Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

본 연구는 현재 국립축산과학원 가축유전자원세터에서 보유 하고 있는 재래닭을 순수화된 품종인 것으로 판단하고, 일반 적으로 이용되고 있는 산란사료 및 사양관리 방법을 적용하여 특히, 동절기에 있어 재래닭의 정자의 보존 기간과 수정률 및 초기배자의 생존율을 각각 비교함으로써 재래닭의 생산성 향 상을 위한 기초자료를 제공하고, 나아가 표준능력을 고찰하고 자 수행하였다. 본 시험에 사용된 공시계는 39주령의 재래닭 6계통 적갈색(R, Red Brown Strain), 황갈색(Y, Yellow Brown Strain), 회갈색(G, Gray Brown Strain), 흑색(L, Black Strain), 백색(W, White Strain) 그리고 오계(O, Ogol Strain)를 대상 으로 하고 대조군으로는 3계통 즉, 외래도입종 중에서 대표적 인 다산종인 White Leghorn (F Strain), Rhode Island (C Strain) 그리고 육용종인 Cornish (H Strain)의 수정률 및 초기배자 생존율을 조사하였다. 단 한번의 인공수정 후, 3 주간 생산된 알의 수정률 확인을 한 결과, 재래닭의 경우, 6 계통간의 유의 적인 차이는 없지만 93.3 ~ 100.0비율로 인공 수정 후, 2일째 부터 수정률이 6일째 동안 최고 높음을 확인했다. 6일째까지 상대적으로 일정하게 높은 수정률을 유지하다가 7일부터 17일 째 까지 점진적으로 감소함을 확인했다. 17일 이후 생산된 알 의 경우 무정란임을 확인 할 수 있었다. 재래닭(R, Y, 그리고 O) 21일간 생산된 알의 배발생정지율의 결과, 인공수정 후, 약 4일째 생산된 알(3 ~ 6일)에서 외래도입종 3품종간의 유의적인 차이는 보이지 않았지만, 배발생정지율이 0%임을 확인하였다. 세 품종 모두 약 7일째 생산된 알부터 배발생정지율이 13.8 ~ 26.7%로 급격히 증가하고 12일부터 감소하는 것을 확 인했다. 재래닭 3 계통의 결과도 인공수정 후, 약 4 일째 생 산된 알에서 외래도입종 3 품종과 유사한 패턴을 나타내면서 배아 사망율이 6 일째까지 0%를 보였다. 금후, 체내 정자보존 기간이 수정률 및 초기배자 생존율에 미치는 영향을 좀더 엄 밀하게 조사하기 위해서는 정액 성상 및 활력 검사와 더불어 암탉의 주령에 따른 변화도 함께 보다 세부적이고 입체적인 방법의 체계적인 조사가 반드시 필요하다고 할 수 있겠다.

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed (37℃) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at 38.5℃ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

In a conventional sense, dried-spermatozoa are all dead and motionless due to the lost of their natural ability to penetrate oocytes both in vivo and in vitro. However, their nuclei are completely able to contribute to normal embryonic development even after long-term preservation in a dried state when the dried-spermatozoa are microinjected into the oocytes. In this sense, dried spermatozoa must still be alive. Thus, defining spermatozoa as alive or dead seems rather arbitrary. Several drying method of sperm including freeze-drying, evaporative/convective-drying and heat-drying were represented in this review. Although the drying protocol reported here will need further improvement, the results suggest that it may be possible to store the male genetic resources.

Artificial insemination is a technique to transfer instrumentally sperm from the male into the female’s reproductive system. A key factor among artificial insemination techniques is gathering sperms. Here, a method for collecting sperm of bumblebee male by pressing was firstly developed. This method has stage of separating reprodutive tract of male, separating accessory testis, separating vas deferens, pressing vas deferens with cover-glass and collecting sperm with syringe. The developed method was 2.8 fold higher in rate of collecting sperm than that of existing method. Among 1M NaCl-, Insect ringer solution-, Phosphate buffered saline(PBS)-collecting sperm solutions, PBS was exhibited the best result as 17.2 sperms/cell, which corresponded to 1.6-1.8 fold increased over the result of other collecting sperm solution. Consequently, we think that this method is very important technique to save time for collecting sperm, to keep activity of sperm and to reduce contamination of sperm.

