본 연구는 한우 체외 수정란의 성 감별과 신선란, 동결란 및 성 감별 수정란을 이식한 후 수태율, 분만율과 유산율, 생시 체중, 임신 기간을 조사하기 위하여 수행하였다 Aspiration과 punching법으로 biopsied한 수정을 24시간 배양 후 생존율은 각각 80.0%와 90.0%로 유의적인(p>0.05) 차이는 없었다. 수정란을 성 감별한 결과, 웅성 수정란과 자성 수정란의 비율은 각각 42.1%와 52.6%였으며, 5.3%는 수정란의 성을

발정 주기와 상관없이 CIDR를 삽입하는 날에 2.5 mg estradiol benzoate, 50 mg progesterone을 주사하였다. CIDR 삽입 후 4일 동안 FSH를 감량법으로 12시간 간격으로 주사하였으며, FSH 주사 5, 6회째에 를 투여했다. 1회째 주사 24시 간 후 CIDR를 제거하고 GnRH를 주사하였다. 공란 우들을 1번째 주사 후 60시간과 72시간에 수정을 시켰다. 인공수정 7일 후 회수된 수정란을 수정란 이식 때 pro

본 연구는 돼지 수정란의 체외 성숙 및 체외 배양액의 retinol 첨가 효과를 규명하기 위하여 체외 성숙 및 체외 배양액에 retinol을 첨가하여 수정란의 체외 발달에 미치는 영향을 구명하고자 수행되었다. 체외 성숙 배양액에 retinol을 첨가한 결과 성숙율은 으로 각 처리구 간의 유의적인 차이가 없었다(p>0.05). 체외 수정 후 배반포로의 발달율은 첨가구에서 의 발달율을 나타내어 타 처리구에 비하여 유의적으로(p<0.05) 높게 나타났으며,

본 연구는 아미노산의 첨가가 돼지 수정란의 체외 발달율에 미치는 영향을 구명하고자 PEF가 함유된 NCSU-23을 기본배지로 체외성숙 및 체외배양액을 조성한 후 EA(Essential amino acid), NA(Non-essential amino acid) 및 EANA(EA+ NA)를 첨가하여 체외성숙, 체외수정 및 체외발달에 미치는 영향을 조사하였다. 체외성숙 배지에 아미노산을 첨가한 결과 MH 단계까지의 체외성숙율은 NA 첨가군이 83.3%로 대조구 70.0%에 비하여 유의적으로(p<0.05) 높았다. 그러나 체외 수정 이후의 배 발달율과 수정율에서는 아미노산 첨가군과 무첨가군 사이에 유의적인 차이는 없었다. 체외배양액에 아미노산을 첨가한 후 배반포의 내부세포괴(ICM) 세포와 영양배엽(TE) 세포의 발달에 미치는 영향을 조사한 결과, ICM에서는 유의차를 발견할 수 없었으나 TE 세포는 EANA 처리구가 18.0±0.5개로 대조구 16.09±0.56개에 비해 유의적(p<0.05)으로 많았다. 총세포수에서도 EANA 처리구가 50.0±1.0개로 대조구 44.2±1.1개보다 유의적(p<0.05)으로 많았다. 이상의 결과를 종합할 때, 돼지의 체외수정란 생상에 있어서 아미노산의 첨가는 배반포로의 발달율에는 영향을 미치지 못하였으나 체외성숙율을 높이고 배반포의 세포수 향상에 도움을 주는 것으로 판단된다. 특히, 영양배엽(W) 세포의 발달율이 높은 것으로 보아 아미노산의 첨가는 돼지수정란의 착상에 도움을 줄 것이라 기대된다.

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 /ml epidermal growth factor(EGF) at 39 under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.75.1 and 96.64.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

This study was carried out to improve a technique of embryo transfer for twin calves production in Hanwoo cattle. Blastocysts for the donor of embryo transfer were classified into three criteria by accessment of morphology; early blastocyst, blastocyst and expanded blastocyst. Tow embryos were introduced transcervically into utrerine horn either of Hanwoo or Holstein by ipsilaterally or contralaterally to the corpus luteum. Thiry-six out of 57 recipients cows were inseminated by artificially on the next day of estrus, and followed by transfer of embryos into contralaterally. The pregnance rates of recipients following transfer of bovine embryos of day 7, 8 and 9 was 43.5, 18.2 and 8.3%, respectively. These results appeared that these was a significant (P<0.05) difference between on day-7 embryos and day-9 embryos, but not between on day-8 and day-9 embryos. Although there was not significant(P<0.05) difference in the pregnancy rates between the blastocysts(11/25, 44%) and expanded blastocysts(2/19, 10.5%) and between the blastocysts and early blastocysts(2/13, 15.4%), the embryos at blastocyst stage are more suitable than others for obtaining higher rate of pregnancy. There was no significant difference on pregnancy of the embryos transferred prior to presence(6/21, 29%) or absence (9/36, 25%) of artificial insemination. On pregnancy of Holstein, 2(15.4%) out of 13 recipients were pregnant in heifer. Similar Pregnancy rates were obtained between 1∼2 parities and 3∼4 parities by 30% (6/20) and 27.3%(3/11), respectively. Taken together, there was not significant difference in pregnancy rate due to small number of recipients used for this experiment. Both of Hanwoo and Holstein introduced the embryos by contralsterally to the corpus luteum were slightly higher pregnancy rate compare to by ipsilaterally (12/41, 29.3% vs, 3/16, 18.8%). The ratio of production of twin and single calves in Holstein was 20% (9/45) and 2.2% (1/45), respectively. However, in Hanwoo cows both of production of twin and single were similar as 8%. This result suggests that Holstein as recipients was superior to Hanwoo cows for production of twin calves. Out of all 15 pregnant, 12(80%) were produced a total of 22 normal calves in which the others composed of abnormal, as judging as 2(13.3%) for abortion and 1(6.6%) for stillbirth during the pregnant period.

