• Journal of Embryo Transfer
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• v.15 no.3
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• pp.271-277
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• 2000

This study was conducted to establish the optimal culture conditions for in vitro production of bovine embryos derived from slaughter house ovaries. Cumulus-oocyte- complexes (COCs) collected by aspiration from follicles of 2~7 mm in diameter were matured in Ham's F-10 medium supplemented with 0.01 $\mu\textrm{g}$/m1 epidermal growth factor (EGF) at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. After 24 hrs of culture, the oocytes were co-cultured with epididymal sperm selected off by Percoll-density gradient in TALP medium for 24 hrs. The presumptive zygotes were cultured in HECM-6 medium for 3 d post-insemination, and followed by cultured in TCM199 medium until 7 to 10d post-insemination. The cultures were compared of their cleavage and development into later stage in culture medium by additions of different protein sources (PVA, BSA and BCS) and by different embryo density. The rates of cleavage and development rates into blastocyst were not significantly (P<0.05) different among the culture media containing with BSA (75.0% and 40.5%), BCS (76.7% and 38.0%) and PVA (72.5% and 42.2%), respectively. Significantly (P<0.05) higher blastocysts rates were obtained in culturing of 30 and 40 embryos in each 50$\mu$l droplets of culture medium than in 5, 10 and 20 embryos. These results indicate that the optimal density of embryos is 30~40 embryos in a 50$\mu$l droplet of culture medium. Furthermore there is no effect of different protein sources on early embryonic development.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.42-42
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• 2001

The objective of this study was to assess the development of porcine follicular oocytes fertilized by ICSI. Cumulus-oocyte-complexes (COCs) were collected by aspiration from follicles of 2-7 mm in diameter from a local slaughterhouse ovaries. Oocytes matured for 40-44 h were centrifuged at 12,000g for 6 min and then injected with sperm prepared by swim-up procedure in the presence or absence of 5 mM dithiothreitol (DTT). Injected oocytes were cultured in NCSU 23 medium during 6 to 8 days. IVF controls were compared to those of resulting embryos. The results obtained were as. follow: 1, The rates of cleavage and development rates into blastocyst by ICSI were not significantly (P<0.05) different between with (53.0% and 19.7%) or without (48.3% and 23.8%) centrifugation, respectively. 2. The cleavage and developmental rates to blastocyst after ICSI with or without 5mM DTT treated-sperm were not significantly (P<0.05) different (60.4% vs 16.4% and 48.5% vs 22.2%, respectively). 3. The cleavage and the developmental rates to blastocyst were not significantly (P<0.05) different between the zygotes obtained by IVF (51.8% vs. 22.4%) and ICSI (51.4% vs. 21.6%). 4. The number of blastomere in blastocyst stages after IVF or ICSI was not significantly different (46.7 $\pm$2.9 and 41.9$\pm$4.6).

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.5
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• pp.629-634
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• 2012

This study was conducted to improve the yield of mature oocytes from in vitro-culture of ovarian primary follicles by optimizing follicle retrieval from neonatal mice of different ages. Primary follicles of 75 to $99{\mu}m$ in diameter were collected daily from 7- to 14-day-old neonatal mice, and subsequently cultured in ${\alpha}$-MEM medium. Number of primary follicles isolated, growth of the follicle during in vitro-culture and maturation of intrafollicular oocytes were monitored. Overall, mean number of preantral follicles per animal was improved from 10.7 to 88.7 as the age of follicle donors was increased from 7 to 14-day-old. Number of primary follicles was increased gradually up to 11-day-old (35.7 follicle per an animal), then reduced to 29 in 14-day-old (p = 0.0013). More follicles retrieved from 10-day-old or 11-day-old females maintained their morphological normality at the end of primary culture than the follicles retrieved from 9-day-old. Of those cultured, primary follicles retrieved from 11-day-old mice yielded largest larger number of early secondary follicles than the follicles retrieved from in the other ages (39 vs. 13 to 29%). More than 3.3-times increase (0.86 to 2.86; p<0.05) in an average number of mature oocytes per animal was observed in the group of 11-day-old, compared with 9-day-old. However, no difference was found in the percentage of primary follicles developing into the pseudoantral stage (21 to 30%; p = 0.5222) and in the percentage of oocytes mucified (32 to 39%; p = 0.5792). In conclusion, a positive correlation between retrieval time and follicle growth was detected, which influences the efficiency to derive mature oocytes by follicle culture.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.3
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• pp.287-295
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• 2017

