• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.6-6
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• 2001

Oocyte freezing has become a prevalent source for related reproductive technologies. This study was carried out to evaluate viability of post-thawed bovine oocyte injected DTT-treated sperm following by two different activation stimuli (Group 1, 5 M ionomycin, 5 min ＋ CR1aa, 3 h . 1.9 mM dimetylaminopurine (DMP), 3 h; group 2, ionomycin ＋ 10 $\mu\textrm{g}$/$m\ell$ cycloheximide(CHX), 5h). The techniques of ultra-rapid freezing used in this study were essentially similar to those of described by Vajta et al (Theriogenology 1999; 52:939-948), Denuded oocytes at 22 h of culture were exposed to cryoprotectant (3.2 M Ethylene glycol, 2.36 M DMSO, 0.6 M sucrose), and followed by freezing in electron microscopic grid. After thawing the oocytes were transferred back into the drop of maturation medium and cultured for additional 2 h before being subjected to ICSI. All eggs were then cultured in CRlaa medium, and transferred into M199+10% FCS on day 4. The culture was maintained until day 9. In Experiment 1, frozen-ICSI eggs were compared on development into blastocyst to those of unfrozen and IVF control. Those eggs were activated with the method of group 2. A higher proportion of unfrozen-ICSI and IVF eggs developed into cleavage and blastocysts than of frozen-ICSI eggs (65% and 13%; 71% and 23% vs. 39% and 8%; P<0.05). In Experiment 2, development and ploidy of embryos made from group 1 were compared to those from group 2. Between groups there did not differ on the rates of development, however, chromosomal abnormality in group 1 was significantly higher than in group 2 (49% vs. 30%; P<0.05). The present result suggests that frozen bovine oocytes can be used for ICSI.

• 한국수정란이식학회지
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• 제15권3호
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• pp.231-236
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• 2000

This study was conducted to examine the effects of exogenous gonadotropins (PMSG+hCG) and an antioxidant (cysteine) on in vitro maturation of bovine follicular oocytes. Cumulus-oocyte complexes (COCs) aspirated from 2 to 5 mm ovarian follicles were cultured for 22 to 24 hours in a modified bovine embryo culture medium (mBECM) supplemented with 3 mg/mL bovine serum albumin, to which PMSG (10 IU/mL) + hCG (10 IU/mL) and/or cysteine (0.6 mM) were added. When examined the expansion of cumulus ce1ls at the end of maturation culture, greater (p<0.05) expansion was found after addition of PMSG+hCG (79 to 96%) to mBECM than after no addition (0%), regardless of the presence or absence of cysteine in the medium. The addition of cysteine did not stimulate cumulus expansion, but a high proportion (92%) of expansion was achieved when COCs were cultured after the addition of PMSG+hCG and cysteine to the medium. No difference in the proportion of oocytes underwent germinal vesicle breakdown (initiation of maturation) was found after the addition of PMSG+hCG and/or cysteine to mBECM. However, nuclear maturation (development to the metaphase-II stage) of oocytes was significantly stimulated by the combined addition of PMSG+hCG and cysteine, compared with no addition. In conclusion, both exogenous gonadotropins and an antioxidant are important for nuclear maturation of bovine immature oocytes and these factors have a cell-specific stimulatory action.

• 한국가축번식학회지
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• 제21권4호
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• pp.381-387
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• 1997

This study was conducted to investigate the effects of fetal bovine serum(FBS), calf serum(CS) and human serum(HS) on in vitro maturation of porcine follicular oocytes. The results obtained were summarized as follows : 1. The maturation rates of oocytes cultured in medium containing FBS 5, 10, 20 and 30% were 47.0, 63.5, 48.4 and 43.2%, respectively. There were significantly higher than those of non-treated group(25.3%). And the highest maturation rate was the 10% treatment. 2. The maturation rates of oocytes cultured in medium containing CS 5, 10, 20 and 30% were 55.2, 56.6, 59.4 and 46.5%, respectively. There were significantly higher than those of non-treated group(25.3%). And the highest maturation rate was the 20% treatment. 3. The maturation rates of oocytes cultured in medium containing HS 5, 10, 20 and 30% were 74.5, 78.2, 73.1 and 68.6%, respectively. There were significantly higher than those of non-treated group(29.6%). And the highest maturation rate was the 10% treatment.

