50 Effects of Cathepsin B Inhibitor E64 on the Survival Rate of Cryopreserved Semen from Korean Brindled Bulls

Abstract

Korean brindled cattle have distinctive coat colour and are regarded as rare cattle in the Korean peninsula. To preserve the line as a genetic resource for diversity in cattle, semen cryopreservation has been used for selection of individuals for breeding between local AI centers. Nevertheless, the survival and viability of frozen semen from Korean brindled bulls is not uniform due to the difference between the individual bulls or experimental techniques. In this study, E64, a cathepsin B inhibitor, was used at final concentrations of 0.05, 0.5, and 1 µM to test the viability of frozen semen. To prepare frozen semen, a triladyl-egg yolk diluent with 6.4% glycerol was used in a two-step freezing method with 4 different bulls with 3 repeats. A total of 12 ejaculates was diluted to a concentration of 50 to 100 × 10⁶ mL⁻¹ at room temperature, and slowly cooled from room temperature to 5°C in 2 to 3 h. The cooled semen was diluted 1:1 with a secondary diluent containing E64 to prepare the experimental group. After loading, 0.5-mL straws were immersed into liquid nitrogen after 10 min exposure at 5 cm above the nitrogen using a styrofoam box. The viability of spermatozoa after thawing at 37°C for 40 s was analysed by Student’s t-test. The rate of surviving sperm in the 1 µM E64 group (82 ± 4.3%) was significantly higher than that of the control group (71.2 ± 2.0%; P < 0.05). However, the 0.05 and 0.5 µM E64 treatment groups lead to similar rates (77.5 ± 2.0% and 75.0 ± 5.0%, respectively; P > 0.05). Based on these results, it is expected that E64 could be used for the improvement of productivity of frozen semen; further results on in vitro fertilization and development are ongoing.