• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.128-128
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• 2003

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.121-129
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• 1999

The objective of this study was to produce calves by transfer of embryos derived from slaughter house(SHD) and ultrasound-guided ovum pick-up (OPU). At 60 hrs after injection of 400 mg FSH dissolved in 25% polyvinylpyrrolidone(PVP) by single dose, ultrasound-guided follicular oocyte aspiration was ferformed. Day-7 and day-8 blastocysts produced by in vitro maturation (IVM), fertilization (IVF) and culture(IVC) of the oocytes derived from SHD and OPU were nonsurgically transferred into recipients. The results obtained were as follows. The cleavage rate and the development rate to blastocysts were not significantly (P<0.05) different between the oocytes obtained by SHD (72.9% vs. 34.1%) and OPU (75.9% vs. 38.4%). The oocyte recovery rate from the number of follicles by ultrasound-guided aspiration were not significantly (P<0.05) different between Holstein (61.7%) and Hanwoo (60.1%), but the rate of oocytes useful for IVF was significantly (P<0.05) higher in Hanwoo (69.3%) than Holstein (59.6%). The cleavage rate and the development rate to blastocysts was not significantly (P<0.05) different between Holstein (74.9% vs. 39.2%) and recipients on day 8 of estrus cycle resulted in 13 pregnancies (34.2%). One of them was sacrificed during gestation period due to mastitis and another was aborted spontaneous. The resulting 14 calves were morphologically normal at birth. Seventy eight fresh OPU-IVF embryos were transferred into 21 recipients on day 8 of estrus cycles, resulting in pregnancy of 12 recipients (41.4%). Two of them were sacrificed during gestation period due to mastitis and the other two were aborted. Nevertheless, the 11 OPU-calves have been born normally.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.149-157
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• 1993

A new technique was established to maximize the numbers of follicular oocytes recovered from the ovaries obtained at the slaughter house. And their further developmental capacity was demonstrated. There recovery techniques were used; aspiration (ASP, control), slicing (SLC) and slicing combining aspiration (ASP+SLC). Recovered oocytes were cultured in TCM 199+15% FCS+gonadotrophins in an atmosphere of 5% CO$_2$ in air at 39$^{\circ}C$ for 24 h. The nuclear maturation was detemined with chromo-some configuration by rapid staining. And cytoplasmic maturation was examined by the formation of female pronuclei with parthenogenetic activation of the matured oocyte after 18 h of co-culture with granulosa cell monolayer. Total 1,641 bovine follicular oocytes recovered from 245 ovaries. The number of oocytcs per ovary was 1.87 in ASP, 11.05 in SLC and 7.88 in ASP+SLC, respectively. SLC would yield 5.9 folds increase, compared with ASP. The rate of maturation were 92.9% in ASP, 79.1% in SLC and 71.7% in ASP+SLC, respectively. Although the maturation rate in ASP was the highest, metaphase II oocytes per ovary in SLC was 5 times higher than that of ASP. The rates of pronuclei formation upon ethanol activation were 75% in ASP, 67% in SLC and 62.5% in ASP+SLC, respectively. The results demonstrate that it should be possible to maximize the number of the follicular oocyte from the ovary for mass production of bovine embryos. Thus the established technique may provide efficient supply of bovine embryos for biochemical and molecular study of early bovine embryos.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.165-176
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• 1994

This study was carried out to investigate the effect of the timing and dose of human chorionic gonadotropin(hCG) on oocyte recovery, in vitro fertilization, and preimplantation development in superovulation of mouse. F1 hybrid($C57BL{\times}CBA$) mice were obtained and superovulation was induced in female mice by sequential intraperitoneal injection of PMSG and hCG. In the first series of experiments, mice received 5 IU of PMSG given intraperitoneally, and 48 hours later were injected 1 IU, 5 IU, or 10 IU of hCG respectively. In the second series of experiments, mice received 5 IU of PMSG given intraperitoneally and were injected 5IU of hCG 36, 48, or 60 hours later respectively. 1. When the mice received 5 IU of PMSG given intraperitoneally and 48 hours later were injected 1 ItT, 5 IU, or 10 IU of hCG respectively, there were no differences in the total number of the oocytes obtained from the three experimental groups. When the cultures were examined 48 hrs after the termination of insemination the proportion of unfragmented oocytes which had developed over two-cell stage was observer to be lowest in 10 IU hCG group. When the cultrues were examined 120 hour after termination of insemination the proportion of embryos which had developed to the blastocyst stage was observed to be significantly higher in 10IU hCG group than 5IU hCG group(p<0.05), but there was no difference between 10 IU hCG group and 1IU hCG group. 2. When the mice received 5 IU of PMSG and were injected 5 IU of hCG 36, 48, or 60 hours later respectively, there were no differences in the total number of oocytes obtained from the three experimental groups. When cultures were examined 48 hour after the termination of insemination the proportion of unfragmented oocytes which had developed over two-cell stage was observed to be significantly lower in 36 hour interval group than 48 hour interval and 60 hour interval group(p<0.05). When the cultures were examined 120 hour after termination of insemination the proportion of embryos which had developed to the blastocyst stage was found to be higher in 60 hour interval group than 36 interval or 48 hour interval group (P<0.05), and the proportion of hatching blastocyst was found to be higher in 60 hour interval group as well. In this study, it was concluded that the administration of adequate dose of hCG, and long (60 hour) PMSG-hCG interval were necessary in superovulation of mice($C57BL{\times}CBA$) in order to get a large number of oocytes which had an early oocytes which had an early embryonic developmental capability when fertilized in vitro, and especially it had better have been avoided to administer a large dose of hCG.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.147-152
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• 2011

