The Jeju Black Cattle (JBC) are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. Semen analysis is the most commonly used procedure to evaluate male fertility potential. This study was to evaluate the quality of 10 JBC bulls belonging to Jeju Special Self-Governing Province Promotion Institute. [JBC A∼J grade]. The freezing medium (20% egg yolk plus 20% triladyl) was added in semen sample to a final concentration of 100×106 sperm/ml. For sperm cooling, diluted semen was filled in 0.5 ml plastic straws and then kept in refrigerator at 4°C for 2 h. They were placed in 7 cm over liquid nitrogen (LN2) vapor for 10 min and then directly plunged into LN2 for storage. Thawing was done by transferring the frozen straws into water bath at 37°C for 30 sec for analysis. The sperm motility, vitality and morphology in each group was assessed using the Sperm Analysis Imaging System (SAIS Plus; Medical Supply Co, Ltd., Korea), eosin-nigrosin stain and diff-quik kit. There was no difference in the motility of the fresh groups (87.4 ~ 100%), while it was difference in the frozen-thawed groups (42.8 ~ 98.6%) (p<0.05). The best motility was shown in JBC-B (100/fresh and 98.6%/frozen-thawed). There was significant difference in the vitality of the fresh group (19.8 ~ 59.2%) and frozen-thawed group (21.2 ~ 49.8%)(p<0.05). The highest vitality was also shown in JBC-B (59.2/fresh and 49.8%/frozen-thawed). Morphologically, in fresh semen the highest normal ratio was indicated in JBC-E (90.9%) and in frozen-thawed group the highest was in JBC-C (90.2%). These results demonstrated that the analysis including motility, vitality and morphology of fresh or frozen-thawed semen is valuable to select the high quality sperm using for reproduction.

Light Mineral Oil is a material generally used as an overlay covering microdrops of culture medium in petri dishes. Although Light Mineral Oil can protect the damage by oxidation in air, it can't completely protect the damage by evaporation and alteration of pH and osmolality in culture medium. To minimize the damage by evaporation and alteration of pH and osmolality, we assumed that Heavy Mineral Oil could be used as an alternative. Heavy Mineral Oil is high purity paraffin oil which has more viscosity and density than Light Mineral Oil, so it can prevent evaporation and maintain stable osmolality and pH in culture medium more than Light Mineral Oil. The objective of this study was to examine whether the effect of Heavy Mineral Oil is superior to the effect of Light Mineral Oil during in vitro cultivation of porcine oocytes. According to the data of repeated six experiments, survival and cleavage rate of porcine oocytes, and cell number of blastocysts were not significantly different between two groups. However, the in vitro development rate of porcine parthenogenetic embryo was significantly higher in Heavy Mineral Oil group than in Light Mineral Oil group (Light, 36.6% ± 3.9%; and Heavy, 52.1% ± 6.4%, p < 0.05). Thus, these results indicated that the treatment of Heavy Mineral Oil can improve the in vitro developmental capacity of porcine parthenogenetic embryos compared to Light Mineral Oil.

Recent research on stem cell conditioned medium (CM) has been revealed that CM could influence on the embryo development when supplemented to in vitro culture medium. However, the optimal basal medium for CM production has not determined although it is the fundamental factor of CM. The purpose of this study is to examine the effect of human derived adipose stem cell CM (hASC-CM) with different basal medium on mice embryo development after parthenogenetic activation (PA). hASC-CM was collected from 2 kinds of serum free basal medium, DMEM and KSFM, respectively on day 5 from the culture of hASC isolated from human fat tissue. Intra-peritoneal injection of PMSG and hCG was conducted into 7-week-old ICR mice for superovulation. The oocytes were recovered from the oviductal ampulla, 18 h after hCG injection, and denuded using 0.1% hyaluronidase. PA of oocytes was conducted with KSOM media including strontium chloride. The parthenotes were in vitro cultured in 3 groups: 100% KSOM (Control), 75% KSOM + 25% DMEM or KSFM without FBS (DMEM or KSFM group) and 75% KSOM + 25% hASC-CM from DMEM or KSFM (DMEM-CM or KSFM-CM group). Cleavage rate was assessed after 2 days post IVC and blastocyst formation rate was evaluated after 6 days post IVC both using stereomicroscope. Total cell number of blastocysts was counted by Hoechst staining. 1way ANOVA from Graphpad prism 5 was used for statistical analysis and the values are presented as means ± standard error of mean. As a result, blastocyst formation rate of DMEM-CM group (16.09±3.32%, P<0.05) was significantly lower than control and DMEM group (34.43±2.89% and 34.49±5.34%, P<0.05) but cleavage rate and total cell number of blastocysts showed no significant difference among groups. In case of KSFM, there was no significant difference in cleavage rate, blastocyst formation rate and total cell number of blastocysts among the control, KSFM group and KSFM-CM group. The sort of basal medium used for the CM collection affected the development of parthenotes during in vitro culture differently. Therefore, further research should be conducted to find out the alternative basal medium of CM able to improve the embryo development. This research was supported by Nature Cell (#550-20170028), Cooperative Research Program of RDA (CCAR, #PJ013954022018), Research Institute for Veterinary Science and the BK21 plus program.