Egg development and morphological change of larvae of the Sakhalin sole Limanda sakhalinensis were studied by observing specimens obtained in a rearing experiment from fertilized eggs to the juvenile stage. The wild broodstock was collected in January 2010 and kept in a circular water tank (${\O}1.5{\times}1m$) at a temperature of $14.5{\pm}0.5^{\circ}C$. Fertilized eggs ranged from 0.72 to 0.82 mm ($0.77{\pm}0.07mm$, $mean{\pm}SD$) in diameter. The eggs were spherical, transparent and adhesive demersal. The egg yolk was divided from the oocyte 10 min after fertilization (AF), and an embryo was formed in 36 h AF. More than 50% of the eggs hatched within 133 h AF. The mouth and anus did not open until $3.5{\pm}0.25mm$ total length (TL). At 4, days after hatching (AH), the fish became larvae 3.7 to 4.2 mm ($4.0{\pm}0.36mm\;TL$), yolk absorption was completed and the mouth began to open. The left eye moved upward and the nostril moved to the right at 39 days AH. These post-larvae ranged from 8.0 to 9.9 mm TL ($8.9{\pm}1.33mm\;TL$). At 50 days AH, the fish became juveniles ($12.4{\pm}1.20mm\;TL$) There were 70-72 dorsal fin rays, 55-56 anal fin rays, 11 pectoral fin rays, and 6 ventral fin rays and the juveniles adopted a benthic life.

• Clinical and Experimental Reproductive Medicine
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• v.15 no.1
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• pp.25-34
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• 1988

The intrafo1licular echoes of cumulus oophoruses within ovarian follicles were assessed with the use of ultrasound in 86 women taking part in an in vitro fertilization(IVF) or gamete intrafallopian transfer(GIFT) program, stimulated with pure follicle-stimulating hormone(FSH)/human menopausal gonadotropin(hMG)/human chorionic gonadotropin (hCG). When intrafo1licular echoes were clearly separated from the follicular wall or relatively dispersed within the follicle, they were considered to be a dissociated cumulus, and when they were only slightly prominent from the follicular wall, they were suspected to be a nondissociated cumulus. A cumulus was seen in 62.1% of the follicles larger than 10 mm diameter and 75.1% of them were dissociated. The larger the follicles in size, the more the cumuluses in number and dissociation. The number of follicles and intrafollicular echoes per woman was not different whether or not she would be pregnant, but the number of dissociated cumuluses was significantly more in pregnant women. The number of observed dissociated cumuluses correlated significantly with the number of recovered mature oocytes. When an intrafollicular echo is seen, it can be taken as evidence of a sign of maturity of that particular follicle and oocyte. Ultrasonographic monitoring of intrafollicular echoes and follicular size is very helpful to predict follicular maturation in ovulation stimulation cycles.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.5
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• pp.632-636
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• 2005

This study was conducted to investigate the effects of removal of all ovarian follicles through repeated transvaginal follicle aspiration (TVFA) on the subsequent follicular populations in buffaloes. This information is crucial for determining the optimum time interval between successive aspirations for recovering oocytes from live buffaloes through Transvaginal Oocyte Retrieval (TVOR). The oestrus of cycling buffaloes (n=5) were synchronized by a single PGF injection schedule. All the ovarian follicles were removed once every 7 days for 6 weeks through TVFA, starting from Day 7 of the oestrous cycle (Day 0 = day of oestrus). The number and size of individual ovarian follicles was recorded at Day 3 and Day 5 (Day 0 = day of TVFA) through transrectal ultrasonography. The follicles were classified on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large ($\geq$10 mm). There was no difference in the number of small and medium follicles, and the number of total follicles between Day 3 and Day 5. However, the number of large follicles was significantly higher (p<0.05) at Day 5 than that at Day 3. There was a significant (p<0.05) decrease in the proportion of small follicles and an increase (p<0.05) in the proportion of large follicles from Day 3 to Day 5, with no change in the proportion of medium follicles. The number of total follicles at Day 3 or Day 5 did not differ during the 6 TVFA sessions. It can be concluded that an interval of 3 days is more suitable than that of 5 days between successive aspirations for recovering oocytes through TVOR in a twice weekly schedule and that repeated removal of follicles through TVFA does not adversely affect the number of total follicles 3 or 5 days after TVFA.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.2
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• pp.83-89
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• 2013

Objective: To investigate outcomes of stimulated IVF cycles in which GnRH antagonist was omitted on the ovulation triggering day. Methods: A total of 86 women who underwent controlled ovarian hyperstimulation with recombinant FSH and GnRH antagonist flexible multiple-dose protocols were recruited and prospectively randomized into the conventional group (group A) or cessation group (group B). The GnRH antagonist, 0.25 mg/day of cetrorelix, was started when the leading follicle reached 14 mm in diameter and was continuously administered until the hCG triggering day (group A, 43 cycles) or until the day before hCG administration (group B, 43 cycles). The maturity of oocytes, fertilization rate, embryo quality, and implantation and clinical pregnancy rates were evaluated. Results: The duration of ovarian stimulation, total dose of gonadotropins, serum estradiol levels on hCG administration day, and number of oocytes retrieved were not significantly different between the two groups. The total dose of GnRH antagonist was significantly lower in group B than group A ($2.5{\pm}0.9$ vs. $3.2{\pm}0.8$ ampoules, p<0.05). There was no premature luteinization in any of the subjects. The proportion of mature oocytes and fertilization rate were not significantly different in group B than group A (70.7% vs. 66.7%; 71.1% vs. 66.4%, respectively). There were no significant differences in the implantation or clinical pregnancy rates. Conclusion: Our prospective randomized study suggested that cessation of GnRH antagonist on the hCG administration day during a flexible multiple-dose protocol could reduce the total dose of GnRH antagonist without compromising its effects on pregnancy rates.

