• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.147-156
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• 1997

Ultrasound-guided follicular aspiration was performed in Holstein heifers once weekly with or without pretreatment of single or multiple decreasing doses using a total of 400 mg FSH. Oocytes were aspirated with a 6.5 MHz convex-array ultrasound trasducer designed for intravaginal use. All the visible follicles larger than 4 mm in diameter were punctured with a 17 gauge, 55 cm needle at each aspiration session and the follicular fluids containing oocytes were obtained by vacuum suction. The results obtained were as follows: As a preliminary experiment, the recovery rates of folicular oocytes by ultrasound-guided aspiration from the isolated ovaries of Korean native cows were compared between suction methods using manual syringe or vacuum pump. The recovery rate of oocytes using vacuum pump (80.7%) was significantly (P<0.05) higher than that using manual syringe (47.1%). The follicles were counted by their size in diameter with ultrasound image, and recovery rates and grades of follicular oocytes collected by ultrasound-guided aspiration were investigated in Holstein heifers pretreated with or without FSH. A group of heifiers were injected with multiple decreasing doses (twice a day for 3 days) of a total of 400 mg FSH. The other 2 groups were injected with a single dose of 400 mg FSH mixed with 25% PVP. Ultrasound observation of follicle population and/or ultrasound-guided transvaginal oocyte aspiration were performed 12 hrs following the last FSH injection in the multiple dose group, and 48 or 60 hrs after FSH injection in the single dose groups. Most of the visible follicles had small size of less than 3 mm in diameter in unstimulated heifers (71.0%), but medium size in all the heifers treated with FSH. (70.5 to 92.8%). The number of OPU follicles per session (4.6$\pm$1.9) were much less, compared to the vilsible follicle counts (9.7$\pm$2.2), in the nustimulated heifers due to the small dominant follicles. Among 4 goups of heifers the most visible as well as OPU follicles were observed in the heifers at 60 hrs following treatment of a single dose of 400 mg FSH (21.2$\pm$2.3 and 21.0$\pm$2.0), and the differences in both the follicle counts between the groups was found significant (P<0.05) The rates of oocyte recovery from the follicles by ultrasound-guilded aspiration were varied 46.3 to 75.0% in the heifers unstimulated and treated with a single dose of 400 mg FSH, but the group difference was not significant. The number of recovered oocytes per session a, pp.ared to be highest at aspiration at 60 hrs following single FSH (10.6$\pm$2.2) than at aspiration at 48 hrs after single FSH (7.8$\pm$2.7) or in the unstimulated heifers (3.4$\pm$3.0). The proportion of grade I and II oocytes to all oocytes collected was varied 31.8 to 64.0% between the groups. However, there was found no significant difference in both the number of oocytes recovered per session and the percentage and the percentage of grade I and II oocytes. From the above results it was concluded that the more oocytes of superior quality might be recovered economically by ultrasound-guided aspiration at 60 hrs following the pretreatment of a single dose of 400 mg FSH and by suction using a vacuum pump system of about negative pressure of 75 to 85 mmHg.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.203-210
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• 1999

