Alpha-linolenic acid (ALA; n-3 18:3), a one of omega-3 fatty acid, is mainly contained in chloroplast of plant and ALA is an essential fatty acid, not synthesized in mammalian body, it must be supplied from foods. Polyspermy is especially high on in vitro fertilization (IVF) in pigs, which is a major obstacle to in vitro embryo production systems. In our previous study, when ALA was supplemented during in vitro maturation (IVM), the methaphase-II rate and gluthathione level was increased. The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) supplementation during IVM and subsequent of IVF in pigs. The cumulus-oocyte complexes (COCs) were submitted to IVM medium containing 0, 25, 50, and 100 μM ALA for 44 h. After 44 h of IVM, denuded oocytes were co-cultured with spermatozoa during 18 h. After 18 h of in vitro fertilization, oocyte were using aceto-orcein method, to evaluated penetration rate, monospermy (number of monospermy oocytes/total oocytes), and the IVF efficiency (number of monospermy/total penetrated oocytes). In results, 25 and 50 μM ALA groups were significantly increased on penetration rate compared with 100 μM ALA group (p<0.05). Similarly, monospermy rate were significantly increased 25 and 50 μM ALA groups than control group (p<0.05). IVF efficiency was no significant difference between control and ALA treatment groups. Our findings suggested that treatment of ALA supplementation during in vitro maturation (IVM) and subsequent of in vitro fertilization in pigs, ALA can increase IVF efficiency by effectively blocking polyspermy and increasing monospermy some mechanism in porcine oocytes. However, the study of mechanism by which ALA blocks polyspermy are needed, and this study suggests that ALA has a positive effect on in vitro production of porcine oocytes by decreasing polyspermy. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

In the present study of this experiment was to understand the expression of apoptotic gene expression in the ovary of miniature pigs and pigs on the 15th day of estrus. Also the compare and analyze of programmed cell death type(Apoptosis and autophagy) expression pattern during mature oocyte on the miniature and normal pig cells. Analysis of mRNA gene expression of ovary in miniature and normal pigs on the 15th day of estrus showed that the expression of genes related to Autophagy (ATG13, MAP1LC3, Beclin1) was high in normal pigs but the expression of ATG1 and ATG5 genes was low. In addition, the expression of genes related to apoptosis (Casp-3, BAX) was high in the mini pigs, and the gene related to the LH hormone was high in the miniature pigs, whereas the expression of the gene related to the FSH hormone was high in the normal pigs. On the other hand, the result of muture oocyte on the miniature and normal pig cells is the expression of Casp-3 protein was moust high from treatment of FL+rapa (FSH+LH and Rapamycin) of the oocyte on the miniature pig cell. However, MAP1LC3 expression was higher in the oocytes of treatment of rapanycine treatment on the nomal pig cells. There was no gene expression in cumulus cells of matured oocytes in mini pig cells, whereas MAP1LC3 expression was higher in oocyte cumulus cells matured in normal pig cells. It was confirmed that the miniature and normal pigs showed different programmed cell death patterns, In the case of oocytes matured in miniature pig cells, MAP1LC3 gene expression was found to be low in spite of treatment with Autophagy regulator.

