미토콘드리아는 세포질 칼슘 항상성 및 ATP 생산에 중요한 역할을 하는 세포 소기관으로 이러한 미토콘드리아의 기능은 성숙과 수정 그리고 배 발달에 매우 중요한 역할을 한다. 미토콘드리아 칼슘 축적은 기능장애를 일으킨다. 그러나 돼지 체외성숙란 및 수정란에서 미토콘드리아 칼슘 변화의 관련성에 관한 연구는 보고된 적이 없다. 본 연구의 목적은 미토콘드리아 칼슘 지시자로 알려진 Rhod-2 염색을 이용하여 성숙란 및 수정란에서 미토콘드리아 칼슘 축적의 변화를 확인하였다. 형태학적 모습의 기준을 통해 난구세포의 세포층과 세포질의 균질도를 바탕으로 G1과 G2로 나누어서 체외성숙을 진행하였다. 이후 두 그룹에서 핵 성숙율을 비교하였을 때, G2가 G1에 비해 낮게 나타났다(p<0.001). 돼지 체외성숙란 및 수정란에서 평균적인 Rhod-2 spot 의 수는 G1보다 G2에서 더 많이 나타났다(6시간째 체외수정란: p<0.05). 다음으로 Rhod-2 spot 수에 따른 난모세포의 비율을 확인하기 위해 Rhod-2 spot 의 수를 4개의 군(n<10, 10≤n<20, 20≤n<30, 그리고 30≤n)으로 나누어 해당 난모세포의 비율을 확인하였다. 체외성숙란 및 체외수정란 모두 G1이 G2에 비해 10개 미만(n<10)인 Rhod-2 spot 의 수를 가지는 난모세포가 많았으며, 체외수정란에서는 유의적으로 높았다(p<0.05). 마지막으로 체외성숙란 및 수정란에서 Rhod-2 intensity 값을 측정하여 두 그룹을 비교하였을 때, G2가 G1에 비해 유의적으로 높은 것을 확인 할 수 있었다(성숙란; p<0.001 그리고 수정란; p<0.05). 본 연구의 결과를 토대로 돼지에서 미성숙 난포란의 형태학적인 품질은 체외성숙 및 체외수정 과정 동안 미토콘드리아 내 칼슘 축적과 관련이 있음을 확인하였다.

In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml IL-1β. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and IL-1β groups than EC without hCG and IL-1β. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and IL-1β groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with IL-1β is beneficial and useful for enhancing the production of porcine blastocysts in vitro.

The objective of this study was to investigate the changes of oxidative stress and antioxidant enzyme during in vitro development with washing culture oil in porcine embryos. During the culture, the four types of culture oil such as paraffin oil with or without washing and mineral oil with or without washing were examined. The oil was washed with PZM-3 during 7 days and collected oil only. The embryos were stained with CellTrackerTM Red, DC-FDA and Hoechst 33342 to confirm the effects of the oil. As a results, Cleavage rates and total cell number were no difference among the four oil groups. However, ≥16 cell embryos were significantly different in fore type oil treat-ment and blastocyst rate was significantly higher washing paraffin treatment than in other group(p<0.05). Also, the expression of free radical were lower in washing paraffin oil than in other groups (p<0.05). On the other hand, the expression of glutathione were not significant different among paraffin oil with or without washing and mineral oil with or without washing, however washing paraffin oil and washing mineral groups were higher than other treat-ment groups. In conclusion, the washing oil was expected with positive effects on in vitro development in porcine embryos.

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.

