The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 106 cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 106 cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.

The increase in the meat quality and milk production of cows, which breed Korean Native Cow (KNC) and Holstein cow, is not improving reproductive efficiency. In this study, we examined the effect of interferon (IFN) supplementation on motility of frozen-thawed semen and pregnancy rate after artificial insemination of KNC and Holstein cow. In experiment 1, we investigated the effect of IFN-tau concentration (10,000 IU and 20,000 IU) on the percentage of total motility (TM) and progressive motility (PM) of frozen-thawed spermatozoa. In experiment 2, KNC were infused 20,000 IU IFN-tau at insemination or after insemination. In experiment 3, KNC or Holstein cow were inseminated with frozen-thawed semen and infused 20,000 IU IFN-gamma or -tau after insemination. In experiment 1, the average of TM (23.9% to 30.9%) and PM (18.5% to 21.9%) were similar between control and treatments. In experiment 2, the pregnancy rates of IFN infusing times were not different from 45.8% to 53.6%. In experiment 3, the pregnancy rates of Holstein cow infused different kinds of IFN were similar (control, IFN-gamma, IFN-tau; 42.9%, 40.5%, 48.0%). In the case of KNC, however, the pregnancy rate of control was 55.6%, which was significantly lower than that of IFN-gamma (68.9%; p<0.05). Thus, IFN is effective on the improvement of reproductive efficiency, but further study is needed.

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 purpose of this study was to improve the frozen-thawed sperm of Korean Native Cattle using magnetized water. The semen was collected by artificial vagina. Before cryopreservation, Triladyl was flowed through magnet [0, 2000, 4000 and 6000 Gauss (G)] for 5min. The semen was diluted to magnetized Triladyl with 20% egg-yolk for freezing. The diluted semen was placed in 0.5ml straws, and freezing process was exposed on ‒120℃ for 10min. Diluted sperm was stored into LN2. Analysis of frozen- thawed sperm was estimated of viability with SYBR14/PI stain, and membrane intact with hypoosmotic swelling test (HOST). The mitochondrial function analysis was conducted by staining with Rhodamin 123 by flow cytometry and was analyzed histogram. The intensity of acrosome damage was analyzed using FITC-PNA stain by flow cytometry. Analysis of rhodamin 123 and FITC-PNA stain were used mitochondria and acrosome membrane intact. As a results, sperm viability was significantly higher in 4000G (76.0±1.2%) group than other groups (p<0.05). However, HOST was significantly higher in 4000 (37.7±0.6%) and 6000G (35.0±1.1%) than 0 (30.3±0.9%) and 2000G (30.7±0.5%) (p<0.05). In addition, mitochondria membrane and acrosome damage were significantly lower in 6000G (1.40±0.08% and 26.7±2.9%) group than other groups (p<0.05). In conclusion we suggest that magnetized water could improve the ability of sperm on cryopreservation of korean native cattle. * This work was supported by Grant No. PJ 907008 from Rural development administration (RDA).

Although the function and utility of RNA transcripts derived from matured spermatozoa remains unclear, they might play important roles in the establishment of a paternal genome and subsequently embryo development. Herein, we investigated the expression of X-chromosome linked RNA transcripts in matured bovine spermatozoa. The total RNA was extracted from the matured spermatozoa, and then converted to cDNA. Autosomal genes (ACT-β and H-2A) and X-chromosome linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) were analyzed for the characterization of X-chromosome linked RNA transcripts and compared to female fibroblasts by RT-PCR. The transcripts of autosomal genes (ACT-β and H2A) and X-chromosome linked genes (ANT3, HPRT, MeCP2, RPS4X and ZFX) were not detected in spermatozoa. However, XIAP (X-linked inhibitor of apoptosis protein) and XIST (X-chromosome inactive-specific transcript, a kind of paternal imprinted gene) transcripts were detected in spermatozoa, and relative levels of XIAP and XIST transcripts were similar and 0.5-fold lower when compared to female fibroblasts, respectively. Based on the findings, it is summarized that the presence of RNA transcripts of XIAP and XIST in the isolated spermatozoa may imply their role in inhibition of apoptosis and induction of X-chromosome inactivation in embryo development.

