본 연구는 체외에서 한우 난포란의 핵성숙과 그 후의 초기 배발달에 있어서 체외성숙 배지에 아미노산의 첨가가 난포란의 제1극체(PB) 출현율, 배발달율 그리고 배반포의 세포수에 미치는 영향을 검토하였다. 첨가하는 아미노산의 종류와 농도는 각각 MEM 배지의 non-essential amino acids (NEAA)와 BME 배지의 essential amino acids(EAA)는 1, 2, 4배 및 유청중의 lactalbumine hydrolysate(LAH)는 1, 5, 10 ㎎/㎖이었다. 그 결과 NEAA와 EAA의 경우 PB 출현율은 1배 첨가군이 미첨가군보다 유의하게 높았지만(p＜0.05), 첨가량이 증가할수록 오히려 감소하였다. 그러나 배반포로의 배발달율은 모든 군에서 비슷한 경향이었다. 그리고 배반포의 총 세포수와 총 세포수중 TE 세포수는 2배 처리군이 가장 높았으며, ICM 세포수는 아미노산 첨가량이 증가할수록 많아졌다. 한편 LAH의 경우 PB 출현율은 5㎎ 첨가군이 가장높았으며, 배반포로의 발달율은 미첨가군과 1 ㎎ 첨가군이 5 ㎎ 첨가군과 10 ㎎ 첨가군보다 각각 유의하게 높았다(p＜0.05). 그리고 배반포의 세포수는 NEAA와 EAA를 이용하였을 경우와 비슷한 경향이었다. 이상의 결과로부터 체외성숙용 배지에 아미노산의 첨가는 생산된 배반포의 품질을 향상시킬 수 있기 때문에 배반포의 체외생산에 이용할 수 있는 새로운 아미노산의 종류 및 농도를 탐색할 필요가 있다고 사료된다.

본 연구는 고품질육의 DNA marker가 규떵된 한우로부터 초음파유노 난포란의 연속적 채취를 통하여 능력이 우수한 한우 수정란온 대량생산하는 방법의 확립과 이를 한우농가에 응용하고자 초음파 난자채취기를 이용하여 등지방층두께, 일당증체량, 근내지방도 및배최장근 단면적에 연관된 DNA marker를 보유하고 있는 한우 5두로부터 개체및 난포수, 채취방법, 회수한 난포란의 등급 등을 조사하였다. 한우 5두의 개체별 난포수는 6, 10, 5, 4 및 11회

This study was carried out to investigate the effect of biological factors on the in vitro production(IVP) of bovine oocytes for development of simple culture methods and medium. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol and this study was examined if there were necessary to co-culture, media change, media type and embryo density. This results were as follows: 1. The development rate according to co-culture with cumulus cells and non co-culture as drop culture was not significantly different in cleavage (68.9 vs 71.7%), 8-cell stage (41.2 vs 44.1%) and blastocyst stage (12.2 vs 13.8%), respectively (p<0.05) 2. The blastocyst development rates in YS and CRIaa were higher than that in TCM199 (12.4, 10.4 vs 3.7%), but the cleavage (69.0, 77.8 and 61.0%) and 8-cell stage (31.7, 37.0 and 35.7%) development accoring to YS, TCM199 and CRIaa ws not significantly different, respectively (p<0.05). 3. There was no significantly different in cleavage (62.6, 59.5 and 61.2%), 8-cell(34.7, 37.9 and 34.0%) and blastocyst (9.5, 11.6 and 12.8%) development among medium change time as control, Group I and Group II, respectively (p<0.05). 4. Blastocyst formation of 8-cell stage according to embryo density was not significantly different in 1, 10 and 25 embryos (27.3, 22.5 and 34.0%), respectively (p<0.05). These results indicated that simple culture system could reduce bovine IVP embryos as drop culture as non co-culture system, high density embryo (25 embryos/50 1 drop). YS defined medium and no medium change for whole culture period, although other biological factors need to examine in order to produce efficient IVP bovine embryos.

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(14.44.7 vs 8.63.4) and transferable embryos(8.94.2 vs 5.42.7) 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 (14.44.7 vs 6.92.7, ; 8.94.2 vs 3.31.6). 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.

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.

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(14.011.54) 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(3.71.57) 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.

This study was carried out to compare the actual size(length and height) of ovaries, follicles and corpora lutea of Korean native cow with those on sonograms. We used 3 different probes(3.5 MHz abdominal probe, 6.5 MHz transvaginal probe and 5.0 MHz transrectal probe) and a calipher for measurements of ovaries, follicles and corpora lutea on sonograms and actual size. Under water immersion, 157 ovaries were scanned with 3 probes and measured in actual size and compared each other. The average height and width of ovaries of Korean native cows were 17.403.99 and 34.236.02mm, respectively. In comparison of height, length of ovaries and preovulation follicles, we found that image with a transvaginal probe was nearly the same as the actual size(p<0.01), but with an abdominal probe the image was appeared larger than the actual size. In measurement(diameter) of preovulation follicles the transvaginal probe was proven to be more accurate to the actual size than other probes and in corpus luteum measurement all probes were accurate. In the comparison of number of follicles by different size ranges, there was no statistical difference in the count of follicles over 10 mm in diameter between the transvaginal probe and naked eyes.

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28 and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35g /ml FSH, 10g /ml LH, 1 g /ml at 39 under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P