돼지 수정란의 체외 생산 효율성 향상을 위해서는 배발생율과 더불어 고품질의 배를 조기에 선별해야 한다. 체외 배 발생율에 대한 보고는 많지만, 고품질의 배를 선별할 수 있는 기술에 대한 연구는 거의 없었다. 본 연구에서는 돼지 난포란 유래 수정란의 체외배양에 있어서 배반포로의 배 발달과 생존에 미치는 Vitamin K1(vit K1) 첨가 농도, 시기 및 시간의 효과를 검토하였다. 1.0 μM, 3.0 μM 및 6.0 μM vit K1을 배양 1일째 24시간 첨가한 결과, 배반포 발달율이 시험군이 14.5 ± 4.3, 0.0 및 0.0%로써 대조군의 35.5 ± 3.2%에 비하여 유의하게 낮았고(p<0.05), 배반포의 생존율도 대조군이 31.8 ± 2.6%로써 시험군의 22.2 ± 2.9, 0.0 및 0.0%에 비하여 유의하게 높았다(p<0.05). 상기 첨가 농도에서 첨가 시간을 달리한 결과, 1.0 μM 농도에서 6시간 처리군의 배반포 발달율과 생존율이 각각 26.5 ± 2.9% 및 47.2 ± 2.8%로써 가장 높았고 특히, 12시간 처리군보다 유의하게 높았다(p<0.05). 3.0 μM 농도에서는 대조군의 배발달율이 36.4 ± 3.1%로 가장 높았으나, 생존율은 3.0시간 첨가군이 41.7 ± 3.2%로 대조군에 비하여 유의하게 높았다(p<0.05). 6.0 μM 농도에서도 배발달율은 대조군(32.0 ± 2.8%), 생존율은 0.5시간 첨가군(42.9 ± 1.8%)이 가장 높았다. 각각의 vit K1 첨가 농도와 시간을 기준으로 서로 다른 배양 시기에 첨가한 결과, 1.0 μM 6시간 첨가군에서는 배반포 발달율은 배양 4일째 첨가군, 생존율은 배양 2일째 첨가군이 가장 높았다. 한편, 3.0 μM 3.0시간 및 6.0 μM 0.5시간 첨가군에서는 배양 4일째 첨가군의 배반포 발달율(59.5 ± 4.1% 및 50.0 ± 3.6%)과 생존율(72.7 ± 5.4% 및 79.2 ± 4.0%)이 대조군과 다른 시험군에 비하여 유의하게 높았다(p<0.05). 한편, vit K1 첨가에 따른 배반포의 세포 수를 조사한 결과, 첨가군(1.0 μM 6시간 배양 2일째, 3.0 μM 3.0시간 배양 4일째 및 6.0 μM 0.5시간 배양 6일째)이 53.4 ± 5.8, 49.4 ± 3.8 및 51.5 ± 4.5개로써 대조군의 40.2 ± 2.3개에 비하여 유의하게 많았다(p<0.05). 그러나 사멸세포 수는 시험군이 3.2 ± 0.9∼3.7 ± 2.1개로써 대조군의 4.2 ± 1.2개보다 적었으나, 유의차는 없었다. 세포 사멸 유도 유전자인 Bax mRNA 발현은 처리군과 대조군은 비슷하였으나, 세포 사멸 억제 유전자인 Bcl-xL mRNA 발현은 처리군이 대조군보다 높았고 특히, 6.0 μM 0.5시간 배양 4일째 첨가군이 가장 높았다. 이상의 결과로부터 돼지 미성숙 난포란 유래 수정란의 체외 배양에 vit K1의 첨가는 배반포의 생존율과 세포수 증가에 효과적이었다. 그 이유에 대해서는 아직 많은 부분이 밝혀져야 되겠지만, 고품질의 배반포 조기 선발에는 활용이 가능할 것으로 생각된다.

The present study was performed to investigate the effect of Hh-Ag1.5, a small-molecule chemical agonist of SMOothened receptor, on the in vitro maturation and development of in vitro fertilized (IVF) embryos in pigs. Oocytes or fertilized embryos were cultured in a maturation or embryo culture medium supplemented with 0 (control), 25, 50 or 100 nM of Hh-Ag1.5, respectively. Although the maturation rate were not different among treatment groups, the blastocyst formation rate in the group treated with 25 nM Hh-Ag1.5 was significantly increased compared to other groups (P<0.05). While the highest dose of Hh-Ag1.5 (100 nM) did negatively affect to the embryo development and cell number in blastocysts compared to other groups (P<0.05), the apoptotic cell index in blastocysts was significantly lower in 25 and 50 nM groups than in control and 100 nM groups (P<0.05). The mRNA expression of the proapoptotic gene Bax and the ratio of Bax/Bcl-XL decreased in among treatment groups compared to control (P<0.05). The embryo quality related genes, Tert and Zfp42, were significantly decreased in 50 and 100 nM groups compared with control and 25 nM groups (P<0.05). In conclusion, the addition of 25 nM Hh-Ag1.5 to in vitro maturation and culture medium can enhance the developmental potential as well as quality of IVF embryos in pig.