The aim of this study was to investigate the role of Src homology 2-containing phosphotyrosine phosphatase SHP2 in intricate signaling network invoked by oocyte to achieve cytoplasmic maturation and also blastocyst development. Activation of SHP2 regulates multicellular differentiation, proliferation and survival through numerous signal pathways. The most prominent pathway is RAS/PI3K and p-AKT signaling cascade, as a result mitogenic effect become enhanced. Oocytes were cultured in cisplatin an anticancer drug, but selective activator of SHP2 and our grouping were SOF medium alone, SOF + EGF, SOF + CISPLATIN 0.3 μM, and SOF + EGF + CISPLATIN 0.3 μM. We evaluated that EGF neutralizes the apoptotic effect of cisplatin as well as maintain the high expression of SHP2, as a result blastocyst development become boosted up. We also found that inhibition of SHP2 with its specific inhibitor PHPS1 5 μM decreases the blastocyst development and neutralizes growth factors effect. The developmental ability and quality of bovine embryos were determined by assessing their cell number, gene expression, immunofluorescence, and immunoblot. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. Our results show that SHP2 have significant effect on MAP kinase pathways which expand the cumulus cells during oocyte maturation and blastocyst development as compare to inhibition of SHP2 with PHPS1. SHP2 not only transduce the signaling of epidermal growth factor but it also has a role in signal transduction of FGF and IGF. The expression of ERK, PI3K/p-AKT and mTOR was increased with EGF, but with the treatment of SHP2 inhibitor the expression of these genes become drop done. So we can conclude from these results that SHP2 is important for oocyte maturation as well as for blastocyst development.

ompared the expression of MMPs in these oocytes and cumulus cell throughout oocytes maturated. In an attempt to investigate the effect of MMP activation and inhibitors in total protein of cumulus cell and, oocytes during oocytes maturation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), TIMPs (TIMP-2 and TIMP-3), as well as their expression profiles (Real-time PCR, Gelatin Zymography and ELISA). Our results that the bovine oocytes MMP-2 and MMP-9 level was significantly associated with the rate of maturity of oocytes (P<0.05). In cumulus cell, MMP-2 was highly expressed in all stages of the oocyte’s maturation. The final oocytes maturation exhibited strong gelatinase activity. There was no significant correlation between cumulus cell MMP-9 and the maturation rate of oocytes. However, for the oocyte cytoplasm MMP-9 expression was significant correlation to the maturation oocytes. There was no significant correlation between cumulonimbus cells MMP-9 and oocyte maturation rates; however, for oocyte cytoplasm, MMP-9 expression was significantly correlated with mature oocyte. However, the TIMP-1 and TIMP-2 protein expression patterns are not correlated with the maturation rate of the oocyte. Our results suggest that MMP different expression pattern may regulate the morphological remodeling of oocyte's in the cumulus cell. Further, the MMP-2 expression has a strong relation with a higher maturation rate of the oocyte.

This study aimed to investigate the changes in the γ-aminobutyric acid (GABA) content of bitter melon (Momordica charantia L.) cultivated from different regions, with different harvest times and at various maturation stages. Methods for observing the changes in GABA content were validated by determining the specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), and precision and accuracy using the HPLC-FLD system. Results showed high linearity in the calibration curve with a coefficient of correlation (R2) of 0.9999. The LOD and LOQ values for GABA were 0.29 and 0.87 μg/mL, respectively. The relative standard deviations for intra- and inter-day precision of GABA were less than 5%. The recovery rate of GABA was in the range of 98.77% to 100.50%. The average content of GABA was 0.93 mg/g and Cheongju showed highest GABA content of 1.88 mg/g. As the time of harvest increased from May to September, the GABA content decreased from 1.56 to 0.86 mg/g. Also, maturation of the bitter melon fruit was associated with a decreased in GABA content.