In this stuyd, the effect of the dominant follicle aspiration for the superovulatory response in HanWoo was investigated. The criterion for the presence or absence of a dominant follicle based on their morphological examination. The dominant follicle was aspirated 48hr before the onset of superovulation treatment by 6.5MHz convex probe connected with a carrier and superovulation induced by FSH (Super-Ov Tyrer, Texas, U.S.A) adminstered twic a day s.c. over 4 day in a decreasing regimen. From 13 HanWoo scanned daily to determine the presence and growth of the dominant follicle, its an average diameter of 15.4mm was measured and an average diameter of corpora lutea was 18.7mm on day of follicular aspiration. In the experiment, a follicular remove by ultrasound-guided aspiration, the ovarian response was significantly enhanced when animals were superovulated in the aspiation of a dominant follicle compare with animals superovulated non-aspiration of a dominat follicle. In the aspiration of a dominant follicle donors yieleded more corpora lutea(14.4$\pm$4.7 vs 8.6$\pm$3.4) and transferable embryos(8.9$\pm$4.2 vs 5.4$\pm$2.7) than control. In cows in which the dominant follicle had been aspirated under sonographical control 2 days before superovuation, the number of corpus lutea and transferable embryos were significantly enhanced compared with animals superovulated in the presence of a dominant follicle (14.4$\pm$4.7 vs 6.9$\pm$2.7, ; 8.9$\pm$4.2 vs 3.3$\pm$1.6). After 7 days of artificial insemination, the embryos at 7 days were cllected by uterine flushing after dominant follicle insemination, the embryos at 7 days were collected by uterine flushing after dominant follicle aspiration and superovulation treatment, and evaluated their quality by morphological criteria. Sixteen embryos with excellent and good grade were transferred into 8 recipient cows. Six pregnancies were identified at 60 and 120 days of gestation by rectal palpations. In conclusion, the present study showed that 1) the presence or absence of a dominant follicle signficicnatly affects superovulatory responses, and 2) ultrasound-guided follicular aspiration of the dominant follicle and superovuation treatment provides an accurate and procedure to increase ovarian responses in HanWoo.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.107-115
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• 1998

The present study was carried out for the comparative study on the collection of bovine follicular oocytes by ultrasound-guided ovum pick-up(OPU) and slaughterhouse-derived (SHD) ovary aspiration and in vitro production of bovine embryos with the follicular oncytes in Korean native cows. Bovine follicular nocytes were observed with a 6.5 MHz convex-array ultrasound transducer designed for intravaginal use and the oocytes were collected with the aspiration equipment attached to the ultrasonograph. Bovine ovaries were collected and transported in phosphate buffered saline from the local slaughterhouse, the follicular oocytes were collected by the aspiration method. The collected follicular oocytes in good quality were matured, fertilized and cultured in the media. The total number of the visible follicles and the recovery rate of follicular oocytes were increased in ultrasonography following follicle stimulating hormone(FSH) treatment in Korean native cows. The mean recovery rate of oocytes was 66.2, 52.8 and 41.7% in the FSH-OPU, non-treatment-OPU and SHD ovaries, respectively. The mean number of recorved oocytes per cow were not significantly(P<0.05) different between the FSH-OPU(14.0$\pm$11.54) and SHD(17.1i6.21) groups, but the numbers in both groups were significantly(P<0.05) higher than the number in the non-treatment-OPU(3.7$\pm$1.57) group. The mean number of usable nocytes in Grade T /11 per ovary was 6.3, 4.8 and 1.3 in the cows of the SHD, FSH-OPU and non-treatment-OPU groups, respectively. The in vitro developmental rate to the blastocyst was not significantly different between the oocytes obtained via OPU(37.1%) and SHD(29.3%). Therefore, the ultrasound-guided OPU technique can be applied to the production of excellent embryos from the high-quality cows, and for the large scale production of in vitro bovine oocytes and embryos, the SHD ovary aspiration method is valuable.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.1-8
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• 2000

The ultrasound-guided oocytes cllection (ovum pick-up ; OPU) has become a substitution for superovlation in cattle. The objective of this study was to examine the effect of OPU frequency on the in vitro production of embryos in Hanwoo cattle. Six cycling Hanwoo cows were distributed into two groups for either once or twice weekly OPU sessions. Oocytes were collected by ultrasound-guided follicle aspiration(SA600) using a 6.5HMz transducer and attached with 18 gauge needle, with vacuum pressure of 40 mmHg. The cumulus-oocyte complexes (COCs) collected from each donor were matured in TCM 199 supplemented with 10% fetal bovine serum at 5% CO2 in air at 38.5$^{\circ}C$ for 22h and in vitro matured oocytes were co-incubated with sperm(separated by Percoll gradient) for 6h. The zygotes were co-cultured on cumulus cell monolayer in 10ul droplets in the same culture medium and conditions used for IVM for 7 days. On Day 7 of culture, development to blastocysts was examined. Although the number of oocytes collected was variable depending on individuals, overall embryo production in the twice per week OPU sessions was better that in the once per week sessions(6~21 vs 2~7 blastocysts produced, respectively). Two cows(E, A) were good oocyte donors and embryo production was superior in cow C ; however, cow F was a poor donor as compared to the others. In conclusion, these results suggest that for embryo production, twice weekly OPU sessions were better than once per week for producing embryos in vitro from Hanwoo cattle.

