This study was conducted to evaluate effect of α-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or 20 μg/mL BSA. Cryopreserved boar sperms were thawed in 37°C water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on sperm capacitation status and sperm survival. Frozen epididymal sperm samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk- Tris-glycerol extender) were thawed in 37℃ water bath for 1 min. To rule out individual variation, 3 sperm samples were mixed after thawing. The mixed samples then were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities (300 X g and 700 X g) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Sperm capacitation status and sperm survival were evaluated using combined Hoechst 33258 and chlortertracycline fluorescence staining assay. The acrosome reacted spermatozoa (AR pattern), uncapaciated spermatozoa (F pattern) and sperm survival were significantly correlated with centrifugation time (p< 0.01). Significantly decreased F pattern observed as centrifugal time increased. As centrifugal time increased, spermatozoa with F pattern decreased and spermatozoa showing AR pattern increased. Moreover, the dead spermatozoa were significantly stimulated in time-dependent manner. However, there were no significant differences in various force of centrifugation and Percoll volume. These results suggest that only centrifugation time significantly affects sperm capacitation status and sperm survival.

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on motile sperm recovery rate and motion kinematics. Frozen semen samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk-Tris-glycerol extender) were thawed in 37℃ water bath for 1 min. After thawing, the mixed semen samples were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities (300×g and 700×g) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Motile sperm recovery rate and motion kinematics were evaluated by computer assisted sperm analyzer using Makler counting chamber. Sperm motility (%) and motile sperm recovery rate showed similar pattern in all treatment groups. However, sperm motility (%) and motile sperm recovery rate were highest at 700×g for 30 min through a discontionous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll. There were no significant differences in motion kinematics after various Percoll washings. These results suggest that force of centrifugation, centrifugation time, and Percoll volume significantly affect motile sperm recovery rate.

This experiment was to investigate whether the mitochondria function assessment can be used for the prediction of sperm fertility through examining the correlation between mitochondria fluoromicroscopic frequency of frozen-thawed eight Hanwoo bull semen using rhodamine123 (R123) and in vitro embryo development following fertilization. Individual sperm were stained in 5ug/mL R123-added calcium-free Sp-TALP for 30 min at 0 h, 6 h, 12 h and 24 h after thawing and examined their mid-piece under an epifluorescence microscope using 495 nm excitation filter (x1,000). Three replications were taken, and at least 300 sperm per individual were examined. When semen samples were separated into two groups (good and poor) by sperm motility and fluorescent frequencies at just after thawing, average fluorescent frequencies were remarkably reduced as time going (0 h; 53.29~72.94%, 6 h; 21.40~58.90%, 12 h; 8.26~25.93%, 24 h; 1.00~13.78%, irrespective of selected group, and there were no differences at 6 h or 12 h after thawing between selected groups but indicated significant difference at 24 h after thawing (p<0.05). In vitro fertilization rates in good and poor groups ranging 70.8~77.8% and 52.1~84.5%, respectively, were not significantly different. However, in vitro development rates of the same groups ranging 25.7~40.0% and 12.9~1.8%, respectively, were significant different (p<0.05). These results demonstrate that mitochondria fluoromicroscopic assessment of frozen-thawed bovine sperm may be used as a criterion to select more fertile sperm.

ICSI시 동결 융해한 부고환 정자의 이용 가능성을 알아보고자 난자의 배양시 체외성숙율과 활성화 처리를 한 난자와 동결 융해한 부고환 정자로 ICSI시 체외발생율을 조사하였으며, 결과를 요약하면 다음과 같다. 1. 난포란을 회수 후 24시간 배양하였을 때 배양 시간에 따른 GV, MI, M II로의 체외성숙율은 각각 7/60(11.7%), 5/60(8.3%), 48/60(80.0%)였고 30시간 배양 시간에 따른 GV, MI, M II로의 체외성숙율은