Cellular cyclic adenosine-3’ 5’-monophosphate (cAMP) modulator is known as meiotic inhibitor and can delays spontaneous maturation in IVM experiment. Among many cAMP modulators, the role of Pituitary adenylate cyclase activating polypeptide (PACAP) on IVM isn’t known. The purpose of this study is to improve the maturation of oocytes derived from follicles ≤ 3 mm in diameter through PACAP as meiotic inhibitor during pre-in vitro maturation (pre-IVM). First, we checked PACAP and its receptors in cumulus cells and, to establish the optimal phase and concentration of PACAP for pre-IVM, we conducted chromatin configuration assessments. As a result, the rate of GV (Germinal Vesicle) according to duration of pre-IVM was significantly decreased 12 h and 18 h after IVM (87.1 and 84.1%, respectively) compared to 0 h (99.4%). When COC was cultured for 18 h, the GV rate in the 1 μM of PACAP treatment group (82.1%) was significantly higher than any other PACAP treatment groups (60.5, 64.1, 74.4 and 69.9 %, respectively). So, we divided into four groups as follows; MF (the conventional IVM group, obtained from follicle from 3 to 6 mm in diameter), SF (the conventional IVM group, obtained from follicle ≤ 3mm in diameter), Pre-SF(-)PACAP (IVM group including 18 h pre-IVM without 1 μM of PACAP, obtained from follicle ≤ 3mm in diameter) and Pre-SF(+)PACAP (IVM group including 18 h pre-IVM with 1 μM of PACAP, obtained from follicle ≤ 3mm in diameter). To examine the effect of PACAP during pre-IVM, we investigated analysis of nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. In cumulus cells, PACAP receptors, ADCYAP1R1 and VIPR1 were detected but were not detected in oocytes. After IVM, the Pre-SF(+)PACAP had the highest Metaphase II rate (91.7%) among all groups (P<0.05). The GSH levels in the MF and Pre-SF(+)PACAP were significantly higher than in the other groups (P<0.05) and ROS levels was no significant difference among all groups. In conclusion, these results indicated that even though the oocytes were derived from SF, pre-IVM application of PACAP improved meiotic and cytoplasmic maturation by regulating intracellular oxidative stress.

The maturation and spawning of black scraper, Thamnaconus modestus were studied using samples collected monthly from March, 2015 to February, 2016 in the coastal waters off Middle East Sea, Korea. The gonadosomatic index (GSI) of female was the highest in June. The spawning periods lasted from March to September based on histological observation of female gonad development. The percentage of sexually mature females estimated from a logistic function was over 50% for the size group 18.02 cm (total length, TL). The size of eggs spawned was between 0.40 to 0.58 mm. Fecundity varied between 185,648 and 9,747,250 eggs. The relationship between the fecundity and TL of the fish was expressed in the fecundity equation as F = 0.0297TL5.4835.

참다래(Actinidia chinensis)의 연화과 발생은 재배 환경과 기후의 영향을 받기 때문에 경작년에 따라 발생 정도에 차이가 있다. 수확전 키토산 살포는 황육계 및 적육계 참다래 과실의 경도를 증가시키는 것 으로 확인되었지만 수확후 침지처리는 유의한 결과를 보이지 않았다. 수확전 키토산 살포는 과실 성숙을 균일하게 만들어 주어 적육계 참다래의 수확기 연화과 발생율을 감소시켰다. 과실내부 조직의 CO2 농도 는 대조구에 비하여 45% 증가하였는데 이는 과피에 키토산 피막이 형성되었기 때문이었다. 수확전 처리 한 키토산에 의하여 과실 건물중과 전분함량이 증가하였고 가용성고형물과 산함량이 높아져 과실 성숙이 늦어졌으며 저장 중에도 과실 경도 및 전분 감소가 지연되었다. 수확전 키토산 처리는 적육계 참다래의 과피 반점 형성을 감소시켜주었다. 결론적으로 수확전 키토산 살포는 참다래 과실의 과피장해 억제, 경 도 저하 지연을 통하여 저장성을 증진시키는데 기여하였다.

