• 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.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 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.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.4
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• pp.627-633
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• 1997

Gonad structure, germ cell development and reproductive cycle of the smallmouth scorpionfish, Scorpaena miostoma were investigated based on histological method. Samples were collected monthly in the vicinity of Suyoung Bay, Pusan, Korea from November 1995 to October 1996. The testis is seminiferous tubule type in internal structure. Seminiferous tubule consists of numerous testicular cysts which contain numerous germ cells in same developmental stage. The ovary consists of several ovarian lamellae originated from ovarian outer membrane. Oogonia originated from the inner surface of the ovarian lamella protrude to the ovarian cavity in oocyte stage, and they are suspended by the egg stalk. Biological minimum size of female and male were 12.5cm in total length. Gonadosomatic index (GSI) of female (3.81) and male (0.23) were the highest in October. Reproductive cycle was classified into the following successive stages: in female, growing stage $(May\~August)$, maturation stage $(September\~October)$, ripe and spawning stage $(November\~December)$, recovery and resting stage $(January\~April)$, and in male, growing stage $(June\~August)$, maturation stage $(September\~October)$, ripe and spent stage $(November\~January)$ and recovery and resting stage $(February\~May)$.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.99-106
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• 2001

This study was undertaken to compare the efficiency of recovery rate and development rate of follicular oocytes collected either by aspiration or by slicing method. The follicular oocytes collected by the two methods matured in TCM199 supplemented with 10% steer serum at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. After 22 h of culture, the oocytes were inseminated with frozen-thawed semen (2$\times$10$^{6}$ sperm/ml of final concentration) prepared with Percoll-density gradient in IVF-TALP medium for 16 h. Later, sets of 15 presumptive zygotes were transferred into 50 $\mu$L, droplets of CR1aa medium. On day 4 of the culture, embryos were transferred to TCM199 until day 9. The percentages of nuclear maturation to pre-metaphase II in the oocytes collected by aspiration are significantly (P<0.05) higher than that by slicing (83% vs. 62%, respectively). The mean number of oocytes recovered by slicing per ovary is significantly (P<0.05) higher than that by aspiration (15.1 vs. 6.7, respectively). Although the rates of cleavage and development to blastocyst of oocytes collected b)\\\\`aspiration are significantly (P<0.05) higher than that by slicing, the number of transferable embryos obtained by slicing method is significantly (P<0.05) higher than that by aspiration. From the results. we may conclude that slicing method is better than aspiration method for obtaining large number of transferable embryos per ovary.

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.83-88
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• 2000

This study was conducted to determine the effect of repeated ultrasound-guided transvaginal retrieval of oocytes recovery, estrous cycle and ovarian adhesion in Korean native, Hanwoo heifers. Heifers were at unknown stages of the estrous cycle at the start of experiments in which all follicles $\geq$6mm in diameter were ablated. The results obtained in this study were as follows; Follicle developing number and oocytes collected number were no effected to repeated OPU to nine session, 4 e.a range oocytes collected to repeated OPU session. Oocytes were observated follicles were 8.7$\pm$4.2 e.a, collected oocytes were 4.1$\pm$3.4 e.a to two times collected per week and observated follicles was 10.2$\pm$6.1 e.a, collected oocytes were 4.3$\pm$2.9 e.a to one times collected per week, but no difference significantly(P＜0.05). Ovaries adhesive percentage to repeated OPU was eight ovaries adhisived(20%) of forty ovaries, three ovaries(7.5%) to 1~3 times oocytes collected, four ovaries(l0%) to 4~6 times, one ovaries(2.5%) to 7~9 times oocytes collected session. To repeated OPU effection, ovaries adhisive heifers were long estrous cycle(＞25 day) to 7 heads(87.5%) of 8 head, non-adhesive ovaries heifers were 5 heads(41.7%) were long estrous cycle to repeated OPU 12 heads. Although, now unknown about the dynamics of follicles wave and about functional changes to repeated OPU ovaries, more question about ovaries adhesive cause remain.

• Korean Journal of Veterinary Research
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• v.34 no.4
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• pp.735-744
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• 1994

Catheters were placed into the external jugular veins of immature female rats. On the following day (day 28 of age), the animals were injected subcutaneously with pregnant mare serm gonadotropin(PMSG): 4IU(control) or 20IU(superovulation). Each animal was sequentially bled at Ohr and 12hr and subsequently at 6hr intervals until sacrifice at 72hr after PMSG. The superovulatory dose of PMSG significantly(P<0.05) increased the ovulatory response by 4.0 fold above controls. On the other hand, superovulated oocytes displayed considerably different stages of meiotic maturation: prophase I (14.7%), anaphase I (36.2%), telophase I (10.3%), metaphase I/II (32.4%), while in control rats a majority of the oocytes examined(94.0%) consistently showed a metaphase II configuration. Serum luteinizing hormone(LH) levels were determined by RIA. Both groups exhibited a similar time relationship with two distinct peaks: an initial slight rise at 0-18hr and a second sharp rise at 54-60hr. However, there was a marked change in the magnitude of LH levels between the two groups. In superovulated animals, prior to the second peak, overall LH levels were significantly(P<0.001) higher than controls. In contrast, at the peak occurring at 60hr, LH concentrations were significantly(P<0.001) reduced by 54% below that of control. Additionally, a maximum increase of mean ${\Delta}LH$ between two peaks was much less in superovulated as compared to control rats. The initial prolonged elevation of serum LH before 54hr in superovulated rats was found to result from actual cross-reaction of the injected PMSG with LH antibody in the assay, while a precipitous second elevation between 54hr and 60hr resulted primarily from an endogenous LH surge. This study clearly defines time-course features of serum LH in PMSG-treated rats. The overall results indicate that, following superovulatory treatment with PMSG, the increased ovulatory response is primarily associated with PMSG-derived intrinsic gonadotropin, and that the recovery of immature or asynchronously mature oocytes at ovulation may reult from the circulatory alteration of LH activity characterized by an initial prolonged elevation of serum LH and its subsequent attenuation.

