Optimal concentration of Rho-associated coiled-coil kinase (ROCK) inhibitor improved sperm membrane functionality and fertilizing ability of cryopreserved-thawed feline sperm

Abstract

Sperm cryopreservation induces irreversible loss of viability and fertilizing ability. This study aimed at examining the effects of Rho-associated, coiled-coil kinase (ROCK) inhibitor on quality of frozen-thawed feline sperm. Ejaculated semen from individual cats (n = 6) was examined for the expression of LIMK1 and LIMK2 mediated ROCK cascade. The effects of ROCK inhibitor during cooling and cryopreservation on sperm quality and fertilizing ability were also examined. Feline sperm were treated with different concentrations of ROCK inhibitor (10, 20 and 40 μM) during cooling at 4 °C and cryopreservation. Sperm cooled and conventionally cryopreserved without ROCK inhibitor (0 μM) served as a control group.The ROCK cascade was confirmed in feline sperm as they expressed mRNA of LIMK1 and LIMK2 genes. Cryopreservation significantly reduced sperm quality in terms of viability (91.63 ± 3.96 vs. 60.11 ± 8.93), progressive motility (91.67 ± 3.54 vs. 46.67 ± 8.66) and acrosome integrity (93.49 ± 3.64 vs. 63.81 ± 5.31) for fresh and frozen-thawed sperm, respectively (p < 0.05). The positive effects of ROCK inhibitor on sperm quality were pronounced at 1 and 3 h post-thaw. ROCK inhibitor at 10 μM significantly improved sperm motility and membrane functionality compared to those observed in a control group (0 μM) (p < 0.05). In vitro fertilization revealed that supplement ROCK inhibitor at 10 μM during cryopreservation significantly improved in vitro fertilizing ability of the frozen-thawed sperm (p < 0.05). However, it did not subsequently increase morula and blastocyst rates (p > 0.05). Increased concentrations of ROCK inhibitor to 20 and 40 μM did not further improve the quality of frozen-thawed sperm. In conclusion, an optimal concentration (10 μM) of the ROCK inhibitor added into cooling medium could improve post-thaw sperm quality.