This study was designed to determine the effect of monosodium glutamate (MSG) on in vitro maturation (IVM) of oocytes and early development of parthenogenesis (PA) embryos in pigs. Each IVM and IVC medium was supplemented with various concentrations (0, 0.1, 0.5 and 5 mM) of MSG and non-essential amino acids (NEAA) depending on the experimental design. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II (MII) stage were electrically activated to induce parthenogenesis (PA). When immature oocytes were treated with MSG in the absence of NEAA during IVM, nuclear maturation (83.1-87.1%), intra-oocyte glutathione content, cumulus expansion, and cleavage (91.4-93.4%) of PA embryos were not influenced by MSG treatment at all concentrations. However, blastocyst formation of PA embryos was significantly increased by 5.0 mM MSG (45.3 ± 6.2%) compared to control (25.6 ± 3.4%). MSG treatment during IVM in the presence of NEAA did not show significant effect on nuclear maturation of oocytes and blastocyst formation after PA while 0.5 mM MSG (89.3 ± 1.9%) decreased (P < 0.05) cleavage of PA embryos compared to 0.1 mM MSG (94.6 ± 1.1%). When PA embryos were treated for 7 days with MSG during IVC, 5.0 mM MSG significantly decreased blastocyst formation (27.8 ± 4.9%) compared to no treatment (41.4 ± 1.9%) while no decrease in blastocyst formation was observed in 0.1 and 0.5 mM (37.4 ± 3.4% and 34.4 ± 2.6%, respectively). Our results demonstrated that 5 mM MSG in a NEAA-free chemically defined maturation medium showed positive effect on PA embryonic development while 5 mM MSG treatment during IVC was deleterious to PA embryonic development in pigs.

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Alpha lipoic acid (ALA) is a biological membranes compound. As the antioxidant, it decreases the oxidized forms of other antioxidant substances such as vitamin C, vitamin E, and glutathione (GSH). To examine the effect of ALA on the in vitro maturation (IVM) of porcine oocytes, we investigated intracellular GSH and reactive oxygen species (ROS) levels, and subsequent embryonic development after parthenogenetic activation (PA). Intracellular GSH levels in oocytes treated with 50uM ALA increased significantly (P < 0.05) and exhibited a significant (P < 0.05) decrease in intracellular ROS levels compared with the control group. Oocytes matured with 50 uM of ALA during IVM displayed significantly higher cleavage rates (67.8% vs. 83.4%, respectively), and higher blastocyst formation rates and total cell number of blastocysts after PA (31.6%, 58.49 vs. 46.8%, 68.58, respectively) than the control group. In conclusion, these results suggest that treatment with ALA during IVM improves the cytoplasmic maturation of porcine oocytes by increasing the intracellular GSH levels, thereby decreasing the intracellular ROS levels and subsequent embryonic developmental potential of PA.

Ferulic Acid (FA) is a metabolite of phenylalanine and tyrosine, a phenolic compound commonly found in fruits and vegetables. Several studies have shown that FA has various functions such as antioxidant effect, prevention of cell damage from irradiation, protection from cell damage caused by oxygen deficiency, anti-inflammatory action, anti-aging action, liver protective effect and anti-cancer action. In this study, we investigated the maturation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) of porcine oocytes by adding FA to the in vitro maturation (IVM) medium and examined subsequent embryonic developmental competence at 5% oxygen through parthenogenesis. There is no significant difference between the control group (0μM) and treatment groups (5μM, 10μM, 20μM) on maturation rates. Intracellular GSH levels in oocyte treated with 5μM of FA significantly increased (P < 0.05), and 20μM of FA revealed significant decrease (P < 0.05) in intracellular ROS levels compared with the control group. Oocytes treated with FA exhibited significantly higher cleavage rates (79.01% vs 89.19%, 92.20%, 90.89%, respectively) than the control group. Oocytes treated with 10μM showed significantly higher blastocyst formation rates (28.3% vs 40.3%, respectively) after PA than the control group. Total cell numbers in blastocyst of 10μM FA displayed significantly higher (39.4 vs 51.9, respectively) than the control group. In conclusion, these results suggested that treatment with FA during IVM improved the developmental potential of porcine embryos by increasing intracellular GSH synthesis and reducing ROS levels. Also, there was an improvement of cleavage rate, blastocyst formation and total cell numbers in blastocysts. It might be associated with Keap1-Nrf2 pathway as an antioxidant regulate pathway that plays a crucial role in determining the sensitivity of cells to oxidative damages by regulating the basal and inducible expression of enzymes which is related to detoxification and anti-oxidative effects, stress response enzymes and/or proteins and ABC transporters.