• Development and Reproduction
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• v.9 no.1
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• pp.23-31
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• 2005

Vitellogenesis during oogenesis, reproductive cycle and first sexual maturity of the female Neptunea (Barbitonia) arthritica cumingii was investigated by light and electron microscope observations. In the early vitellogenic oocyte, the Golgi complex and mitochondria were involved in the formation of lipid droplets and yolk granules. In late vitellogenic oocytes, the rough endoplasmic reticulum and multivesicular bodies were involved in the formation of proteid yolk granules in the cytoplasm. A mature yolk granule was composed of three components: main body(central core), superficial layer, and the limiting membrane. The spawning season was between May and August and the main spawning occurred between June and July when the seawater temperature rose to approximately $18{\sim}23^{\circ}C$. The female reproductive cycle can be classified into five successive stages: early active stage(September to October), late active stage(November to February), ripe stage(February to June), partially spawned stage(May to August), and recovery stage(June to August). The rate of individuals reaching the first sexual maturity was 53.1% in females of 51.0 to 60.9mm in shell height, and 100% in those over 61.0mm.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.3
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• pp.338-346
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• 1995

Studies on activities of hepatocytes and changes of protein and total RNA contents in liver and muscle tissues associated with the gonadal maturation were performed using the adult rockfish, Sebastes schlegeli collected monthly from the adjacent waters at Poryong, Chungnam, Korea. Hepatosomatic index (HSI) of female increased from September and reached the maximum value (2.58) in February (growing stage of ovary). HSI of male began to increase from October (early maturation stage) and revealed the maximum value (3.20) in April (recovery stage). During the period of yolk formation in oocyte, the amount of glycogen particles and lipids in the hepatocytes of female gradually decreased, but total RNA contents increased. In the hepatocytes of male, a number of lipid droplets remained until the mature stage of testis. With the gonadal development of both sexes, granular endoplasmic reticula (Er) became abundant in the hepatocyte. These Ers might be supposed to have the leading role for the syntheses of protein and vitellogenin in the cytoplasm of the hepatocytes. Protein and total RNA contents in the liver and muscle tissues of female began to increase from January (growing stage) and reached the maximum value in early April (maturation stage), and then decreased in late April (gestation stage). In male, their contents began to increase from October (early maturation stage), thereafter reached the maximum value in December (maturation and copulation stage), and decreased in January (degeneration stage).

• Clinical and Experimental Reproductive Medicine
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• v.22 no.2
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• pp.211-220
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• 1995

Though the endometriosis is not always related with infertility, endometriosis causes infertility in some patients. There are many treatment modalities of infertile patients who have endometriosis. In recent years, Assisted Reproductive Technology(ART) have been widely accepted as being a useful tool for the treatment of infertile endometriotic patients. The objective of this study was to evaluate the outcome of ART in infertile endometriotic patients who have been carried out IVF-ET from Jan, 1992 to Dec, 1994 and to compare the results between COH/IUI and IVF-ET in the patients with endometriosis stage I. Tubal disease only patients were grouped(308 patient, 956 cycles) as a control. Endometriosis group was subdivided into 4 groups according to American Fertility Society classification; endometriosis stage I (45 patients, 61 cycles), stage II (26 patients, 39 cycles), stage III (26 pateitns, 37 cycles), stage IV (33 patients, 50 cycles). The outcomes of IVF-ET in endometriosis patients were as follows; The oocyte recovery rates were significantly lower in stage III, IV endometriosis. In case of stage III endometriosis, the fertilization rate was significantly lower than other stages of endometriosis. Clinical pregnancy rates per cycle were not different between the tubal group(22%) and the endometriosis group(25%). According to endometriosis stage, the implantation rate and clinical pregnancy rate were significantly lower in stage IV (5.6%, 16%) compared with other stages (I; 10.0%, 26%, II;9.8%, 31%, III;12.6%, 32%). It suggests that some factor like autoantibodies may inhibit implantation of embryos in stage IV endometriosis. To evaluate the possibility that simply increasing the number of gametes at the site of fertilization might account for pregnancies attributed to IVF-ET, the authors retrospectively analyzed the outcome of couples undergoing IUI during hMG cycles and CC cycles between 1992 and 1994 in the women with endometriosis stage 1. In case of stage I endometriosis, though the COH/IUI group showed lower FSH level and lesser age profile than IVF-ET group, IUI group has resulted in lower pregnancy rates(19.2%) compared with the IVF-ET group(26.2%). In conclusion, endometriotic infertile patients can get comparable pregnancy rates with the tubal factor infertility patients during IVF-ET program. Moreover even in stage I endometriosis, IVF-ET may be an more effective treatment modality than COH/IUI.