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.103-109
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• 1996

The aim of this study was to evaluate the effect of recombinant bovine somatotropin (bST; Boostin-S, LG Chem) treatment with FSH (Super OV) or PMSG on superovulatory response for transvaginal ultrasound-guided oocyte retrieval (TVR) in calves. Eight Korean Native Cattle(KNC) heifer calves; 150 to 240 days old; were randomly assigned to four treatment groups: 1) FSH(75 mg); 2) FSH (75 mg) + bST(500mg) 3) PMSG(1;000 IU); 4) PMSG(1, 000 IU) + bST(500 mg). Experimental calves in group 1 (n=2) and 2(n=2) were weekly superovulated for 4 consecutive weeks with daily injection of FSH for 3days and the next day subjected to TVR session. Animals in group 3 (n=2) and 4(n=2) were weekly stimulated for 4 consecutive weeks with a single dose of 1, 000 IU PMSG. TVR was performed on 72 hours after PMSG injection. Calves in group 2 and 4 was received injection of 500 mg of bST every 10 days. At each TVR session, follicle number and size were recorded; the oocytes collected and graded according to cumulus and cytoplasm investment. Collected oocyte were determined viable oocyte according to morphological quality with granulation of oocyte and number and status of cumlulus cells. IVM and IVF were performed and assessed cleavage rate on day 3 after fertilization. A Sonovet 600(Medison, Co., Ltd) realtime ultrasound scanner with a 6.5 MHz convex transducer, fixed at the tip of 500 mm estended handle equipped with a needle guide was used in collecting oocyte. Differences between groups were analysed by chi-square test. The population of large follicle ($\geq$5 nun) and aspiration rate were not significant different among the 4 groups. But, the number of small follicles (<5 mm) and aspirated oocyte in the KNC calves treated with bST were 1.3~1.6 times higher than in KNC calves treat with FSH or PMSG alone. In conclusion, the administration of bST with FSH or PMSG at superovulation for TVR in calves was increase the nurnber of small follicle which was influenced the number of aspiratable follicle.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.195-202
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• 1997

The present study was carried out to evaluate the effect of superovulation treatments on ovarian responses, oocyte recovery rates and grades of collected oocytes using an ultrasound-guided transvaginal approach in Korean native cows. Superovulation in cows was induced with two different regimenes: 1) FSH-decreasing dose(n=8): the cows were received twice per day for three days of the total dose of 400 mg of FSH-p, 2) FSH-single dose(n=9): the cows were administrated a single dose of 400 mg of FSH-p in 25% PVP. The Observation of visible follicles and collection of oocytes were performed 12 hours following the last FSH in FSH-decreasing dose group and 48 hours after the FSH-single dose injection. All visible follicles larger than 6 mm were punctured and aspirated with a 6.5 MHz convex-array ultrasound transducer designed for intravaginal use. The mean number of visible follicles(> 6 mm) was significantly(P<0.05) higher in the FSH-decreasing dose treatment (22.811.9) and FSH-single dose treatment (20.612.0) groups than the non-treatment group(7.0$\pm$8). The mean recovery rate of oocytes was not significantly(P<0.05) different between the treatment and control groups, but the mean number of collected oocytes was significantly(P<0.05) higher in the FSH-decreasing dose treatment( 12.611.5) and FSH-single dose treatment (11.813.6) groups than the non-treatment group(3.7$\pm$0.5). In conclusion, the FSH-single dose treatment at superovulation in cows for ultrasound-guided aspiration might increase the number of aspiratable follicles and the recovery rate of follicular oocytes as the FSH-decreasing dose treatment.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.233-240
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• 1994