• 한국가축번식학회지
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• 제22권1호
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• pp.1-9
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• 1998

본 연구는 체외에서 성숙된 소 미수정란을 electron microscope grid와 동해제인 EFS30을 이용하여 초급속 동결하였을 때 정상적인 배 발달의 유도 가능성 여부를 조사하고 동해제 및 동결방법의 유해성 여부를 indirect immunocytochemistry방법으로 확인하고자 실시하였다. 동해제는 30% ethylene glycol, 0.5 M sucrose, 18% ficoll과 10% FBS가 들어 있는 PBS로 된 EFS30을 사용하였다. 본 연구에서 얻어진 결과는 다음과 같다. 동해제와 동결과정이 난자의 microtubule, microfilament 및 chromatin의 형태에 미치는 영향을 indirect immunocytochemistry방법으로 조사하였던 바, 동해제 노출 뿐 아니라 동결에 의해서도 대조군과 차이를 나타내지 않았다. 초급속 동결이 소 미수정란의 체외수정에 미치는 영향을 검토했을 때, 총 정자침투율(96.7%, 90.0%), 정상 자응전핵 형성율(74.6%, 68.9%)과 난자당 정자수(1.50, 1.44)가 동결군과 대조군에 있어서 차이는 볼 수 없었다. 또한, 초급속 동결-융해 후의 체외발달능을 조사했던 경우, 85.5%의 높은 난자 생존율과 74.5%의 난할율, 그리고 31.4%의 배반포 형성율을 얻었다. 이러한 결과는 난자의 생존율을 제외한 수정율과 배반포 형성율에 있어서 대조군(76.0%, 34.6%)과 노출군(77.9%, 33.0%)의 결과와 매우 유사한 것이었다. 이와 더불어, 각 처리군에서 얻어진 배반포기배를 Hoechst 염색방법으로 총세포수를 조사하였을 때도 그 차이는 확인할 수 없었다. 따라서 체외에서 성숙된 소 미수정란은 EM grid와 EFS30 동결액을 이용한 초급속 동결방법으로 동결하였을 때 정상적인 배발달을 유도할 수 있다는 것을 알 수 있었다.(收量)은 양공시두부(兩供試頭部) 모두 괴근장(塊根長)과 정(正)의 상관(相關)이, 분기수(分岐數)와는 부(負)의 상관(相關)이있었다.(發芽率)이 98.1%이던 것이 68.8%로 감소(減少)하였다. 3. 온도변화(溫度變化)에 따른 수수의 발아소요일수(發芽所要日數)는 12일(日)($15/10^{\circ}C$), 6일(日)($25/20^{\circ}C$) 및 3일(日)($40/35^{\circ}C$)로 온도(溫度)가 상승(上昇)함에 따라 비례적(比例的)으로 단축(短縮)되었다. 옥수수 수는 16일(日), 7일(日) 및 3일(日)이 소요(所要)되었다.量)은 $24.6{\sim}36.7%$로서 건엽중(乾葉中)의 함량(含量)보다 월등히 높았고 조단백질함량(粗蛋白質含量)은 $2.0{\sim}5.3%$로서 건엽중(乾葉中)의 함량(含量)보다 현저히 낮았다. 특(特)히 P.931의 건경중(乾莖中)의 조섬유함량(粗纖維含量)은 다른 작물(作物)에 비해 현저(顯著)히 높은 편이었다.적차이(量的差異)를 나타냈다.間)에는 부(負)(-)의 상관(相關)이 있다.($P{\leq}0.01%$). 5. NEL 및 starch value 환경온도(環境溫度)가 상승(上昇)됨에 따라 감소(減少)된다. 4 엽기(葉期) sorghum식물(植物)의 환경온도(環境溫度)를 달리 하였을 때 NEL가치(價値)는 각각(各各) 4.87MJ($30/25^{\circ}C$), 5.46MJ($25/20^{\circ}C$) 및 5.81MJ/kg($18/8^{\circ}C$)로 변(變)하여 고온(高溫)에서 net energy lactation 축적(蓄積)이 크게 감소(減少)되었다.다. 그러나 기온(氣溫)이 낮은 조건(條件)