Internal sperm storage after mating is important for insect reproduction, because it permits delayed fertilisation, and post-copulatory mate choice in polyandrous females. The polyandry is common in many animal taxa including insects, because it increases female fitness by reducing the risk of infertility and providing opportunities for sperm competition and choice. The reproductive success of males, on the other hand, often depends upon avoidance of sperm competition by preventing mated females from copulating and receiving sperm from other male suitors. A widespread strategy used by males is the use of the male seminal fluid proteins (SFPs) that form the mating plug and alter female behaviors, for example by suppressing mating receptivity and elevating egg-laying. Under these circumstances, females are expected to evolve mechanism(s) to control exposure to the male SFPs in order to maximize fitness by balancing the positive and negative impacts of polyandry. Here, we discover that Drosophila melanogaster females eject male ejaculates 1-6 h after mating with a stereotypic behaviour, and that this is regulated by a brain neuropeptide pathway composed of diuretic hormone 44 (Dh44), and its receptor Dh44R1. We showed that suppressing Dh44 or Dh44R1 signals in the brain expedites sperm ejection, whereas enhancing Dh44 or Dh44R1 signals delays sperm ejection. This study uncovers a molecular mechanism by which females can influence sperm competition and selection, and counter actively the negative impact of polyandry.

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM (61.1 ± 1.5%, 64.7 ± 2.0%) of nicotinic acid than other groups (0 mM, 52.1 ± 2.3%; 20 mM, 47.8 ± 5.1%, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid (26.1 ± 1.8%, 24.9 ± 1.5%) than other groups (0 mM, 35.3 ± 0.8%; 20 mM, 36.5 ± 1.9%, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM (84.2 ± 3.6%, 88.4 ± 2.3%) of nicotinic acid than other groups (0 mM, 77.3 ± 4.4%; 20 mM, 73.3 ± 3.6%, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM (17.0 ± 1.3%) of nicotinic acid than other groups (0 mM, 9.4 ± 0.5%; 5mM, 12.6 ± 0.8%; 20 mM, 5.0 ± 1.0%, P<0.05). Moreover, total cell number was higher in 5 and 10 mM (53.6 ± 2.9%, 57.9 ± 2.8%) of nicotinic acid than other groups (0 mM, 41.0 ± 1.4%; 20 mM, 23.2 ± 2.8%, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid (0.7 ± 0.1%) than other groups (0 mM, 1.0 ± 0.1%; 10mM, 0.9 ± 0.0%; 20 mM, 1.4 ± 1.0%, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2∼3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above LN2 for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at 50℃ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions’ groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.

The objective of this study was to determine the effects of E. coli on boar sperm quality and reproductive performance in sows after artificial insemination. Three different levels of E. coli were artificially inoculated to semen with following concentrations; Control, 500, 5,000 and 50,000 colony forming unit (cfu)/ml. Semen samples were preserved at 17℃ for 5 days. Sperm motility was significantly decreased (p<0.05) on day 3 in the group inoculated with 5,000cfu/ml compared to control groups. In all treatment groups, sperm motility was gradually decreased as storage time increased, but the decline pattern was more drastic in the groups inoculated with 5,000 and 50,000 cfu/ml groups from day 3 (p<0.05) compared to control group. After 3 day of storage at 17℃, sperm viability in sample inoculated with the highest concentration (50,000 cfu/ml) of bacteria was less (p<0.05) than that of control group. The pH of semen sample pH was maintained 7.2～7.5 in all groups during the experimental period. No differences (p>0.05) were found for both storage time and bacterial concentration. The pregnancy rate and live born piglets tend to decrease by increasing the concentration of E. coli in semen. In particular, the rate of pregnancy was lower in the group inoculated with 50,000 cfu/ml (58.3%) compare to the other groups (81.8, 75.0, 76.5%). These results suggest that the contamination of E. coli in boar semen negatively affects fertilizing ability of boar sperm and the reproductive performance obtained from sows after artificial insemination.

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was eva-luated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental com-petence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, 0.1 μg/mL, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of pene-trated oocytes but ROS intensity was significantly decreased by 0.01 μg/mL compared with other groups (p< 0.05). Moreover, treatment with 0.01 μg/mL of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with 0.01 μg/mL of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supple-mentation of Humulus japonicus extract in IVF medium may have important implications for improving early embryo-nic development in bovine embryos

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein 50 μM for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and 50 μM and genestein 1 and 50 μM for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and 50 μM, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin 1 μM for 3 hr incubation. In mitochondrial activities, addition of quercetin 50 μM for 6 hr incubation increased mitochondrial activity but decreased at 100 μM concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin (1∼10 μM), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin (25∼50 μM). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin 10 μM (p<0.05). The developmental rates to morula or blastocysts at low (0.01∼1 μM) and high (5∼10 μM) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and 10 μM) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations (0.01∼10 μM).

