• Asian-Australasian Journal of Animal Sciences
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• v.31 no.9
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• pp.1420-1430
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• 2018

Objective: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. Methods: Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and $200{\mu}M$), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. Results: Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the $50{\mu}M$ EGCG-treated group compared with the control group. Adding $50{\mu}M$ EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the $50{\mu}M$ EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the $50{\mu}M$ EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the $50{\mu}M$ EGCG-treated oocytes. Conclusion: In conclusion, $50{\mu}M$ EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property.

• Ocean and Polar Research
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• v.31 no.3
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• pp.231-238
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• 2009

A study was carried out to investigate the effect of water temperature on daily pattern and rate of total ammonia nitrogen (TAN) excretion in juvenile dark-banded rockfish Sebastes inermis (mean body weight: $14.8{\pm}0.3g$) under fasting and feeding conditions. Fish were acclimated over 10 days under three different water temperatures (15, 20 and $25^{\circ}C$). After 72 hours of starvation, fasting TAN excretion was measured at each temperature. To investigate post-prandial TAN excretion, fish were hand-fed with a commercial diet containing 47.7% crude protein for 7 days, two times daily at 09:00 and 17:00 hr. Water was sampled from both the inlet and outlet of each chamber every 2 hrs over a 24 hr period. Both fasting and post-prandial TAN excretion increased with increase in water temperature (P<0.05). Mean fasting TAN excretion rates at 15, 20 and $25^{\circ}C$ were 8.1, 9.0 and 9.2 mg TAN kg $fish^{-1}h^{-1}$, respectively. The value of $15^{\circ}C$ was lower than those of 20 and $25^{\circ}C$ (P<0.05), but there was no significant difference between $20^{\circ}C$ and $25^{\circ}C$ (P>0.05). Mean post-prandial TAN excretion rates at 15, 20 and $25^{\circ}C$ were 20.1, 22.9 and 23.4 mg TAN kg $fish^{-1}h^{-1}$, respectively. A peak post-prandial TAN excretion rate occurred after 12 hrs from the first feeding at $15^{\circ}C$ (mean 28.7 mg TAN kg $fish^{-1}h^{-1}$), $20^{\circ}C$ (33.7 mg TAN kg $fish^{-1}h{-1}$) and $25^{\circ}C$ (36.8 mg TAN kg $fish^{-1}h{-1}$), respectively. The TAN loss for ingested nitrogen at $15^{\circ}C$ (36.2%) was lower than that of $20^{\circ}C$ (40.8%) and $25^{\circ}C$ (41.7%). Based on overall results, water temperature exerts a profound influence on the nitrogen metabolism of juvenile dark-banded rockfish.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.11-19
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• 1998

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 $\mu$M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 $\mu$sec) in 0.3 M mannitol solution supplemented with 100 $\mu$M CaCl$_2$ and MgCl$_2$. The nuclear donor embryos of 8-cell stage were synchronized to G$_1$ phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

• Journal of Life Science
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• v.20 no.11
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• pp.1634-1639
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• 2010

This study was conducted to increase the efficiency of farming practice in rainbow trout, Oncorhynchus mykiss, by sex reversal and chromosome-set manipulation techniques. To obtain phenotypic males, hormonal sex reversal was carried out using an exogenous hormone treatment method. 5 mg of 17 alpha-methyltestosterone per kg diet was supplied for 82 days after first feeding at $10^{\circ}C$ and $13^{\circ}C$. More than 93% of the male population was produced by this method and growth of hormone-treated fish at $13^{\circ}C$ was faster than that of untreated bi-sexual groups. Induced diploid gynogenesis was carried out using artificial insemination of UV-irradiated sperm into haploid eggs. Based on the appearance of the rate of haploid syndrome and survival of embryo, a UV ray dose of at least $3,600\;erg/cm^2$ was required to inactivate rainbow trout sperm genetically. Haploid embryos were restored to diploid by blocking the extrusion of the second polar body using heat shock treatment at $28^{\circ}C$ for 20 min, 10 min post insemination. Gynogenetic diploid sex ratios were confirmed after maturation of the fish erythrocyte measurements and chromosome counts.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.11-20
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• 1997