• 한국가축번식학회지
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• 제16권3호
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• pp.239-246
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• 1992

미성숙 나포란을 20% FCS가 첨가된 TCM-199으로써 24시간 동안 5% CO2, 8% O2의 공기 조건하에서 체외성숙시킨 다음 난자의 위란강내로 정자를 미세주입하여 체외수정을 실시하였다. 미세주입 하기전 정액을 0.75% BSA가 첨가된 Ham's F-10 배양액으로 2시간 동안 5% CO2, 8% O2의 공기 조건하에서 배양함으로써 수정능 획득을 하도록하였으며 이어 30분 동안 12mM의 dbcGMP와 10mM의 imidazol이 함유된 Ham's F-10 배양액으로 배양함으로써 첨체반응을 유도하였다. 본 실험에서 정자의 미세주입 후 제 2극체와 전핵형성이 일어난 난자의 비율은 각각 9.5, 5.4%였으며 2세포기 이후로 발달한 난자의 비율은 4.1%이었다.

• Journal of Animal Science and Technology
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• 제63권2호
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• pp.272-280
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• 2021

Cumulus-oocyte complexes (COCs), which contain immature oocytes, are matured in vitro for in vitro embryo production. Oocyte and cumulus cells are then separated using hyaluronidase. To date, there have only been a few reported cases of the toxic effects of hyaluronidase on porcine oocytes. The aim of this study was to compare the effects of bovine testis-derived hyaluronidase and recombinant human hyaluronidase on oocyte denudation and quality. Porcine COCs were matured for 44 h and denuded using different hyaluronidase concentrations and exposure times. Then, oocytes were activated by electrical parthenogenesis. In experiment 1, COCs were denuded using bovine-derived, ovine-derived (Hirax), and human recombinant (ALT-BC4) hyaluronidases for 10 and 20 min. In experiment 2, bovine-derived and human recombinant (ALT-BC4 and ICSI Cumulase®) hyaluronidases were used to denude the COCs for 2 and 20 min. In both experiments the oocytes were all completely denuded, and there was no degeneration. Rate of embryo development was significantly increased in group treated ALT-BC4 for 2 min and not significantly different in other treatment groups. In general it slightly decreased with longer exposure times. These results have confirmed that different sources of hyaluronidase do not have detrimental effects on the quality of porcine oocytes and suggest that the human recombinant hyaluronidase ALT-BC4 is suitable for oocyte denudation with an increased blastocyst rate.

• 한국수정란이식학회지
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• 제9권2호
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• pp.145-152
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• 1994