The main purpose of this study is to estimate the effect of adding Tea-N-Tris (TES) to the freezing buffer for miniature pig sperm. In particular, we attempted to identify the association between the MMPs expression and the fertility and viability of frozen sperm from each extender (LEY (Lactose Egg-Yolk), TLE (TES + LEY), TFGE (TES + Fructose + Glucose Egg-Yolk)). In accordance with this, Hypoosmotic Swelling Test (HOST) respond test was the lowest among sperms frozen in LEY while the highest HOST respond was observed among sperms frozen in TLE. Furthermore, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. LEY group showed lower rate of blastocyst development than the TES supplement group, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE group were used for IVF. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer only suppresses MMPs protein activation but also maximize in-vitro fertility, providing a means to improve the success rate in the in vitro manipulation of miniature pig sperm.

The objective of this study was to compare the effect of semen extenders on the sperm motility, viability, acrosome integrity and functional integrity of plasma membrane (HOST: hypo-osmotic swelling test) during liquid preservation of Korean Native boar semen. In this experiment, semen was diluted in Androhep plus, Beltsville Thawing Solution (BTS), ModenaTM, Seminark and Vitasem LD. Sperm-rich fractions were collected from three Korean Native boars and sub-samples were diluted (30×106 spermatozoa/ml) in different semen extenders. Semen samples were stored at 17℃ for 96 hours. On everyday (0, 24, 48, 72, 96 h) after storage, the sperm characteristics relevant for fertility, such as sperm motility, viability, acrosome integrity and HOST positive were evaluated. The motility of spermatozoa stored in different extenders was no significantly different among other extenders (P>0.05). Also, no difference was observed among samples processed with different extenders in the percentage of sperm viability, acrosome integrity and HOST positive. All extenders maintained a high percentage (70%) of sperm motility, viability and acrosome integrity through 96 h of storage. The result of this study show that there was no significant differences among extenders in their capacity to preserve motility, viability and membrane integrity of spermatozoa from normal, fertile Korean Native boars for 96 h of liquid preservation at 17℃.

The objective of this study was to investigate the effect of bacterial contamination on elapsed time after preservation on boar semen. Known numbers of Escherichia coli (E. coli) were inoculated to freshly ejaculated semen and sperm parameters such as viability, motility, agglutination, acrosome integrity and hypo-osmotic swelling test were perform-ed during 7 days of liquid preservation. Semen samples were prepared using antibiotic free BTS extender and 4 di-fferent levels of E. coli were treated to semen with following concentrations; 3,000, 5,000, 7,000, 10,000 CFU/ml of sperms. Semen samples were preserved at 17℃ for 7 days in semen storage until analyzed. Aliquots were subjected to measure the sperm viability, motility and agglutination using computer assisted sperm analysis (CASA) system, acrosome integrity was performed using chlortetracycline (CTC) staining method and hypo-osmotic swelling test was performed using hypotonic solution from day 1 (day of semen collection) to 7. Detrimental effects on sperm motility and viability were observed 3 days after preservation at the level of 5,000 CFU/ml (p<0.05). Percentage of sperm abnor-mality was higher (p<0.05) in over 5,000 CFU/ml groups. Sperm agglutination rate was also significantly higher (p< 0.05) in groups of 5,000 and 7,000 CFU/ml. The rate of acrosome reacted sperm was higher as preservation time goes in all the samples but the pattern was clearly higher among E. coli contaminated groups (p<0.05). The sperm mem-brane integrity in terms of hypo-osmotic test, E. coli affects little compared to other sperm parameters. The deleterious effects observed due to the bacterial contamination in semen suggest that importance of hygiene protocol to minimize the bacterial contamination during semen collection and processing.

Sperm capacitation refers to polymerization of filamentous (F)-actin from globular (G)-actin. While the role of ac-tin-related protein 2/3 (Arp2/3) complex in actin polymerization is well appreciated, the underlying mechanism(s) and its relationship with capacitation are poorly understood. Therefore, to evaluate the potential role of Arp2/3 complex on capacitation, bovine spermatozoa were incubated with multiple doses (1, 10 and 100 μM) of CK-636, an inhibitor of Arp2/3 complex with heparin. The cellular localization of the Arp2/3 complex in spermatozoa was identified by immunohistochemistry, whereas western blot was also applied to detect the protein tyrosine phosphorylation of sperm proteins. Additionally, sperm motility and kinematic parameters were evaluated using a computer-assisted sperm analysis system. CK-636 resulted in significant changes in the ratio of Arp2/3 complex localization between acrosome and equatorial region of the spermatozoa. Short-term exposure of spermatozoa to 100 μM of CK-636 significantly decreased sperm motility, however a non-detectable effect on protein tyrosine phosphorylation was observed during capacitation. On the basis of these results, we propose that Arp2/3 complex is associated with morphological changes during capacitation and compromised sperm motility.