This study was conducted to examine whether efficiency of oocyte production from superovulated prepubertal goats. Fifteen prepubertal and twenty adult goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen implanted CIDR for 10 days. Superovulation treatment of the goats received twice daily intramuscular injection of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotrophin treated goats were injected with 10 mg $PGF_2{\alpha}$ on Day 8 and 400~600 IU hCG in the afternoon on Day 10. Oocytes were recovered by follicle aspiration or oviduct flushing at 35 to 40 h after hCG injection through mid-ventral incision. The in vivo matured oocytes was activated by ionomycin (5 min) and 6-DMAP (3.5~4 h). The activated oocytes were cultured in mSOF medium containing 0.8% BSA at 38.5$^{\circ}C$ in an atmosphere of 5% CO$_2$, 5% O$_2$, 90% N$_2$ for 7~8 days. There was no significant difference in the mean number of CL and in vivo matured and follicular oocytes recovered. But, quality of I + II grade follicular oocytes was lower (p<0.05) in the prepubertal goat (25.0%) than the adults (52.4%). The same results were also observed in the cleavage and blastocyst rate of activated oocytcs. The cleavage and blastocyst rate from prepubertal derived oocytes were lower (p<0.05) in the prepubertal goat (54.5%, 23.3%) than the adult goat (86.8%, 46.6%). Considering overall these results, we suggest that maturation of donor goats is a major factor affecting recovered oocytes quality and in vitro development of activated goat oocytes.

• Animal cells and systems
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• v.5 no.1
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• pp.37-44
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• 2001

Gonadal development, reproductive cycle, gonad index, meat weight rate, and first sexual maturity of the top shell, Omphalius rusticus were Investigated monthly by histological observations. Specimens were collected from the west coast of Korea during the period from January to December in 1999. O. rusticus is dioecious and oviparous. The gonad is widely situated on the surface of the digestive g1and located in the posterior spiral meat part in the shell. The ovary and the testis were composed of a number of oogenic follicles and several spermatogenic follicles, respectively. Ripe oocytes were approximately 120-130 $\mu$m in diameter. The meat weight rate peaked in June (27.7%), and then rapidly decreased in September (19.5%). Monthly changes in the gonad index in both sexes reached the maximum in June, and then sharply decreased in September. Percentages of first sexual maturity of female and male snails ranging from 9.0 to 9.9 mm in shell heights were 58.3% and 54.5%, respectively, and 100% in those over 11.0 mm in both sexes participated in reproduction. Reproductive cycle of this species can be categorized into five successive stages: in females, early active (October to April), late active (December to June), ripe (April to September), spawning (July to September) and recovery (September to January): in males, early active (November to March), late active (December to June), ripe (April to September), spawning (July to September) and recovery (September to December). Gonadal development, gametogenesis, reproductive cycle, and spawning were closely related to the seawater temperature.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.183-191
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• 2001

This study was designed to establish the superior method for IVF embryos from DNA marker-proved Hanwoo cattle. DNA markers related to marbling score were identified using DNA fingerprinting with Ml3 probe and restriction enzyme Hae III. Oocytes were aspirated from unstimulated. immature ovarian follicles using a combined method of rectal ovarian-palpation and transvaginal ultrasound-guidance(6.5MHz) under local abesthesia. The aspirated oocytes were washed twice with fresh D-PBS containing 5% FBS and were rewashed 4 to 5 times with TCM-199 containing 5% FBS. A morphological grade of I to IV was assigned to each oocyte. Data were analysed using the GLM procedure of SAS. Mean number of follicles identified on ultrasound was 5.5 $\pm$2.9 in right and 4.3 $\pm$2.8 in left ovaries, respectively. The highest follicles(16.6$\pm$2.6) were found in 5101 cow compared to others. Recovery rate of follicular oocytes in individual cow was highest in 5101 cow with 89.3% in > 2mm and 94.0% in $\leq$ 2mm follicles. Total recovery rate was significantly(P<0.01) higher in $\leq$ 2mm(85.7%, 130/154) than > 2mm follicles(74.2%, 201/271). Significantly more oocytcs of Grade IV were recovered from > 2mm follicles. Mean number follicles recovered was 4.8$\pm$3.7. 3.0$\pm$3.4 and 0.3$\pm$0.6 in $\leq$2mm, 2~6mm and $\geq$6mm follicles. respectively. Our results imply that the more fertilizablc oocytes can be recovered from invisible-immature follicles by the combination of simultaneous rectal ovarian-palpation and ultrasound-guided approach in Hanwoo cattle.