The suitable electric stimulation is essential for activation and fusion of oocytes before or after nuclear transplantation The present study was undertaken to determine the optirnal condition for the parthenogenetic activation of in vitro rnatured(IVM) bovine oocytes by electric stimulation. Different direct current(DC) electric voltage of 1.0, 1.5 and 2.0 kV/cm and pulse duration of 30, 60 and 120 $\mu$sec were applied to the JVM nocytes in 0.3 M mannitol solution containing each 100 $\mu$M CaCl$_2$ and MgCl$_2$. IVM occytes at 24, 28 and 32 hours Post-maturation(hpm) were also electrically stimulated at 1.5 kV /cm, for 60 $\mu$ sec. The stimulated nocytes were then co-cultured in TCM-199 solution containing 10% fetal calf serum with bovine oviductal epithelial cells for 7~9 days in a 5% $CO_2$ incubator at 39$^{\circ}C$ ~ Their activation and in vitro development to morula and blastocyst were assessed under an inverted microscope. The higher activation rates 62.8 and 63.4% and in vitro de- velopment rates to morula and blastocyst 5.1 and 10.9% were shown in the oocytes stimulated at the voltage of 1.0 and 1.5 kV/cm than 2.0 kV/cm, respectively. No signifi- cantly(P<0.05) different activation rate was shown in JVM oocytes stimulated for 30, 60 and 120 $\mu$sec, but developmental rates to morula and blastocyst was significantly(P<0.05) higher in the oocytes stimulated for 30 $\mu$sec(6~3%) and 60 $\mu$sec(10~0%) than 120 $\mu$sec(0~ 0%). The aged oocytes at 28 and 30 hpm showed significantly(P<0.05) higher activation rates(72~7 and 79.7%) than the oocytes at 24 hpm(50~9%)~ Also, their developmental rates to morula and blastocyst were significantly(P<0.05) higher in the nocytes at 28(14.3%) and 32 hpm(15.9%) than 24 hpm(3.6%). From these results, it can be suggested that the optimal electric stimulation for IVM bovine occytes is a DC voltage between 1.0 and 1.5 kV/cm, pulse duration of 30 or 60 $\mu$sec, and the optimal age of IVM oocytes for electric activation is at 32 hpm.

• 한국수정란이식학회지
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• 제8권2호
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• pp.143-149
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• 1993

The objective of this study was to produce calves derived from in vitro fertilization of in vitro matured follicular oocytes. Oocytes aspirated from small antral folicles of ovaries obtained at a local slaughter house were matured and fertilized in vitro. At l8hrs after insemination with Korean native cattle semen, oocytes were co-cultured for 6~7 days by utilizing co-culture system with bovine oviduct epithelial cell. After co-culture, good or excellent quality late morulae or early blastocysts were selected by morphological criteria under stereo microscope. Selected embryos were transferred to recipients on day 6 or 7 (estrus = day 0). Recipients were monitored by observation for estrus and rectal palpation after 60 days from embryo transfer. One of them went to term with the birth of a calf. This case is the first production of calf derived from in vitro fertilization in Korea.

• Asian-Australasian Journal of Animal Sciences
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• 제1권3호
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• pp.153-156
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• 1988

Bovine blastocysts were obtained by in-vitro culture of embryos derived from in-vitro fertilization of oocytes matured in-vitro. These blastocysts and blastocysts from inseminated donors were bisected by a simple method (without a holding pipette) using a microblade operated by a micromanipulator. A pair of demi-embryos was transferred nonsurgically into each uterine horn of a recipient cow 6 or 8 days after estrus. Pregnancy resulted from the third transfer. Ultrasound examination done 52 days after estrus (46 days after transfer) confirmed the presence of at least one fetus in the each uterine horn. This is the first report to show the viability of bisected bovine blastocysts obtained from in-vitro culture of embryos derived from in-vitro fertilization of oocytes matured in-vitro. In addition, a simple method to bisect bovine embryos is described.

• 한국가축번식학회지
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• 제18권2호
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• pp.121-126
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• 1994

This study was conducted to examine the condition of activation of the nuclear transplant bovine embryos. In vitro fertilized(IVF) and nuclear transplant embryos(NTs) were co-cultured with bovine oviduct epithelial tissue(BOET). NTs were treated with cycloheximide(CHXM) for 0 to 6 h after electrofusion to investigate the activation conditin of recipient ooplast. Then, the infljence of the CHXM treatment timing on the cleavage and development of NTs were investigated in relation to the nuclear transplant time. The cleavage rates of NTs were increased with the increasing time of the CHXM treatment from 0 to 6 h (54.7 to 91.3%, P<0.01). Similar trend was shown in the development into the morula or blastocyst stage, but very limitted. Activation of enucleated oocytes prior to fusion enhanced development of NTs compared with that post fustion. This result suggests that the frequency of activation of NTs can be greatly enhanced by treating with CHXM for 6 h. The result also suggests that if blastomeres of unknown cell cycle stage are used, activation of enucleated oocytes prior to fusion enhances development of NTs.