This study was carried out to improve a technique of cloned animal prodcution by preactivation of nuclear recipient oocytes with ionomycin and 6-dimethylaminopurine (6-DMAP) in rabbits. The oocytes were collected from the oviduct of superovulated rabbit at 19∼20 hours post hCG injection. The collected oocytes were preactivated and self-enucleated by treating 5 uM ionoycin for 5 min, and 2.0 mM 6-DMAP for two hours. Microsurgical removel of the chromation complex in the second polar bodies was effectively performd and single blastomere separated from 32-cell stage rabbit embryos was injected into the perivitelline space of the enculeated recipient oocyts. Follwoing electrofusion and in vitro culture for 18 hours, the nuclear transplant(NT) embryos were transferred into the uterine horns of naturally mated or synchronized recipient does. When 32 NT embryous reconstituted with preactivated oocytes were transferred to 2 recipient does, one foster doe delivered two offspring (6.3%), while not a offspring was delivered from three foster does which received 17 NT embryos reconstituted with non-preactivated oocytes. A total of 68 NT embryos reconstituted with preactivated oocytes were transferred into the uterine horns of 7 synchronized ecipient does. Among them, two recipients were pregnant and delivered three offspring(5.9%).

도축장에서 채취한 돼지난자를 직경별로 간격으로 나누어, 난자의 크기에 따른 체외성숙과 발육능을 구명코자 체외성숙과 체외수정 후의 배 발달율을 조사하였다. 채취한 난자의 투명대를 제외한 평균직경은 이었으며, 직경별 분포는 < 105, 105~110, 110~115,115~120, 120~125, <이 각각 3.0, 11.0, 31.2, 41.5, 13.0 및 0.4%로 72.7%가 110부터 사이였다. 체외성숙율에 있어서 직경 미만난자는 66.7%인 반면

본 연구는 토끼난자에 있어서 배란 후 경과시간에 따른 탈핵률을 조사하고 ionomycin과 DMAP을 사용하여 활성화와 아울러 자체 탈핵을 유도하는 방법을 고안하였으며 아울러 이들의 탈핵효율과 핵이식후 체외발달을 확인한 결과는 다음과 같다. 1. hCG 주사후 15∼16 시간에 채란된 토끼난자는 73.4%의 탈핵률을 보였고, 16∼17시간에는 75.8%, 19∼20 시간에는 58.5%의 탈핵률을 보여 토끼난자의 탈핵은 hCG 주사후 17시간 이내에 실시

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.

The pronuclear injection of metallothionein-human growth hormone (MT-hGH) gene into rabbit zygotes was performed to establish in vitro developmental system and to detect the presence of the injected gene by nested PCR. Mature female New Zealand White rabbits were superovulated by eGG and hCG treatments. The rabbits were mated and the zygotes were collected from the oviducts 18-22 h after hCG injection by flushing with D-PBS. Two to three picoliters of MT-hGH gene was microinjected into male pronuclei. The foreign gene-injected zygotes were cultured in TCM-199 or RD mediurn containing 10% FCS with a monolayer of rabbit oviductal epithelial cefls in a 5% incubator. The presence of injected DNA in rabbit embryos or blastomeres at different developmental stages .vas detected by a nested PCR analysis. The results are summarized as follows ; 1.The developmental rate of the MT-hGH gene-injected zygotes to blastocyst was significantly higher in TCM-199 medium (68.1%) than in RD medium (42.9%). 2.The gene injection into pronuclei at 18 or 22 hours post hCG treatment during pronuclear stage did not much affect on the in vitro development of the rabbit embryos. 3.The rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased when they developed to blastocysts. The results indicate that the screening of transgene in rabbit embryos by nested PCR analysis could be a prornisible method for the preselection of transgenic embryos. Furthermore, the preselection of transgenic embryos would greatly reduce hoth the cost and effort of production of transgenic animals.