• Clinical and Experimental Reproductive Medicine
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• v.15 no.2
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• pp.135-147
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• 1988

To compare the stimulation effect of the ratio in follicle stimulating hormone and luteinizing hormone in induction of multiple follicular growth, the serum $E_2$ level, the diameter of follicle, number of aspirated follicles and cleavage rate of in vitro fertilized preovulatory oocytes as well as the pregnancy rate were evaluated. Forty one patients with irreparable tubal disease were stimulated by hMG(n=24) or FSH/hMG(n=17) for the purpose of in vitro fertilization and embryo transfer. The following results were obtained. 1. Serum estradiol($E_2$) levels on the day of hCG administration were $921.0{\pm}353.3\;pg/ml$ in hMG group and $1272.9{\pm}1060.6\;pg/ml$ in FSH/hMG group. The serum $E_2$ value of hMG group was significantly lower than that of FSH/hMG group. 2. The diameter of leading follicle by ultrasonogram on the day of hCG administration were $16.2{\pm}2.0\;mm$ in hMG group and $16.2{\pm}2.6\;mm$ in FSH/hMG group. No significant difference of follicle diameter between two groups was demonstrated. 3. The number of follicles with diameter above 10 mm by sonogram on the day of hCG injection were $3.91{\pm}2.32$ in hMG group and $6.52{\pm}3.86$ in FSH/hMG group. There was significant difference of number of follicles between two groups, (p< 0.01). 4. The number of oocytes found per patient at aspiration were $2.59{\pm}1.00$ in hMG group and 3. $76{\pm}2.31$ in FSH/hMG group. There was significant difference of number of aspirated oocytes between two groups. (p< 0.05). 5. The detection rate of preovulatory oocyte at aspiration were 68.4%(39/57) in hMG group (n=22) and 77.6%(38/49) in FSH/hMG group (n=13). 6. The cleavage rate of preovulatory oocyte at 44 hours after insemination were 74.4%(29/39) in hMG group(n=22) and 81.6%(31/38) in FSH/hMG group (n=13). When only hMG was used, one pregnancy was established in 15 patients to whom 29 zygotes were transferred. And a full term normal female baby was delivered by elective cesarean section. In the FSH/hMG group, five pregnancies out of 9 transferred patients were confirmed by serum ${\beta}-hCG$. Two pregnancies were spontaneously aborted before the 6th week of pregnancy. One patient aborted her baby at the 18th week of pregnancy because of incompetent internal os of the cervix. Two patients delivered two full term babies by elective cesarean section. From the above findings, paralell with the increase in the ratio of exogenous follicle stimulating hormone to luteinizing hormone, an increase in oocyte recovery was observed as well as an improvements in pregnancy rate. It was concluded that FSH enrichment early in the follicular phase had a beneficial effect in the controlled ovarian hyperstimulation.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.187-192
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• 1997

The survival rate and motility recovered after cryopreservation of testicular spermatozoa in testicular sperm extraction (TESE)-ICSI program is low. The purpose of this study was to assess the availability and efficiency of mouse empty zona pellucida in cryopreserving human TESE spermatozoa. Mouse empty zonae pellucidae were obtained by extraction of cytoplasm with or without cytochalasin B treatment. Motile sperm from proven-fertile donor and two azoospermic patients after TESE were individually inserted into empty zona pellucida and cryopreserved. Two to five days after cyropreservation, the frozen sperm were thawed and the rates of recovery and motility were observed. The ooplasmic extraction rates of control (N=80) and cytochalasin B treated oocytes (N=80) were 94.0% and 96.2%, respectively (p>0.05). The post-thaw recovery rates of spermatozoa and rates of motility recovery of ejaculate (N=70) and testicular (N=70) sperm were 97.1%, 97.1% and 95.7%, 94.3%, respectively (p>0.05). The results of this study showed that the mouse zone pellucida is useful for cryostorage of single testicular spermatozoa.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.293-293
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• 2004

본 실험은 도축전 암소에 성선자극호르몬(Gonadon, 동방(주) : 3 ㎖ 목근육 주사, Gonadorelin 100 ㎍/㎖)을 처리하여 인위적으로 새로운 발정주기를 유도하여 난자 회수율 및 수정란 생산성을 향상시키고자 실시하였다. ① 실험대조군으로 85두(T1), 호르몬처리 37두를 대상으로 처리시간(T2 ; 72hrs, T3 ; 96 hrs, T4 ; 120. T5 ; 148 hrs.)에 따라 도축 후 난소로부터 혈통수정란 생산을 위해 개체별로 난자 회수량을 확인하였다. 회수된 난자를 이용하여 체외성숙 및 체외수정을 실시하였고, 이들 난자의 발육상태, 난발달 및 동결가능 수정란수를 각각 확인하였다. (중략)