Alpha-linolenic acid (ALA) is one of n-3 polyunsaturated fatty acids and found mainly in the chloroplasts. Many studies have been reported that intracellular reactive oxygen species (ROS) in mammalian oocytes were reduced by supplementation of ALA in in vitro maturation (IVM) medium. Based on these reports, we expected that ALA acts as an antioxidant during IVM of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidant effect of ALA supplementation during IVM in porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated in IVM medium containing 200 μM H2O2 or H2O2 with 50 μM ALA for 44 h. Nuclear maturation stage of oocytes was evaluated using aceto-orcein method. For measurement of oxidative stress state, intracellular ROS and glutathione (GSH) levels were measured using carboxy-DCFDA and cell tracker red, respectively. In results, oocytes in metaphase-II (MII) stage development was significantly reduced in H2O2 group compared to non-treated control group (61.84±1.42% and 80.00%, respectively; p<0.05) and it was slightly recovered by treatment of ALA (69.76±1.67%; p<0.05). The intracellular GSH levels was decreased in H2O2 groups compared with control groups, but it was enhanced by ALA treatment (p<0.05). On the contrary, H2O2 treatment increased intracellular ROS level in oocytes and H2O2-induced ROS was decreased by treatment of ALA (p<0.05). Our findings suggested that ALA treatment under oxidative stress condition improve oocyte maturation via elevated GSH and reduced ROS levels in oocytes. Therefore, these results suggest that ALA have an antioxidative ability and it could be used as antioxidant in in vitro production system of porcine embryo.

The induction of regeneration and the maturation of the free-living gametophytes of Undaria pinnatifida were studied at temperatures (5, 10, 15 and 20℃), irradiance (5, 10, 20 and 40 μmol photons m-2 s-1) and photoperiods (14 : 10, 12 : 12 and 10 : 14 h L : D). Female gametophyte fragments were maintained in active regeneration without them reaching sexual maturity under conditions of 15℃, 20 μmol photons m-2 s-1, 10 : 14 h (L : D); by contrast, the conditions for male gametophytes were slightly different at 15℃, 5 μmol photons m-2 s-1, 14 : 10 h (L : D). The sexual maturation of female and male gametophytes took place under 5℃, 20-40 μmol photons m-2 s-1, 10 : 14 h (L : D) and 15℃, 40 μmol photons m-2 s-1, 10 : 14 h (L : D), respectively. These results provide basic information for controlling the regeneration and maturation of free-living gametophytes for srain improvement as well as the cross breeding of U. pinnatifida.

국내에서 육성된 중생종 동양배 품종인 ‘창조’는 성숙 기간 중 지속적으로 과실이 비대하여 600g대의 편원형 과실로 발달하였다. 성숙기간 중 경도는 경시적으로 저하되어 만개 후 153일인 9월 13일에 32.58N으로 조사되었고 만개 후 160일에는 26.44N으로 급격히 경도가 저하되었다. 식감과 관련된 품질요인 중 과실의 전분함 량은 만개 후 153일에 0.737, 만개 후 160일에 0.451로 전분함량이 급속히 감소하였고, 과육의 세포벽함량을 조사하였던 결과, 과실비대가 증가하고 과실성숙의 진행과 더불어 알코올불용성물질 함량은 낮아지는 경향을 보였다. 과피색차 중 적색도(a*)는 만개 후 139일 이후 양의 값을 보여 외관상의 과피녹색 발현이 소실되었는데 과피의 SPAD 값을 조사한 결과에서도 동일하게 엽록소의 감소를 확인할 수 있었다. Hue angle 역시 성숙과 연화 기간 중 유의하게 감소하는 경향을 보여 수확시기의 지 표로 활용 가능하다고 판단되었다. 성숙기간 중 과육 내에 축적되는 당 성분을 분석한 결과, 만개 후 153일에 총량이 최대치를 보였는데 전 생육시기에 걸쳐 전체 당 성분 중 과당이 우점하고 있었고, 과실성숙도가 높아질 수록 자당의 비율이 유의하게 증가하여 만개 후 160일 에 수확한 과실에서는 자당이 과당을 넘어 우점하여 과숙단계로 생각되었다. 따라서, 배 신품종 ‘창조’의 적정 수확시기는 만개 후 153일인 9월 13일 이전으로 평가되었고 수확시기를 놓친 경우에는 경도 등 과실의 품질이 급격하게 하락하는 것으로 사료되었다. 대만 지역 수출 을 모의하여 단기수송온도 설정을 위한 수확 후 저온순화 실험을 실시한 결과, 만개 후 146일에 수확한 과실이 만개 후 153일에 수확한 과실에 비하여 25oC 유통 후 높은 경도를 유지하는 경향을 보였고 최종 수송 목표 온도인 5oC로 저하되는 속도가 느릴수록 과육장해의 발 생심도 및 에틸렌발생량이 높은 경향을 보였으므로 ‘창조’ 품종에 있어 동남아 지역 수출 시 생리장해 및 과실 품질 유지를 위해서는 수확시기를 만개 후 146일 이전 에 설정하는 것이 바람직할 것으로 판단되었다. 본 연구 결과를 바탕으로 ‘창조’ 배의 목적별 수확시기 결정 및 과실의 선별 기술에 일조하고 수출 시장에서의 한국산 배의 신인도 확보에 기여할 것으로 기대된다.