본 연구는 돼지의 난포란을 체외성숙하여 세포질내정자주입(ICSI)에 의해 생산된 체외수 정란의 체외발달율을 평가하기 위하여 실시하였다. 세포질내정자주입에 의한 체외수정란의 발달율은 서로 다른 보존상태의 정자인 신선정자, 액상정자 및 동결-융해된 정자를 이용하 더라도 수정율과 배발달율에는 영향을 미치지 않는 것으로 나타났다. 세포질내정자주입 후 난자의 전기적 활성화를 처리한 실험군이 활성화를 처리하지 않은 실험군에 비해 수정율과 배반포기배로의 발달율에 있어서는 높은 경향을 나타내었으나, 체외수정 실험군 및 전기적 활성화를 처리한 실험군과 전기적 활성화를 처리하지 않은 실험군간 배반포기배의 할구수는 유의적인 차이를 나타내지 않았다. 또한 각각의 실험군에서 얻은 배반포기배의 염색체를 분 석한 결과, 정상 이배체 염색체상의 비율에 있어서도 유의성을 나타내지 않았다.

돼지 핵이식 복제수정란의 체외 발달율을 개선하고자, 핵이 주입된 수핵난자를 전기자극 에 의한 융합 후 demecolcine으로 세포질 활성화 처리를 실시하였다. 2-세포기로 분할 전 까지 핵의 이상분열을 억제하여 정상적인 핵형 유지여부 및 demecolcine이 핵의 방출을 유 도하여 탈핵의 전 처리, 핵이식 후 활성화의 후 처리 및 탈핵 전․후 처리를 모두 한 핵이 식 수정란의 체외발달율을 조사하였다. 융합이 이루어진 복제 수정란에 demecolcine을 4시간 처리하였을 때 정상적인 핵 모양인 CLC(clustered chromosomes) 상태는 43.2%로서 무 처리구의 15.0%보다는 높았으며, 1PN (single pronucleus) 상태는 46.9%로서 대조구의 35.0%와는 차이가 없었다. 비정상적인 핵 형인 ≥2PN, PPB+CLC or PN 및 ≥2PN 7.4, 0 및 2.5%로서 무 처리구의 17.5, 22.5 및 10%로서 처리구가 낮았다. 융합 이후 16시간에는 1PN이 85.0%로서 대조구의 56.7%보다 는 높았다. 분할율은 전․후 모두 처리구에서 86.4±2%로서 후 처리(81.3±6%)와 대조구(79.6±6%) 보 다 높았으며, 전 처리의 85.7±2%와는 차이가 없었다. 배반포기로의 발달율에 있어서도 전․후 처리가 18.1±3%로서 전 처리(11.0±3%)와 대조구(11.3±1%)에 비하여 높았으나, 후 처리의 15.5±1%와는 차이가 없었다. 배반포기 수정란의 할구수도 전․후 처리가 21.6±6% 로서 전, 후 처리 및 대조구의 각각 18.8±8%, 19.4±3% 및 19.7±2%보다 많았다. Demecolcine을 돼지 복제 수정란에 처리하면 분할 전까지 핵의 이상 분열을 억제하는 것 이 확인되었으며, demecolcine 처리에 의한 탈핵은 수핵난자의 세포질 손상을 감소시키고 보다 용이하게 할 수 있다. 본 연구에서와 같이 탈핵 및 활성화를 목적으로 탈핵 전․후 처 리를 하였을 때 체외발달율이 다소 개선되는 경향을 보였다.

