• Journal of Embryo Transfer
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• v.27 no.4
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• pp.265-270
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• 2012

In canine, oocytes are ovulated at the GV (germinal vesicle) stage and they have to fulfill maturation phase before reaching metaphase II stage. The efficiency of in vitro maturation is still very low. Therefore, the aim of this study was to investigate the effect of in vitro maturation on nuclear changes of immature canine oocytes recovered from different reproductive stages ovaries and different culture conditions. The oocytes were cultured in TCM-199 with supplement at 5% $CO_2$ and $38.5^{\circ}C$ for 72 h. The nuclear maturation of canine oocytes was evaluated with Hoechst 33342 stain under fluorescence microscope (Fig. 1). The results of this study detected differences in in vitro maturation rate between oocytes recovered from follicle status and non-follicle status ovaries. However, these differences were not significant as indicated in Table 1 and Fig. 2. In regard to the effect of culture condition with supplements, we did not found significant differences compared with control group (Table 2, Table 3). One of the reasons for this data could be the conditions that ovaries were exposed during slaughtering process or the long distant transportation of the ovaries. Although these data have not shown clearly significant differences results compared with control, furthermore the different reproductive status ovaries was beneficial for maturation of oocytes in vitro and can be a basic part of knowledge to improve in vitro maturation of canine oocytes.

• Development and Reproduction
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• v.22 no.4
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• pp.297-307
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• 2018

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and $100{\mu}M$ ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in $50{\mu}M$ ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by $50{\mu}M$ ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and $50{\mu}M$ ALA groups (p<0.05), especially, treatment of $50{\mu}M$ ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and $50{\mu}M$ ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

• The Korean Journal of Food And Nutrition
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• v.34 no.1
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• pp.79-87
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• 2021

This study was conducted in order to investigate the quality characteristics of MBA wine resulting from treatment with different oak barrel maturation methods. This study focused on the maturation of wine in five different types of barrels, including a stainless-steel maturation barrel, a foreign medium-toasted oak barrel, and domestic light, medium and heavy toasted oak barrels, and looked at the resulting differences in quality characteristics between the wines. All oak barrels used for this study had a capacity of 100 liters. The results of the study revealed that the pH content increased by up to 3.86~3.93% after 9 months, and then decreased after this point. The total anthocyanin content increased up to 152.52~174.95 mg/L during a 6 month maturation period, and thereafter began decreasing in concentration, with overall anthocyanin levels tending to be higher after maturation in foreign oak barrels. Overall, functional elements tended to measure higher after maturation in foreign oak barrels as opposed to maturation in domestic oak barrels. Therefore, these results indicate that it is necessary to improve the production of domestic oak barrels in the future in order to reliably produce wines with higher levels of functional elements.

• Korean Journal of Organic Agriculture
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• v.9 no.1
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• pp.17-27
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• 2001

To clarify the proper and safe duration of maturation periods for sawdust-pig manure composts, leaf lettuces were applied with pig manure composts fermented for 15, 30, 45, 60, 75 days and 1 year (control) and cultivated in a plastic house with or without additional PE film tunnel. The changes in physiochemical properties of soil and leaf lettuce growth were measured. Shorter duration of maturation periods enhanced the generation of N $H_3$ gas from the composts and resulted in significant decrease in seed germination, growth and yield of leaf lettuce. Under with and without PE tunnel conditions the concentration of N $H_3$ from compost over 3.8 and 2.1 mg/100g composts, respectively induced growth reduction. In proportion to the increase of maturation duration saw-dust containing pig manure exhibited decrease in C/N ratio, N $H_4^{+}$-N, N $H_4^{+}$N $O_3^{-}$ ratio but increase in N $O_3^{-}$-N contents. In case of applying pig manure compost without PE film tunnel condition the minimum maturation period of pig manure composts for safe leaf growth was 60 days, while minimum 75 days of maturation was required when applied with PE film tunnel. tunnel.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.777-784
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• 2001