Secretory leukocyte protease inhibitor (SLPI), also known as neutrophil elastase and cathepsin-G protease inhibitor, functions in protection of epithelial cells from proteases. SLPI is expressed and secreted by many mucosal tissues, including lungs, seminal vesicles and cervix in women. SLPI plays an important role in protection of endometrial epithelial cells during pregnancy from degradation by degradation by proteases derived from trophoblast at the maternal-conceptus interface. In pigs, SLPI mRNA is known to be expressed in endometrial tissues, but the expression of SLPI in the endometrium throughout the estrous cycle and pregnancy has not been determined. Therefore, we analyzed the expression and regulation of SLPI mRNA in the endometrium throughout the whole stages of the estrous cycle and pregnancy in pigs. We obtained endometrial tissues from gilts on Days 0 (day of estrus), 3, 6, 9, 12, 15, and 18 of the estrous cycle and on Days 10, 12, 15, 30, 60, 90, and 114 of pregnancy. Real-time RT-PCR analysis showed that the expression of SLPI mRNA in the endometrium increases during midt-o late pregnancy. During the estrous cycle, levels of SLPPI mRNA in estrus and proestrus were higher than those in diestrus and metestrus. In situ hybridization analysis showed that SLPI mRNA was specifically localized to the glandular epithelial cells in the endometrium during pregnancy with strong signal intensity during mid-to late pregnancy. SLPI mRNA was not detectable in conceptus tissues on Days 12 and 15 of pregnancy, but SLPI mRNA was expressed in chorioallantoic tissues during mid-to term pregnancy with increasing levels toward term pregnancy. To determine the effects of steroid hormones, estrogen and progesterone, on the expression of SLPI mRNA, endometrial explant tissues from immature pigs were treated with increasing doses of estradiol-17β (E2) and progesterone (P4). Increasing doses of E2 and P4 increased the expression of SLPI mRNA in endometrial tissues. These results showed that SLPI was expressed in the endometrium in a pregnancy stage-and cell type-specific manner and the expression of SLPI was regulated by E2 and P4 in endometrial tissues, suggesting that SLPI may play an important role in regulating the endometrial epithelial cell function during mid-to late pregnancy in pigs. Further analysis to determine the roles of SLPI at the maternal-conceptus interface is still needed.

Preimplantation embryonic production in vitro is important in human assisted reproductive technology and animal embryo engineering. Icariin (ICA) is one type of flavonoids and a main component isolated from the stem leaf of Epimedium brevicornum. Flavonoids, which are among the best well-studied natural antioxidants, have been demonstrated to be active in clearing reactive oxygen species (ROS). The purpose of this study was to investigate the effects of ICA treatment during porcine oocyte in vitro aging and their in vitro developmental competency after parthenogenetic activation (PA). Porcine oocytes were matured in vitro for 44 h (control) and for an additional 24 h in the presence of 0, 5 μM ICA (aging, ICA-5), respectively. This study investigated the effects of ICA on nuclear maturation, ROS level, apoptosis index, and the developmental capacity of aged porcine oocytes. Oocyte survival was not different in aging group compared to control or ICA-5 group. The increased ROS level during in vitro aging was prevented in ICA-5 group, while GSH level was not decreased. The decrease of normal spindle formation during in vitro aging was prevented in ICA-5 group. After PA, although the cleavage rate was not different among treatment groups, the blastocyst formation was significantly higher in control and ICA-5 group than in aging group. However, there was significantly difference in the apoptotic index of the ICA-5 group, while it was no difference in the total cell number of the ICA-5 group. (p<0.05). Therefore, this result demonstrated that the ICA is an effective agent to prevent the deterioration during in vitro aging of porcine oocytes.

