Cryopreservation of ram spermatozoa in the presence of cyclohexanhexol-derived synthetic ice blocker

• SIBs can reduce cryoinjuries on ram spermatozoa.
• The protective effects of 1,3-CHD are similar to those of 1,4-CHD.
• SIBs cannot reduce cryoinjuries on ram sperm membrane.
• SIBs can inhibit cell apoptosis caused by freezing.
• The protective mechanism of SIBs needs further research.

The effects of cyclohexanhexol-derived synthetic ice blockers (SIBs) including 1,3-cyclohexanediol (1,3-CHD) and 1,4-cyclohexanediol (1,4-CHD) on chilled or frozen Yunnan semi-fine wool ram spermatozoa were systematically assessed and compared in this study. The collected semen was pooled and equally divided into two parts. In the first part, in order to evaluate the effect of SIBs on chilled spermatozoa before freezing, the semen was divided into nine groups according to the concentrations of SIBs (0, 25 mM, 50 mM, 100 mM, or 200 mM). In the second part, the treatment of semen was similar to the first part. However, except for the cooling and equilibrating procedures, spermatozoa were preserved in liquid nitrogen for one month. Motility, moving velocity, acrosome status, membrane integrity, and mitochondrial membrane potential (MMP) of frozen/thawed spermatozoa were determined using a computer-assisted spermatozoa analysis system (CASAS), fluorescence staining, and flow cytometry. The data indicated that when the concentrations of SIBs were less than or equal to 100 mM, there is no significant difference among the treating groups as concern spermatozoa motility and moving velocity following equilibration at 5 °C. However, when the concentrations of SIBs were increased to 200 mM, the motility and moving velocity of chilled ram spermatozoa were significantly decreased compared to the control spermatozoa (P < 0.05). With elevation of the SIB concentrations, the motility of frozen/thawed spermatozoa was firstly increased and then reduced. With a concentration of 50 mM or 100 mM, the spermatozoa motility was approximately 50% and significantly more than the other groups (P < 0.05). Additionally, when the SIB concentrations were less than or equal to 100 mM, there is no significant difference between the treating groups as concern moving velocity. Moreover, when the concentration of 1,3-CHD or 1,4-CHD was 100 mM, the percentage of frozen/thawed ram spermatozoa with intact acrosome was 82.57 ± 8.97% and 79.51 ± 13.49% respectively, which were significantly higher than that of the control spermatozoa (70.21 ± 13.24%, P < 0.05). Additionally, with elevation of the SIB concentrations, the percentages of frozen/thawed ram spermatozoa with damaged membrane showed a steady increase. Finally, the presence of 100 mM SIBs can significantly improve the MMP status of frozen/thawed ram spermatozoa compared to spermatozoa frozen in the absence of SIBs. In conclusion, SIBs can improve the viability of frozen/thawed ram spermatozoa as concern motility, acrosome status, and MMP. However, SIBs cannot improve moving velocity and membrane integrity of frozen/thawed spermatozoa. Finally, the protective effect of 1,3-CHD is not superior to that of 1,4-CHD on chilled or frozen ram spermatozoa.