Effects of conventional slow freezing on the freezability of the Buffalo Epididymal Spermatozoa

Abstract

The isolation and cryopreservation of cauda epididymal spermatozoa (ES) open new horizons for assisted reproductive technologies and animal genetic resource banking. However, variations in sperm freezability challenge the significance and potential applications of these techniques. Therefore, this groundwork in swamp buffalo (Bubalus bubalis) distinguished high (HF) and low (LF) freezability ES by analyzing conventional sperm parameters and protein expressions following slow-freezing procedures. ES were recovered from the cauda epididymis of 18 individual swamp buffaloes after their humane slaughter. After cryopreservation, ES samples were categorized into HF and LF using post-thaw sperm motility. Sperm motion, kinematics and velocity and membrane integrity were assessed using computer-assisted sperm analysis (CASA) and hypo-osmotic swelling test (HOST), respectively. Accordingly, reactive oxygen species (ROS) generation and lipid peroxidation were also measured via fluorescence staining and thiobarbituric reactive substances (TBARS) assay. Proteins of interest, namely ATP synthase subunit beta 1 (ATP1B1) and glutathione S-transferase Mu 3 (GSTM3), were also examined for their presence in fixed cauda tissues via immunohistochemistry and relatively quantified from ES through Western blot. Results revealed that all CASA parameters except wobble, linearity, and straightness were significantly higher in HF as compared to LF. However, there were no substantial differences in plasma membrane integrity among freezability groups after a hypo-osmotic state. Nevertheless, the amounts of ROS and MDA were considerably greater in LF than HF. ATP1B1 and GSTM3 proteins were also found to appear in the luminal ES and the epididymal tubular epithelium. While ATP1B1 levels were comparable between freezability clusters, the abundance of GSTM3 significantly differs between HF and LF, perhaps due to the protein's protective role against oxidative stress. These findings suggest that the variabilities in slow-freezing responses may be used to differentiate freezability phenotypes in the swamp buffalo ES.