Sperm cryopreservation is well known as a valuable method to preserve the genetic traits. Although many studies have established semen cryopreservation protocols, lack of studies were conducted to discover the differences of sperm proteome and functions between ejaculated and epididymal spermatozoa following to cryopreservation. Therefore, the objective of this study was to (i) evaluate the effect of cryopreservation on bull epididymal spermatozoa and (ii) discover the potential biomarkers, which have highly tolerance to freezing on bull epididymal spermatozoa. Our preliminary study demonstrated that spermatozoa from each bulls have different resistance on freezing during cryopreservation. We divided spermatozoa into two groups according to sperm motility following to cryopreservation; high freezing-tolerant spermatozoa (HFS) and low freezing-tolerant spermatozoa (LFS). Several sperm functional parameters, i.e. sperm motility/motion kinematics, speed parameters, viability, mitochondrial membrane potential, and capacitation status. Our results showed that all parameters except for motion kinematics and capacitation status had significant differences between HFS and LFS. Subsequently, two dimensional electrophoresis were conducted to compare the expression levels of sperm proteome between both groups. Three proteins {glutathione s-transferase mu 5 (GSTM5), voltage-dependent anion-selective channel protein 2 (VDAC2), and ATP stynthase subunit beta (ATP1B1)} were differentially expressed. Based on these results, we propose that epididymal spermatozoa from individual bull have different freezability upon cryopreservation and three differentially expressed proteins might be selected as a biomarker to predict high freezing-tolerant epididymal spermatozoa.

Sperm cryopreservation preserves genetic resources for animal breeding and for human patients who suffers from permanent testicular damage. Although the sperm cryopreservation has been used for many years, the addition of cryoprotective agent (CPA) during cryopreservation negatively affects sperm function and quality. Our previous study reported that the addition of CPA reduced bull sperm physiological functions. However, the sperm cells collected from individual bulls presented different sensitivity to the damage induced by CPA. In the present study, we examined if CPA affect sperm cells acquired from individual bulls. Individual bull spermatozoa were divided into two groups based on motility parameters; high CPA-tolerant sperm (HCS) and low CPA-tolerant sperm (LCS). Our results showed that the HCS group presented good physiological function after CPA exposure, whereas the LCS group showed a significant decrease in the sperm function. We also found differentially expressed five proteins between the HCS and LCS groups, which refer to cytosolic 5′-nucleotidase 1B (NT5C1B), fumarate hydratase (FH), F-actin-capping protein subunit beta (CAPZB), voltage-dependent anion-selective channel protein 2 (VDAC2), and cytochrome b-c1 complex subunit 1 (UQCRC1). NT5C1B and FH showed abundant expression in the HCS group, while the expression of CAPZB, VDAC2, and UQCRC1 was relatively lower in the HCS group than in the LCS group. The current results suggest that NT5C1B, FH, CAPZB, VDAC2, and UQCRC1 can be used as potential markers to predict CPA-tolerable spermatozoa. Those markers provide a reliable tool to select animals and breeds with CPA tolerance.