The influence of cryopreservation of donor embryos on the in vitro developmental potential in the nuclear transplant rabbit embryos was evaluated. The embryos of 16-cell stage were collected and cryopreserved with EFS solution by vitrification method. The frozen embryos were thawed and synchronized to S and G$_1$ phase of 32-cell stage. The recipient/ cytoplasms were obtained by removing the first polar body and chromosome mass from the oocytes collected by non-disruptive microsurgery procedure. The separated S and G$_1$ phase blastomeres of 32-cell stage were injected into enucleated recipient cytoplasms by micromanipulation. After culture until 20 hrs post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. The fused nuclear transplant embryos were co-cultured with rabbit oviduct epithelial cells. After in vitro culture for 120 hrs, the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. The electrofusion rate was significantly (P<0.05) reduced in the frozen nuclear donor,compared with fresh donor nuclei as 80.0 vs 62.8% in S phase and 81.7 vs 64.8% in G$_1$phase, respectivley. The in vitro developmental rate to blastocyst stage with the S and G$_1$phase of fresh embryos(26.3 and 61.1%, respectively) was found significantly (P<0.05) higher, compared to the S and G]phase of frozen embryos(11.9 and 34.6%, respectively). When frozen as well as fresh donor embryos were synchronized to G$_1$ phase, the in vitro developmental rate to blastocyst stage was significantly (P<0.05) higher, compared with S phase donor nuclei. The cell counts of nuclear transplant embryos developed to blastosyst stage were significantly (P<0.05) more in G$_1$ phase of fresh or frozen embryos (180.1 and 125.7 cells, respectively), compared with S phase nuclear donor (145.1 and 103.7 cells, respectively). From the above results it was concluded that the rabbit embryos cryo- preserved by vitrification might be available as nuclear donor, though the developmentalpotential and cell counts of nuclear transplant rabbit embryos were decreased significantly.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.62-62
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• 2003

This study was carried out to investigate the effects of activation agents on parthenogenetic activation of pig oocytes matured in vitro. The medium used for oocyte maturation was tissue culture medium (TCM) 199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin $B_{l2}$, 25 mM Hepes, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Cumulus-free oocytes involving first polar body were activated by exposure to various concentrations of ethanol and exposure time of ethanol in Hepes-buffered NCSU23 medium. Also, oocytes were activated by electric pulse alone or combination with ethanol. For electrical activation, oocytes were rinsed twice in 0.3 M mannitol solution supplemented with 0.1 mM CaC1$_2$, 0.2 mM MgC1$_2$, 0.5 mM Hopes and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1 mm apart which was overlaid with the same activation solution. Oocytes were activated with a single DC pulse of 1.3 ㎸/cm for 30 $\mu$sec. After activation treatments, oocytes were washed three times with Hepes-buffered NCSU23 medium and were washed twice with NCSU23 culture medium containing 0.4% BSA, and then cultured in 500 ${mu}ell$ of the same medium for 20 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. The activation rates of oocytes were higher in 6, 7 and 8% ethanol concentrations compared with 0, 5, 9 and 10% ethanol concentrations. Significantly more oocytes (29.3~33.7%) were activated in the exposure for 8, 10, 12 and 15 min than those in the exposure for 0 and 5 min, but there was no difference due to exposure to 8% ethanol for 8 to 15 min. Electric pulse treatment followed by exposure to ethanol significantly improved the rate of oocyte activation (61.9%) compared with that of other 3 treatments. In conclusion, the optimal activation treatment of ethanol exposure alone for the in-vitro matured pig oocytes was 8% ethanol for 8 to 15 min. Electric pulse treatment followed by ethanol exposure significantly improved the rate of activation.n.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.133-139
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• 1999

This study was carried out to investigate in vitro/in vivo development of vitrified-thawed immature mouse oocytes. Immature mouse oocytes were vitrified with EFS40 (40% ethylene glycol, 18% ficoll and 0.5 M sucrose). Thawed oocytes were matured for 16 hr in vitro. Matured oocytes with the first polar body were fertilized with the concentration of 1~2$\times$10$^{6}$ $m\ell$ of epididymal sperm. After fertilization, cleavage ($\geq$ 2-cell) and in vitro/in vivo development rates were examined. $\pi$ Ie results were summarized as follows: in vitro maturation rate of immature mouse oocytes in vitrified-thawed group was similar to that in exposed group (67.5%) and control (66.3%), but cleavage rate of vitrified-thawed oocytes (64.9 %) and blastocyst formation rate (59.0%) were significantly different compared to those of exposed group (83.7 and 74.7%) and control (90.7 and 83.7%) (p＜0.05). However, when the blastocysts derived from immature mouse oocytes vitrified-thawed were transferred to pseudopregnant mouse, total implantation (31.3%) was slightly lower than that in control (40.8%), but live fetus formation rate (66.7%)was slightly higher than that in control (58.1%), there was not significantly different. Therefore, when the blastocyts produced in vitro were transferred into recipients, although the development in vitro of oocytes vitrified-thawed was decreased, live fetus formation rate was similar to that of control group. The present results indicate that immature mouse oocytes can be frozen successfully by vitrification with EFS40.

