The effects of extenders, cryoprotectants and conditions in two-step cooling method on Varicorhinus barbatulus sperm

In this study, we developed an optimal cryopreservation procedure for Varicorhinus barbatulus sperm. To this end, we optimized (1) the types and dilution ratios of extenders; (2) types and final concentration of cryoprotectants; and (3) freezing conditions, including equilibration time, height above the surface of liquid nitrogen (LN), and the cooling times in the two-step cooling method. The optimum result was obtained when the sperm was diluted at a 1:9 ratio in D-17 with 10% methanol, equilibrated at 4 °C for 10 min, held at 7 cm above LN for 2 min, and finally stored in LN. After storage for 12 h in LN, the sperm was thawed in a water bath at 40 °C for 6s, the post-thaw sperm motility was 66.10 ± 7.12%, while the corresponding rate for fresh sperm was 87.08 ± 2.38%. Using computer-assisted sperm analysis, we found a significant decrease in the motility parameters of post-thaw sperm, especially the parameters related to velocity. To evaluate the effects of cryopreservation on the structural integrity of sperm, transmission electron microscopy and scanning electron microscopy were employed, which showed the defects in frozen sperm, including: abnormal heads, damaged plasma membranes, broken tails, and the disappearance of the mitochondrial internal crest. In addition, we determined the mitochondrial membrane potential to assess the functional integrity of frozen sperm. Our results showed a decrease in the mitochondrial function of frozen sperm. This procedure could be used alongside cryopreservation of V. barbatulus and supports its commercial-scale production and species conservation.