-selective channel protein 2, and ATP synthase subunit beta, differed between both groups. Thus, our research highlighted differences in bull epididymal spermatozoa freezability upon cryopreservation and these proteins might be useful markers to select high freezing-tolerant epididymal spermatozoa.