• Parasites, Hosts and Diseases
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• v.31 no.1
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• pp.57-66
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• 1993

The potential of fumagillin dicyclohexylamine salt to treat and prevent intestinal giant-cystic disease in Israel carp, Cyprinus carpio nudus, was monitored in field experimental studies. In experiment 1 (therapeutic), most fish were already naturally infected with more advanced stage of Relohqnellu: kitnuet. Fumagillin was administered to ash (mean body weiht of 830 g for a Penod of one month at a dose of 10.62 mg in the first group and 5.3 mg in the second group per fi sh per day. In experiment 2 (fprophylactlcl), most flesh also were already naturally infected with an early developmental stage of the protozoa and fish (average body weight of 484 g) were administered fumagillln for 45 days at a dose of 3.95 mg per fish per day. In both experiments, the cumulative mortalities of fish and the extrusion rates of the polar filaments of the spores were significantly decreased in a dose-independent fashion. In experiment 2 no dead fish were observed. No adverse side effects of the drug were observed among fish from any dosage group. In experiment 2, an oval or dot-like concave lesion of most cysts developed at the 7th day and the vegetative form was never observed at the 17th day postmedication and the cysts were grossly reduced in size as compared with the control group, beginning at the 24th day until the end of the study. In contrast, it was scarcely effective to the cysts in experiment 1. Taking the seasonal development of the protozoa into consideration, the above results revealed that oral administration of fumagillin at 3.95 mg/500 g body weight/day for a month Is the optimal dose for the treatment and prevention of thelohanellosls caused by T kitnuei among C. carpio nudus.

• Journal of Aquaculture
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• v.11 no.3
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• pp.371-380
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• 1998

This study has been conducted to develop the techniques for spat production of the sun and moon scallop from January 1995 to December 1996. With the adult scallops collected from the Sogwipo area, spawning induction and larvae rearing were attempted several times and monthly changes of GSI were also monitored during the experimental period. The results obtained wre as follows. 1. GSI started to increase from June and showed the maximum value of 22.17 and 14.98 in female and male respectively in November, and then gradually decreased from December. 2. Spawning induction by heating method turned out to the most efficient way showing the responding rate of 64.8~91.5%. The responding temperature was $21.4~26.4{\circ}C$ which was $3.1~8.5{\circ}C$ increased from the rearing temperature of $16.3~18.3{\circ}C$. An average number of eggs spawned was $9.2{\times}10^5$ 3. the average size of eggs after fertilization was about $72{\mu}m$ in diameter. The first polar body discharge, blastula formation, and trochopore larvae appearance occurred 30 mininutes, 18 hours, and 22 hours after fertilization respectively. 4. Settling rates in various collectors were similar one another, whereas pouring larvae in the mesh was the most efficient way for larval setting. 5. The spates grew to be 1mm in their shell length for the first 50 days after fertilization and 9.6mm in 135days. 6. Correlation between shell length (SL) of the spat and the number of days (X) after spat settlement could be expressed as $SL=257.75e ^{0.0272x}$(r=0.9100).

• Parasites, Hosts and Diseases
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• v.28 no.3
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• pp.183-194
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• 1990

In an attempt to develop prophylactic and therapeutic measures of the intestinal giant-cystic disease caused by Thelohanellus kitauei in the Israel carp, Cyprinus carpio nodus, pathological observations were conducted upon the carps which were hatched in May 1988 and raised in a net cage fish farm at the Soyang lake, managed by Horim Fisheries for the period of 21 months with 1~2 months interval. After a gross inspection of the carps, necropsy was carried out periodically in order to clarify the pathological changes in various internal organs and muscular tissues. Also. the prevalence of the disease was checked during the period from 1988 to 1990. Gross inspections revealed that the infected carps showed some degree of fading in body and gill color, back-emaciation symptoms, reddish anus accompanying erosion and relaxation and pot-belly, as well as discharge of yellowish white mucoid material from the anus. However, most carps died eventually of intestinal obstruction. Other significant necropsy fadings included cyst formation of various size in the intestinal mucosa, ascites, anemic condition through internal organs and muscular tissues, hyperemia and dilation of intestines with decreased tension, thinness and fragility, and full contents of semi-fluid or yellowish white mucoid material in the intestinal canals. Based on the morphological characteristics of the spores found in the cysts, parasitic location in the intestines, macro- and microscopic findings of the lesions, the parasites were identised as Thelohanellus kitauei Egusa os Nakajima, 1981. Although monthly changes of water temperature were distinct, the extrusion rates of the polar filaments of the spores stayed constant throughout the year with an exception of a lower rate in July, The lesions initiated from mucosa and submucosa in early July became large swellings and then complete mature (orms following the peracute course. From late August the upper cysts were gradually opened and most of the spores were dispersed from anus into the surrounding water through December but only a few lasted until next April. The cysts were completely recovered until next September. Comparing the incidence and prevalence of the disease by year tremendous infection and death rates were checked in the first prevalent year, 1988, but the rates were significantly decreased in the second year, and showed an almost normal status in the third year, 1990. As the above summarized results showed, the disease entity might come to an end in three years after the first prevalent year, however, the spores must be strictly prevented because they could be infective in the water for one year.