Generally, in vivo, primary oocytes are grown and matured into secondary oocytes in the ovarian follicles. Quality of the oocytes matured in vivo is higher than that of oocytes matured in vitro, indicating the importance of materializing the microenvironment of ovarian follicles for production of high quality oocyte. Therefore, we tried to mimic the stiffness of ovarian follicles using an agarose as a biocompatible natural polymer. Unfortunately, to date, there are no many reports on whether the quality of porcine oocytes can be increased effectively under the soft matrix. Accordingly, we tried to evaluate the effects of IVM using different mechanical properties of agarose substrate on developmental competence of porcine oocytes. Agarose substrate was constructed and cumulus-oocyte-complexes (COCs) retrieved from porcine medium antral follicles were matured on non-coated (control) culture dish or dishes coated with 1% and 2% (w/v) agarose substrate. Then, cumulus expansion, embryonic development after parthenogenetic activation, and gene expression level were analyzed and compared. As the results, significant increase in blastocyst formation and cumulus expansion were detected in COCs matured on 1% (w/v) agarose substrate compared with control. Moreover, oocytes of COCs matured on 1% (w/v) agarose substrate showed significantly higher BMP15 expression level compared with control. Pro-apoptotic gene BAX expression was significantly increased in oocytes of COCs matured on 2% (w/v) agarose substrate compared with control. In the glycolytic enzyme phosphofructokinase (PFKP) gene expression, cumulus cells of COCs matured on agarose substrate showed significantly higher PFKP expression than control while they showed significantly lower BAX expression than control. These results demonstrated that quality of porcine oocytes could be increased efficiently by the IVM of immature oocytes on the soft culture matrix using agarose.

This study was conducted to evaluate the effects of insulin and epidermal growth factor (EGF) in a in vitro growth (IVG) medium on oocyte growth, in vitro maturation (IVM) and embryonic development of pig oocytes derived from small antral follicles (SAF) less than 3 mm in diameter. SAF oocytes were cultured for 2 days to induce IVG in alpha-minimal essential medium supplemented with 1 mM dbcAMP and 15% (v/v) fetal bovine serum. After IVG culture, oocyte maturation was induced by culturing IVG oocytes in IVM medium for 44 h. IVM oocytes that extruded the first polar body were selected and induced for parthenogenesis (PA) by applying electric stimulus. SAF oocytes cultured under the insulin treatment showed a significantly increased (P < 0.05) nuclear maturation (73.8%) compared to those cultured with insulin and EGF (59.8%). After PA, the proportions of blastocysts based on the number of metaphase II oocytes were significantly higher (P < 0.05) in oocytes that were cultured for IVG with insulin, EGF, and insulin + EGF (32.4%, 35.2%, and 34.8%, respectively) than in control (22.9%). IVG oocytes treated with insulin showed an increased oocyte diameter (116.3 μm) compared to those treated with insulin and EGF (114.0 μm) (P < 0.05). Intra-oocyte GSH content significantly increased (1.07 pixels/oocyte) by insulin treatment during IVG compared to that of oocytes treated with insulin + EGF (0.78 pixels/oocyte). These results demonstrate that IVG culture of SAF oocytes under insulin or/and EGF treatment supports oocyte maturation and improves embryonic development to the blastocyst stage after PA in pigs.

U0126 is a highly selective inhibitor of both MEK1 and MEK2, a type of MAPK/ERK kinase. This study was conducted to evaluate the effect of U0126 treatment during in vitro maturation (IVM) on nuclear maturation, intra-oocyte glutathione content, and embryonic development after parthenogenesis (PA). U0126 (5 μM) was supplemented to IVM medium during the first 0 (control), 2, and 4 h. The basic medium used for IVM was medium-199 supplemented with 10% (v/v) porcine follicular fluid (standard), 0.6 mM cysteine, 0.91 mM pyruvate, 75 μg/ml kanamycin, and 1 μg/ml insulin. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II stage were electrically activated to induce PA. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) fatty acid-free BSA. When immature oocytes were treated with U0126 during the first 0, 2, 4 h of IVM culture, nuclear maturation was significantly (P < 0.05) increased by the U0126 treatment for 4 h (96.2 ± 1.3%) compared to standard IVM (90.6 ± 2.1%). Cleavage of PA embryos was significantly increased by 4 h- treatment (90.6 ± 2.2%) compared to standard medium (83.9 ± 1.8%). In addition, blastocyst formation of PA embryos was significantly (P < 0.05) increased by the treatment for 4 h (55.8 ± 5.7%) compared to 2 h (38.1 ± 6.1%). The glutathione contents in IVM oocytes were not altered by the U0126 treatments for 0, 2, and 4 h (1.28 ± 0.10, 1.16 ± 0.09, and 1.10 ± 0.09, respectively). Our results demonstrated that 5 μM U0126 treatment during the first 4 h of IVM showed positive effects on nuclear maturation, cleavage, and embryonic development in pigs.