The study aims to assess viability, acrosomal menbrane, integrity and mitochondria membrane potential of sperm separated using a percoll density gradient(45% and 90%) and swim-up methods using Hanwoo epididymal sperm frozen semen. Briefy, motile sperm separated using a percoll gradient and swim-up. 25 μl of sperm dilution from droplets were transferred to 1.5 mL tube and incubated with fluorescent probes at 39°C in dark as follows. After incubation, 75 μl of 5,5',6,6'-tetrachloro-1,1',3,3'- -JC-1; Mitochondrial Membrane Potential Detection kit, Cell Technology Inc., USA) working solution was mixed with sperm dilution and incubated for 30 min. One μl of Hoechst 33342 (H1339; Molecular Probe, Eugene, OR, USA) stock solution was mixed with sperm dilution and incubated for 10 min. And then, 0.5 μl of propidium iodide (PI) stock solution and 0.5 μl of fluorescein peanut agglutinin FITC conjugate (FITC-PNA; Vector Laboratories, FL-1071) were mixed with sperm dilution and incubated for 8 min. After mixing with fluorescent probes and sperm dilution, 5 μl of stained sperm dilution was mounted on a slide glass and covered with cover glass. More than 200 sperms in a slide glass were counted with × 400 magnification by fluorescent microscope (Eclipse Ci_L, Nikon, JAPAN) and evaluated viability, acrosomal membrane integrity, and mitochondrial membrane potential of sperm with triple band filter (DAPI/FITC/TRITC; Nikon, Tokyo, Japan). Live sperm were stained with Hoechst 33342(blue) and dead sperm were stained with PI (red). Damaged acrosomal membrane of sperm was stained with FITC-PNA (green) and intact acrosomal membrane of sperm was not stained. Both of sperm swim-up method with or without BSA separated to Live intact mitochondria (15.39±4.31 vs, 12.58±3.74, and) without significant difference. and percoll density gradient method also similar (7.29±6.54), swim-up method of sperm preparation appeared to be more efficacious in percentage recovery of motile sperm concentration compared to Density gradient method.

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY (; EG and ; 7% G) than 8% LDL (; EG and ;G). Treatment of 4% LDL + 5% EY-EG () has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG () among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.