The objective of this study was to evaluated the efficiency on sperm cryosurvival and ability of in vitro fertilization using Triladyl and Lactose Egg-Yolk(LEY) as extenders for cryopreservation of separated sperm by 65% percoll in miniature pig. Sperm viability was measured with SYBR-14/PI double stained sperm by flow cytometry. Ability on embryo cleavage rate and blastocyst development were observed by in vitro fertilization after frozen-thawing of sperm separated by 65% percoll. The experimental groups were designed that separated sperm by 65% percoll with Triladyl (ST) or LEY(SL) and unseparated sperm with Triladyl(UT) or LEY(UL) for cryopreservation. As a results, the viability was significantly(p<0.05) higher in ST(55.1%), SL(63.1%), UL(58.8%) than UT(38.2%) group. Sperm viability in SL(63.1%) group was significantly(p<0.05) higher than other experimental groups. On the other hand, embryo cleavage rate was significantly(p<0.05) higher in ST(79.1%), SL(83.2) than UT(74.1%) and UL(75.7%) groups at 96h after in vitro fertilization. Blastocyst development was also significantly(p<0.05) higher in ST(21.5%), SL(20.9%) than UT(17.0%) and UL (18.8%) groups. In conclusion, cryopreservation of miniature boar sperm separated by 65% percoll were beneficial to viability and capacity on in vitro fertilization.

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 porcine FSH, 0.5 equine LH, 1.0 17 -estradiol () and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at in humidified atmosphere of 5% in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at in 5% , 5% for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

The brilliant cresyl blue (BCB) has been used to select the developmental competent oocytes in pigs, goats and cows. Growing oocytes have a higher level of active glucose-6-phosphate dehydrogenase(G6PDH) compare to mature oocytes and are rarely stained compared to mature oocytes, because G6PDH converts BCB to colorless. First polar body extrusion regard as a guideline of meoisis completion. Selection of polar body extrude oocyte is more developmental competent to blastocyst than unselected. This study was conducted to compare the BCB test to the polar body extrusion on selection of developmental competent porcine oocytes for the production of blastocyst. Cumulus-Oocytes complex were exposed to 26uM BCB stain diluted in NCSU-23 for 90 min. There was no significant difference embryo development to blastocysts between BCB treated and not treated(19.58±1.99 vs 18.75±2.27 %), which means there was no detrimental effect of BCB exposure to oocytes. Normal fertilization is not differed among treatment groups from 70.0 to 78.4% development to blastocyst, beside polyspermy did not. To compare two different selection methods, BCB test and polar body extrusion, evaluate the developmental competent of IVP embryos. BCB+PB+(blue stained and polar body extruded, 20.71±0.45%) and BCB－PB+(colorless and polar body extruded, 20.04±1.29%) groups are significantly (p<0.05) higher developed than those of BCB+PB－(blue stained and no polar body, 13.24±0.73%) and BCB－PB－(colorless and no poladbody, 7.25±0.77%). These results showed that selection of polar body extruded oocytes method is more efficient than that of BCB test.

This study was performed to confirm the microtubule assemblies and methylation patterns of porcine IVF and parthenogenetic embryos. Cumulus-oocyte complexes were collected and matured in vitro for 42 hr. Oocytes were fertilized by prepared fresh sperm or activated parthenogenetically by exposure to electric stimulation and 6-dimethylaminopurine. Porcine IVF and parthenogenetic embryos were cultured in vitro for 6 days. Embryos were stained by immunofluorescence staining method to observe the dynamic of nucleus and microtubules in the first mitotic phase and the methylation patterns in different developmental stages. After then, samples were confirmed and analyzed through a laser-scanning confocal microscope. IVF embryos had a centrosome originated from sperms, which was shown a ɤ-tubulin spot. However, ɤ-tubulin spot was not observed in parthenogenetic embryos. A lower methylation level was observed in IVF embryos compared to parthenogenetic ones at the morula and blastocyst stages. In conclusion, it is considered that microtubule assemblies and genetic regulation mechanism differ between parthenogenetic and IVF embryos.