The purpose of this study was to improve of frozen-thawed sperm using magnetized water in Korean native cattle. Before cryopreservation, without egg-yolk Triladyl® solution was flowed though magnet [0, 2000, 4000 and 6000 Gauss(G)] for 5 min. The freezing of dilluted semen added with Triladyl containing 20% egg-yolk. Analysis of frozen-thawed sperm was estimated viability with SYBR14/PI double stain, membrane intact with hypoosmotic swelling test (HOST), acrosome reaction with FITC-PNA, mitochondria membrane function with Rhodamin 123 by flow- cytometry. Sperm viability was significantly higher in 4000G group than other groups (p<0.05). However, the Hypoosmotic Swelling Test(HOST) was significantly higher in fresh, 4000 and 6000G than 0 and 2000G (p<0.05). In addition, mitochondria membrane damage and acrosome damage were significantly lower in 6000G group than other groups (p<0.05). in conclusion we suggest that magnetized water could be improve ability of sperm on cryopreservation in Korean native cattle.

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

The object of this study was to find simple and effective methods for the speculation of vitality and scorsome status of bovine spermatozoa. The eosin-nigrosin staining, trypan blue staining, and naphthol yellow S-erythrosin B staining was ofter used for the speculation of vitality and/or acrosome status of bovine spermatozoa, respectively. This study has shown that the combined trypan blue-naphthol yellow S-erythrosin B staining is more accurate and effective for the examination of acrosome status and vitality of bovine spermatozoa.

난관상피세포와 그 배양액에서 분비된 고분자 분획이 소정자의 수정능력에 어떠한 영향을 미치는지 알아보고자 실시한 실험에서 다음과 같은 결론을 얻었다. 1. 난관상피세포배양액으로부터 MW 5 kDa cut-off bucket을 사용하여 탈염 및 농축을 실시, 단백질/고분자분획을 회수하고 이를 체외수정용 배양액에 첨가하고 난관상피세포 monolayer와 공배양을 통해 체외수정을 실시한 결과 고분자분획첨가 및 난관상피세포 공배양(OM+OEC)군이 고분자분획첨가

The present study was performed to investigate the effects of caffeine and heparin on capacitation and acrosome reaction of bovine spermatozoa, effects of antisperm antibodies on acrosome reaction of bovine spermatozoa. The rates of acrosome reaction in control group, caffeine treated group, heparin treated group, caffeine-heparin complex treated group were 40.3, 54.3, 63.3, 72.3%, respectively and there were significant differences among the groups(p<0.01), especially higher in caffeine-heparin complex treated group than the others. The rates of acrosome reaction of antisperm antibodies serum supplemented groups(5, 10 and 20%) were 60.4, 48.9 and 37.1%, respectively and there were significant differences among the groups(p<0.0l), and the more increases in serum concentrations, the more decreases in acrosome reaction, but this phenomenon was not seen in fetal calf serum supplemented group and heifer serum group. When the serum concentration was 5%, the rates of acrosome reactions were significantly lower in fetal calf serum supplemented group than heifer serum group and in antisperm antibodies serum group(p<0.01), and there were no significant differences between heifer serum group and antisperm antibodies serum group(p<0.01). When the serum conecntrations were 10%, 20%, the rates of acrosome reactions were significantly lower in antisperm antibodies serum supplemented group than in fetal calf serum group and in geifer serum group(p<0.01), and there were no significant differences between fetal calf serum group and heifer serum group(p<0.01). These results indicate that caffeine-heparin complex treatment is very effective for inducing acrosome reaction of bovine spermatozoa and that antisperm antibodies block acrosome reaction.