• Ocean and Polar Research
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• v.38 no.1
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• pp.21-33
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• 2016

Gametogenesis of the comb pen shell, Atrina pectinata (Linnaeus, 1767) (Bivalvia: Pinnidae) on the southern coast of Ulleungdo Island, Korea was assessed monthly (November 2013 to October 2014) using histology. Gametogenesis commenced in January when the surface water temperature was $12.6^{\circ}C$ and pen shells evidenced an early development phase with small oogonia from January to April, although few females exhibited ripe eggs in their follicular epithelium. In April, the oocyte diameter increased rapidly, and fully mature eggs appeared in May. First spawning males and females were observed in June as the surface water temperature reached $19.3^{\circ}C$ and July ($23.2^{\circ}C$) respectively. The spawning activity continued until the end of September. Histology indicated that the spawning peak of the females in Ulleungdo Island was July to August. During October to January, most of the pen shells were in spent and resting stages. Our data suggested that A. pectinata is a summer spawner, and their annual gametogenesis is closely associated with the seasonal variation in the surface water temperature. The present study is the first provided fundamental information on the life history of A. pectinata in Ulleungdo Island, and this can be put to good use in the management of this pen shell in the study area.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.63-70
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• 2009

Objective: This study was performed to investigate the effectiveness of minimal stimulation using rhFSH and GnRH antagonist compared with GnRH antagonist multidose protocol (MDP) in IVF treated patients with aged 40 and above. Methods: Seventy-five patients with aged 40 and above were equally randomized to minimal stimulation group (n=37) or GnRH antagonist MDP group (n=38). For minimal stimulation group, ultrasound monitoring was started on cycle day 7 or 8. Daily injections of 0.25 mg cetrorelix together with 150 IU rhFSH were started from the day at 13${\sim}$14 mm of a leading follicle diameter. For GnRH antagonist MDP group, daily injections of 225 IU rhFSH were initiated from cycle day 2 and GnRH antagonist was started at a dose of 0.25 mg/day on rhFSH stimulation day 6 or the day at 13${\sim}$14 mm of leading follicle diameter. In both groups, transvaginal ultrasound-guided oocyte retrieval was performed. According to cleavage and morphologic characteristics of embryos, embryos were transferred 3 to 5 days after oocyte retrieval. Results: There were no differences in patients' characteristics and cycle cancellation rate between the two groups. Total dose and duration of rhFSH used were significantly fewer and shorter in minimal stimulation group than those in GnRH antagonist MDP group. The numbers of oocytes retrieved, mature oocytes and transferred embryos were also lower in minimal stimulation group. However, there were no significant differences in the clinical pregnancy rate and miscarriage rate between the two groups. Conclusions: This study demonstrates that minimal stimulation protocol provides comparable pregnancy rates to GnRH antagonist MDP with fewer dose and days of rhFSH used, and thus can be a cost-effective alternative in women aged 40 and above.

• Journal of Aquaculture
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• v.10 no.2
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• pp.133-141
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• 1997

Gonadal maturation and annual reproductive cycle of the cultured scallop, Patinopecten yessoensis from eastern waters near of Kangwon-do province, Korea were studied on the basis of monthly variation of gonadosomatic indices (GSI) and histological observations of gonadal tissue. During the experimental period, water temperature at the depth of 20m and sunshine duration per day were ranged from $5.3^{\circ}C\;to\;18.0^{\circ}C$ and 9.4 to 14.6 hours, respectively. GSI values of femal were in a wide range from $2.8\pm0.37(August)\;to\;22.66\pm4.38(April)$. GSI values began to increase in March and reached the maxium in April, then decreased repidly. GSI values of male were in a range from $2.04\pm0.80(August)\;to\;20.46\pm1.49(April)$ and were same tendency with female's. Digestive diverticula indices (DDI) of both sex reached the maximum values in December, then deceased gradually until September. Contrary to GSI, adductor muscle indices (AMI) of both sex were the minium values in April, but began to increase rapidly until July and reached $47.71\pm3.17(female)\;and\;48.70\pm3.55(male)$. In the scallp collected hermaphroditic gonads were found. Monthly changes of oocyte diameter were in a range from 11.3 um(October) to 73.3um (April) and nuclear diameters were in a range from 8.3um (September) to 35.3um (April), similar tendency with each other. The changes in number of ovarian tubules were reciprocal tendency with those of oocyte diameter and monthly number of ovarian tubules per $\textrm{mm}^2$ in the tissue perpatation was in a range from 51 (April) to 175 (August). As the results, the annual reproductive cycle of the cultured scallolp from eastern waters of Korea could be classified into five successive stages : multiplicative (October), growing (November to February), mature (March and April), spawning (April to June) and recovery (July to September) in female ; multiplicative (October and November), growing (December and January), mature (February to April), spawning (April to June) and recovery (July to September) in male.