Cryopreservation and in vitro fertilization (IVF) protocols are important in genetic studies and applications to transgenic animals. Various studies about boar sperm cryopreservation have been studied for a long time. Those were about the use of extenders, the choice of sugars, the cooling and warming rates. The factors that influence the boar sperm are the dramatic changes in temperatures, osmotic and toxic stresses, and reactive oxygen species (ROS) generation. Among these factors, ROS generation is the main damage to DNA which is a principal genetic material and the most important for the practical applications. So we wondered whether ROS generation could be reduced. In previous study, monothioglycerol (MTG) was essential for the culture of embryo stem cells. Therefore we added MTG in the freezing extender based on lactose-egg yolk (LEY) with trehalose. For the assessment of the frozen-thawed sperm, we focused onmotility, membrane integrity and DNA damage. First, we used a computer-aided sperm analysis system for overall conditions of sperm such as motility and viability. Then we performed the sperm chromatin structure assay for DNA integrity and hypo-osmotic swelling test for membrane integrity. And our result showed the existence of MTG in the freezing extender caused less damage to DNA and higher motility in frozen-thawed boar sperm. Also we checked a relative antioxidant activity of MTG in modified Modena B extender. We concluded that this reagent can activate sperm mitochondria at MTG 0.2 μM, contribute to sperm motility and DNA integrity but there was no significant difference on membrane integrity. Also antioxidant activity of MTG in modified Modena B extender was proved.

This study was designed to evaluate the effect of L-cysteine on sperm characteristics and oocyte cleavage in vitro in Korean native cattle. For this study, the freezing of diluted semen were added with Triladyl containing 20% eggyolk and/or 0, 5, 10 and 20 mM L-cysteine before cryopreservation. The viability in frozen-thawed sperm were estimated by SYBR14/PI double stain, acrosome damage with FITC-PNA, mitochondria intact with Rhodamin123 and hydrogen peroxide(H2O2) level with carboxy-DCFDA by flow-cytometry. The developmental capacity was also assessed with cleavage rates in oocytes fertilized in vitro by frozen-thawed sperm. In results, the sperm viability was significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). In addition, acrosome damage was significantly decreased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). The mitochondria intact was also significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). On the other hand, the cleavage rates were significantly increased in 0 mM, 5 mM and 10 mM groups than 20 mM concentration of L-cysteine (p<0.05). The oocyte degeneration of oocytes were significantly decreased in 0 mM, 5 mM and 10 mM groups than in 20 mM L-cysteine group (P<0.05). However, there are no significantly differences among the L-cysteine treatment groups. We suggest that concentration of 10 mM L-cysteine have beneficial impact for sperm cryopreserved in Korean native cattle. This result also could be recommended for artificial insemination program if supported by an improvement in the fertility results and required further study.

The objective of this study was to develop of semen transport system for cryopreservation and fertility in bull sperm. The ejaculated semen were diluted with Triladyl containing 20% egg-yolk for transportation. Diluted semen was transported by three methods that there were wrapping tissue (Tissue), sinking under 30℃ water (Water) and sinking between warm water and air (Air) methods. Semen was transported within 2 hours in 0.3℃. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk. And frozen-thawed sperm were estimated with SYBR14/PI double stain for viability, FITC-PNA/PI double stain for acrosome reaction analysis and Rhodamine123 double stain for mitochondrial intact assessment. In results, live sperm (SYBR+/PI-) in Air treatment group (43.3±4.7%) was significantly (p<0.05) higher than other treatment groups (Tissue: 16.3±2.7% and Water: 27.5± 3.1%), dying sperm (SYBR+/PI+) in Air treatment group (55.6±4.7%) was significantly lower than other treatment groups (Tissue: 77.6±3.2% and Water: 67.6±3.3%) (p<0.05). Acrosome reaction in Air treatment group (0.2±0.1%) within live sperm (PI negative region) was significantly (p<0.05) lower than other treatment groups (Tissue: 0.7±0.2% and Water: 0.5±0.1%), the acrosome reaction in Air treatment group (28.6±2.8%) within all sperm also was significantly lower than other treatment groups (Tissue: 44.2±1.8% and Water: 36.2±2.0%) (p<0.05). And mitochondrial intact in Air treatment group within live (97.1±0.4%) and all (61.9±3.3%) sperm were significantly higher than other treatment groups (Tissue: 85.2±3.3%, Water: 87.8±2.9% within live sperm and Tissue: 49.28±3.7%, Water: 42.0±3.1% within all sperm) (p<0.05). Therefore, we suggest that transportation by sinking method between warm water and air was beneficial to improvement of fertility in frozen-thawed in bull semen.