Evaluation of the granulosa cell (GC) monolayers developed from small (<5 mm) and large (>5 mm) follicles on the meiotic competence of caprine oocytes during in vitro maturation was done in this study in comparison to the granulosa cell coculture. Ovaries were collected from the local abattoir and follicular contents were aspirated for the monolayer culture. For IVM the oocytes were collected by puncturing the nonatretic follicles (>4 mm). Results revealed that at the same seeding rate, small follicular granulosa cell monolayer achieved confluence 24-48 h earlier than large follicular granulosa cell monolayer. GC monolayers significantly p (<0.05) improved the rate of meiotic resumption and nuclear maturation (84.76% vs 74.74%) after 27 h of culture in comparison to GC coculture. Statistically there was no significant difference in the maturation rate between the caprine oocytes matured over small or large follicular GC monolayers. It is concluded from the present study that GC monolayers support better nuclear and cytoplasmic maturation of growing caprine oocytes which is evident by better maturation rate over GC monolayer as compared to the oocytes matured with GC coculture. Granulosa cells from small and large follicles can be used for IVM with more or less in the same efficiency after conditioning them with maturation media in 18-24 h before the onset of culture.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.21-25
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• 2010

The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% $CO_2$, 95% air, $38^{\circ}C$. The in vitro maturation rate to MII stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44~72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.139-146
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• 2017

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.

• Korean Journal of Animal Reproduction
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• v.14 no.4
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• pp.263-271
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• 1990

This study was carried out to investigate the factors affecting maturation in vitro of follicular oocytes in Korean Native Cattle. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follicles of 3~6mm. The bovine oocytes were matured in vitro in TCM-199 containing FCS and hormones. The effects of TCM-199 salt type, number of oocytes per drop, incubation time and co-culture with granulosa cells on maturation of oocytes, were investigated. The results obtained are summarized as follows. 1. The maturation rates of follicular oocytes cultured for 22, 25 and 28 hours in Hank's TCM-199 or Earle's TCM-199 were 59.3, 59.6, 80% and 80.0, 90.0, 93.7%, respectively. The maturation rate of follicular oocytes in Earle's TCM-199 was faster and higher than in Hank's TCM-199(P<0.05). 2. The maturation rates of oocytes were 54.5, 62.5 and 62.0% when cultured the oocytes number 10, 20 and 40 per 200${mu}ell$ drop for 18 hours. 3. The maturation of follicular oocytes in the Korean Native Cattle was induced at 14 hours culture, by giving the maturation rate of 90.0% after 20 hours. 4. The maturation rates were 63.3% and 66.7%, respectigely when the oocytes were co-cultured with granulosa cells from medium-size follicles used immediately after recovery in the presence or absence of hormones added(0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH and 1$\mu\textrm{g}$/ml estradiol 17-$\beta$). When the oocytes were co-cultured with granulosa cells from medium-size follicles cultured for 3 days, the maturation rates were 20.8% and 76.2%, respectively(P<0.05). 5. The maturation rate were 88.0% and 70.0%, respectively when the oocytes were co-cultured with granulosa cells from large-size follicles used immediately after recovery in the presence or absence of hormones added(0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH and 1$\mu\textrm{g}$/ml estradiol 17-$\beta$)(P<0.05). When the hormones added(0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH and 1$\mu\textrm{g}$/ml estradiol 17-$\beta$)(P<0.05). When the oocytes were co-cultred with granulosa cells from large-size follicles cultured for 3 days, the maturation rates were 41.2% and 73.3%, respectively(P<0.05).

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.1
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• pp.10-19
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• 2019

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: $79.9{\pm}3.8%$ vs G2: $57.5{\pm}4.6%$) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO ($0.1{\mu}M$, MT) treatment (G2: $68.4{\pm}3.2%$ vs G2 + MT: $73.9{\pm}1.4%$). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.53-53
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• 2002

This study was carried out to verify the incidence of oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughter and then divided into five groups: control group(unvitrified oocytes), 0 hr. group(composed of oocytes vitrified before the onset of maturation) and 10, 14, and 20 hrs groups (vitrified respectively at 10, 14 and 20 hrs after the onset of maturation). (omitted)