It is not easy for porcine embryos produced by in vitro systems to develop into blastocysts with high quality. To solve this problem, many researchers have developed novel culture methods. However, the formation of blastocysts with high quality is still low. In this study, we aimed to produce piglet following transfer of in vitro produced early embryos ( cell stage embryos) or morula and blastocyst. The cell stage embryos were transferred to five estrus-synchronized recipients (200 embryos per recipient). One of the five sows farrowed three piglets, which contain two live piglets and one dead piglet, 114 days after embryo transfer. However, two recipients transferred with morula and blastocysts did not farrow. Microsatellite analysis confirmed that the genomic DNA of two live piglets were not genetically identical to that of the recipient. These results indicate that it is possible to obtain piglets by transfer of early embryos produced by in vitro production (IVP) systems.

The aim of the present study was to investigate the cleavage pattern, its developmental ability and apoptosis of porcine embryo in vitro. Morphology data on a total of 919 embryos were analyzed retrospectively. Forty-eight hours after insemination, embryos were classified into five groups based on the cleavage state as follows; 1 cell, 2 cell, 4 cell, 5 to 8 cell and fragmentation. These groups were cultured another 120 hours and then evaluated for blastocyst formation. Blastocyst formation rates were significantly higher in 4 cell (42.5%) and 5 to 8 cell (48.6%) cleaving groups than in other groups (p<0.05). On the other hand, 2 cell and fragmentation groups produced 4.9% and 3,9% blastocysts, respectively. And we could verify that in the event of 2 cell block and fragmentation of embryo. To analyze the apoptotic frequency in preimplantation development of porcine IVF embryos, all cells of each blastocyst were performed by TUNEL assay. There were no significantly differences in the total cell numbers of embryos and apoptotic cell rate in blastocysts among the each classified groups. Data suggest that 4 cell and 5 to 8 cell cleaving embryos at 48 hour after insemination have high developmental competence, and may be an useful parameter to predict the development of preimplantation embryos and to study using preimplanation embryonic research.

This study was performed to elucidate the effects of addition of and bovine serum albumin (BSA) in vitro maturation (IVM) and in vitro culture (IVC) medium on porcine embryo production. The development rate to the 2 cell () and blastocyst stages () with different BSA concentrations in IVM medium were similar among treatment groups. Blastocyst hatching rate was significantly higher in the control group (0.0mg/ml) than in the group of 1.0mg/ml supplement (20.0% vs. 0.0%; p<0.05). The development rate to the 2 cell () and blastocyst stages () with different concentrations in IVM medium was similar among treatment groups. The development rate to the blastocyst was significantly higher in the group of 1.0mg/ml(15.3%) than in the group of 0.5mg/ml supplement (7.6%, p<0.05). The development rate to the 2 cell and blastocyst stages following the first addition of in IVM medium was significantly higher in the control group (77.0% and 18.9%) and was (77.2% and 16.9%) greater than that observed in other treatment groups (p<0.05). The development rate to the 2 cell stage () and blastocyst stages () with different BSA concentrations in IVC medium was similar among treatment groups. However, blastocyst hatching rate was significantly higher in the group of 3.0mg/ml supplement (30.0%) than in the control group (0.0%; p<0.05). The development rate to the 2 cell stage (), blastocyst () and hatching stages () were not different. The development rate to the 2 cell stage (), blastocyst () and hatching stages () at the different culture periods were similar among treatment groups. This study suggested that if the addition level and periods of addition are adjusted, it is possible to replace BSA in the in vitro porcine embryo production.

This study was conducted to investigate the effect of periods of sperm preincubation, concentrations and storage periods of miniature pig sperm on in vitro penetration of porcine follicular oocytes. High concentration (1×105, 2.5×105, 5×105, 1×106, 5×106 and 1×107) did support sperm penetration than low concentrations (P<0.05). However, polyspermic oocyte rates were increased with high concentrations of sperm. On the other hand, sperm preincubated during 1, 2 or 5h could be penetrated than sperm preincubated during 0, 3 or 4h (P<0.05). When sperm were storaged with different periods, in vitro pentration rates were significantly higher 0～2 days than 3～4 days of sperm storage (p<0.05). These results indicate that sperm treatment factors can effect in vitro penetration in miniature pig.