An artificial vagina was used to extract fresh ejaculates from four Gaoqing bulls (4-6 years) from the Aohang farm in Shandong Province, China. To assess progressive motility, 3 uL of the fresh ejaculate was immediately evaluated through computer-assisted sperm analysis (CASA) (CASA, Nikon, Eclipse E200, 10X, Basler, acA780-75gc, SCA sperm class analyzer)). Only the ejaculates with a volume of >2.0 mL and demonstrating >70% motility were selected for the analysis. A Biladyl® extender (Minitube, GER) was used to dilute the samples in a two-step procedure. Initially, the samples were diluted with a glycerol-free formulation, which constituted 50% of the final volume estimated, and refrigerated at 5°C for 2 hours. Subsequently, the samples were diluted with a pre-cooled (5ºC) glycerol-containing formulation and refrigerated at 5ºC for 2 h. The sperm samples were frozen to a final concentration of 140–200 x 106/mL. A cooling curve of -5 ºC/min was used from 5 ºC to 4 ºC, then -3 ºC/min was used from 4 ºC to -10 ºC, then -40 ºC/min was used from -10 ºC to -100 ºC, then -20 ºC/min was used from -100 ºC to -140 ºC. All samples were stored in liquid nitrogen until further use. Sperm samples were thawed at 37ºC for 30 s and added to 1.5 mL of 45% Percoll and 1.5 mL of 90% Percoll in a 15-mL conical plastic tube. To separate HMS and LMS, sperm suspensions were stacked on top of the gradient and centrifuged at 700 x g for 10 min. The morphologically abnormal spermatozoa, seminal plasma, and extender were recovered from the top layer consisting of 45% Percoll; LMS were recovered from the 45–90% Percoll interface; and HMS were recovered from the bottom layer consisting of 90% Percoll. CASA was used to assess sperm quality according to the World Health Organization (WHO) standards.