This study was conducted to compare the endocrine milieu, and pregnancy rates in In Vitro Fertilization and Embryo Transfer(IVF-ET) program employing combined with gonadotropin releasing hormone agonist(GnRH-a) and pergonal(LH 75lU+FSH 75lU) when either human chorionic gonadotropin(HCG) or progesterone were used for luteal phase support. A total number of 40 IVF-ET treatment cycles were prospectively studied. Ovarian hyperstimulation method was modified ultrashort protocol using GnRH-a. All patients started Decapeptyl at menstrual cycle day # 2, and HMG was started at # 3 days. When leading follicle was ${\geqq}$18mm or at least two follicles were ${\geqq}$14mm in diameter, HCG 10000lU intramuscularly was injected. After 36 hours HCG administration, oocytes were retrieved as usual guided by transvaginal ultrasound. Embryo were transfered 36-48 hours later. The patient's cycles were prospectively randomized to receive HCG(20cycles) or Progesterone (20cycles) for luteal support. The progesterone group received 25mg 1M starting from the day of ET. The HCG group received 1500IU 1M. on days 0, +2, +5 after ET. Estadiol($E_2$) and Progesterone($P_4$) were measured on the day of oocyte aspiration, ET day, and every 6 days thereafter. Results were follows as; 1. Estradiol, progesterone and LH levels on the day of HCG trigger, retrieved oocytes and number of transfered embryo were not significantly different in both groups. 2. On the day of aspiration and embryo transfered day, $E_2$, $P_4$ level were significantly higher in progesterone group than HCG group(p<0.01). 3. $E_2$, $P_4$ level on 6 days after ET were significantly higher in progesterone group than HCG group(p<0.01). But, $P_4/E_2$ ratio was not different in both groups. 4. $E_2$, $P_4$ level 12 days after ET were decreased abruptly in both groups and higher hormonal level appeared in HCG group(P<0.01). 5. The total pregnancy rate in the HCG group was 40% (8/20) and in the progesterone group 15%(3/20). 6. Comparing the pregnant and nonpregnant cases progesterone group was not different the hormonal status. In HCG group, pregnant cases appeared in higher $P_4$, $P_4/E_2$ ratio than nonpregnanct cases(P<0.01).

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.191-197
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• 2016

Historically, Korea old cattle had been consisted with various lines of coat color brindle, black and white-brown breeds or more. The two rare lines of black and white coat color are maintained for animal resources and preserved critically. The present study was carried out to evaluate potential usage of cysteamine supplementation during in vitro matration (IVM) and in vitro culture/production of embryo (IVP) by transvaginal ultrasound-guided follicle aspiration (Ovum Pick-Up: OPU) for the establishment of cryo-banking system. Immature slaughterhouse-derived cumulus-oocyte complexes (SL-COCs) were matured in IVM medium supplemented with 0, 0.1, 0.3 or 0.9 mM cysteamine, and then cultured in mSOF-BAS for 8 days after in vitro fertilization. The treatment of 0.1 mM cysteamine on SL-COCs showed higher rate of blastocyst, so OPU-derived COCs from rare breeds were matured in TCM media supplemented with or without 0.1 mM cysteamine, FSH and 5% FBS. The embryos were evaluated their developmental stages on day 8. During IVM, cysteamine treatment significantly increased the embryo production rate of slaughterhouse-derived COCs (19.6% vs. 30.5%). The presence of cysteamine during IVM of OPU-derived COCs from rare Korean cattle breeds (albino white and black line) also increased embryo production rates than those from SL-COCs (27.4% vs. 41.9% and 36.4%). With these results, cysteamine treatment during IVM is one of key factors IVP of blastocysts to establish banking system of endangered rare Koarean cattle with OPU derived transferable blastocysts.