This study was conducted to investigate the effects of alpha-lipoic acid (aLA) as an antioxidant that decrease the reactive oxygen species (ROS) in bovine embryonic development. Slaughterhouse derived bovine immature oocytes were collected and 4 different concentrations (0, 5, 10 and 20 mM) of aLA was supplemented in bovine in Vitro maturation (IVM) medium. After 20 hrs of IVM, maturation rates, levels of ROS and glutathione (GSH), and further embryonic development after parthenogenetic activation (PA) and in Vitro fertilization (IVF) was investigated according to aLA concentrations. Maturation rate was significantly higher in 10 mM group than other groups (80.5% vs. 62.9, 73.9, 64.2%; P<0.05). In the levels of ROS and GSH in matured oocytes as an indicator of oocyte quality, significantly better results were shown in 5 and 10 mM groups compared with other 2 groups. After IVM, significantly higher rates of blastocyst formation were shown in 10 mM groups in both of PA (27.9% vs. 18.8, 22.3, 14.2%; P<0.05) and IVF (32.6% vs. 23.9, 27.3, 16.2%; P<0.05) embryos. In addition, significantly more cell total cell number and higher inner cell mass ratio in 10 mM PA and IVP blastocysts showed developmental competence in 10 uM groups. Therefore, based on the entire data from this study, using 10 μM of aLA confirmed to be the optimal concentration for bovine oocyte maturation and embryonic development.

The objective of this study was to determine the effect of fructose that was supplemented to a chemically defined in Vitro maturation (IVM) medium on oocyte maturation and embryonic development after parthenogenesis in pigs. The base medium for in Vitro maturation (IVM) was porcine zygote medium (PZM) that was supplemented with 0.05% (w/v) polyvinyl alcohol (PVA) or 10% (v/v) porcine follicular fluid (pFF). In the first experiment, when immature pig oocytes were matured in a chemically defined medium that was supplemented with 5.5 mM glucose or with 1.5, 3.0 and 5.5 mM fructose, 3.0 mM fructose resulted in a higher nuclear maturation (91.5%) than 1.5 and 5.5 mM fructose (81.9 and 81.9%, respectively) but showed a similar result with 5.5 mM glucose (94.2%). However, there was no significant differences among groups in the embryo cleavage (89.4-92.4%), blastocyst formation (37.5-41.1%), and mean cell number of blastocyst (30.8-34.2 cells). Fructose at the concentration of 3.0 mM (1.08 pixels/oocyte) resulted in a higher intra-oocyte glutathione (GSH) content than 1.5 and 5.5 mM fructose (1.00 and 0.87 pixels/oocytes, respectively) while the cumulus cell expansion was not influenced. In the second experiment, effect of individual and combined supplementation of a chemically defined maturation medium with 5.5 mM glucose or 3.0 mM fructose was examined. No significant effect was found in the nuclear maturation (86.3-92.6%). Embryo cleavage was significantly increased by the combined supplementation with glucose and fructose (95.2%) compared to that with 3.0 mM fructose only (85.7%) while blastocyst formation (37.3-42.8%) and embryonic cell number (33.3-34.1 cells) were not altered. Effect of supplementation of pFF-containing medium with glucose and fructose + glucose was examined in the third experiment. No significant effect by the supplementation with glucose and fructose or glucose alone was observed in the nuclear maturation of oocytes (90.7-94.1%) and blastocyst formation (51.0-56.5%). Our results demonstrate that 3.0 mM fructose was comparable to 5.5 mM glucose in supporting in Vitro oocyte maturation and embryonic development after parthenogenesis and could be used as an alternative energy source to glucose for in Vitro maturation of pig oocytes.

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in Vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

The elevated temperature and high humidity has been known as main reason for heat stress on animals and cause detrimental effects on productivity of organisms and physiological conditions of normal bioactivities. The aims of this study were to evaluate the relationship between time of heat shock simulation during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. Heat shocked cumulus-oocyte complexes (COCs) of Korean native cattle were subjected to normal conditions for 22, 21, 18 and 12 h respectively and transferred to heat stress inducing condition at 40.5 °C in other incubator for 0 (control), 1 and 4 h. After maturation for 22 h, the oocytes were fertilized and cultured in mSOF media for 8 d and examined the developmental capacity of embryos. There were no differences in maturation and cleavage rates between 0, 1 and 4 h heat socked oocytes, but blastocysts formation were lower in the 4 h heat stressed oocytes. The apoptotic cells of developed blastocysts were also increased in at day 8 with 4 h heat shocked oocytes. These results indicate that heat shock on oocytes during maturation could cause negative effects on the developmental competence of embryos.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated a specific gene transcription levels in oocytes and cumulus cells (CC) after IVM by realtime PCR arry, and specific protein expression and activation levels in matured CCs by western blotting. Each concentration (0, 1, 10, and 100 ng/ml) of GDF8 was added in maturation medium (TCM199) during process of IVM. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science). Data are presented as the mean and Differences were considered significant at P < 0.05. After 44 h of IVM, oocytes are mechanically denuded from CCs with 0.1% of hyaluronidase, and then the separated oocytes and CCs were sampled following each group. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, the realtime PCR array was performed. In CCs the 1- and 10 ng/ml of GDF8 supplement group showed the transcription co-factors CBP and SP1, cell metabolic regulator MAPK1, and cumulus expansion related genes Has2, Cox-2, Ptx3 and Areg transcription levels were significantly distinguished with control when hierarchically clustered by Euclidean distance with average linkage method after IVM. In matured oocytes the 10- and 100 ng/ml of GDF8 supplement group showed the maternal factors JMJD3 and Zar1, transcriptional regulator FOXO1, Sirt1 and Sirt2, mitochondrial activity factor Sirt3, ACSL3 and ACADL, anti-apoptosis gene BCL-2, and oocyte secrete factor BMP15 mRNA transcription levels were significantly distinguished compared with control. To determine effect of GDF8 supplement during IVM, the GDF8 down steam canonical regulator SMAD2/3 protein phosphorylation levels analyzed in CCs by western blotting. The 10- and 100 ng/ml supplement groups showed significantly increase phosphorylated (P)-SMAD3 (1.56 and 1.34 times higher than control) protein levels (P < 0.05). In conclusion, supplement of GDF8 during IVM activates FOXO homolog transcription and induced cumulus cells expansion via activation of SMAD3 signaling in CCs. While process of IVM, the transcriptional landscape changes in CCs may consequently result maternal factors accumulation and mitochondrial activation in oocytes.