• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.163-168
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• 1992

This study was carried out to set up the ovum bank for ovum donation and to determine the best freezing method for human immature oocytes. Human immature follicular oocytes were cryopreserved by slow freezing and rapid thawing method. Immature follicular oocytes were treated by propanediol(PROH) solution by 2 and 4 step method in protocols A & B, respectively. In protocol C, immature oocytes were exposed to sucrose prior to treatment of PROH by 4 step method. We compared survival rate, maturation rate, and fertilization rate of immature oocytes among three protocols. Results were as follows. 1. Oocytes treated by the protocol C showed the highest survival rate( 70.3 %) and maturation rate(34.6%) after thawing. 2. Survival rate of oocytes treated by the protocol C was significantly higher than that of the protocol B after thawing(p<0.05). In conclusion, treatment of oocytes with sucrose prior to expose PROH was the best freezing method. Sucrose may have reduced the toxic effect of cryoprotectant to oocytes. We failed to induce fertilization of oocytes, which were treated by any protocols, by conventional insemination method, but obtained 28.8% fertilization rate by using partial zona dissection(PZD) method. This result suggests that micromanipulation(PZD) of the thawed oocytes before insemination will improve the fertilization rate.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.5-5
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• 2001

This study was to test whether Hanwoo in vitro matured oocytes can be successfully cryopreserved by a new vitrification procedure using MVC method. For the vitrification, oocytes were pretreated in 10% ethylene glycol (EG10) for 5-10 min, exposed in EG30 for 30 sec, each oocytes were individually put on the inner wall of 0.25 $m\ell$ straw, and then straws were directly plunged into L$N_2$. Thawing was taken by 4-step procedures [1.0 Msucrose (MS), 0.5 MS, 0.25 MS, and 0.125 MS] at 37$^{\circ}C$. In vitro developmental capacity (survival, cleavage ($\geq$2-cell) and blastocyst rates) in vitrified group was no significant difference compared to that in other treatment groups (exposed; 100.0, 74.4, 32.3% and control; 100.0, 78.3, 36.3%): high mean percentage of oocytes (91.2%) was survived, 69.4% of them were cleaved and 27.9% of cleaved embryos were developed to blastocyst. Especially, after transfer of in vitro developed embryos in vitrified group, four of six recipient animals were found to pregnant and three of them were ongoing pregnant by manual palpation at 250 days after transfer. However, among them, two healthy female calves (23 and 25kg) were born. This result demonstrates that MVC method is very appropriate freezing method for the Hanwoo in vitro matured oocytes and that ovum bank can be maintained efficiently by MVC cryopreservation method.

• Clinical and Experimental Reproductive Medicine
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• 제48권1호
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• pp.85-90
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• 2021

We present data from three Caucasian men with Zinner syndrome who attended our center for the treatment of primary couple's infertility. Each patient was scheduled for conventional testicular sperm extraction (cTESE) and cryopreservation. Sperm analysis confirmed absolute azoospermia. Patient 1 had right and left testis volumes of 24 mL and 23 mL, respectively; left seminal vesicle (SV) agenesis, severe right SV hypotrophy with right renal agenesis. Follicle-stimulating hormone (FSH) was 3.2 IU/L. Patient 2 exhibited right and left testis volumes of 18 mL and 16 mL, respectively; a left SV cyst of 32 × 28 mm, ipsilateral kidney absence, and right SV agenesis. FSH was 2.8 IU/L. Patient 3 showed a testicular volume of 10 mL bilaterally, a 65 × 46 mm left SV cyst, right SV enlargement, and left kidney agenesis. FSH was 32.0 IU/L. Sperm retrieval was successful in all patients. Nevertheless, cTESE should be performed on the day of oocyte retrieval.

• Clinical and Experimental Reproductive Medicine
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• 제25권3호
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• pp.287-292
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• 1998

본 연구는 동해방지제인 EFS35를 이용한 생쥐 미수정란의 초자화동결이 cytoskeleton과 염색체에 미치는 영향을 indirect immunocytochemistry와 염색체 분석으로 확인하고자 실시하였다. 생쥐 미수정란은 M2 배양액에 35% ethylene glycol, 18% ficoll, 0.3 M sucrose와 10% FBS가 함유된 EFS35를 이용하여 초자화동결되었다. 본 연구에서 얻어진 결과는 다음과 같다 초자화동결된 미수정란의 융해후 생존율은 89.3%로써 노출군 (97.7%)과 차이가 없었다. 초자화동결이 미수정란의 microtubule과 microfilament에 미치는 영향을 조사하였던 바, 동결군 (95.5, 100%)의 microtubule과 microfilament의 정상적인 형성율은 대조군 (97.5, 100%) 또는 노출군 (92.3, 100%)의 결과와 비교하였을때 차이가 나타나지 않았다. 또한, 체외수정 후 동결 군에서 정상적인 염색체수를 가진 난자의 비율도 73.5%로써, 대조군 (79.5%)과 노출군 (78.7%)의 결과와 차이가 없었던 바, 생쥐 미수정란을 EFS35에 노출하고 동결하는 것이 미수정란의 cytoskeleton과 염색체 성상변화에 영향을 미치지 않는다는 것을 알 수 있었다. 따라서, 본 연구에서 사용된 동해방지제인 EFS35를 이응한 초자화동결법은 생쥐 미수정란의 동결에 적합하다는 것을 시사한다고 하겠다.

• Clinical and Experimental Reproductive Medicine
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• 제29권3호
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• pp.201-207
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• 2002

Objective: To observe the capability of fertilization and embryo development including blastocyst formation of the oocytes in simple media after thawing of the cryopreserved cumulus-free mouse oocytes by vitrification method. Methods: Oocytes were collected from 5 to 6 weeks old ICR female mice, and were denuded from the cumulus cells by 0.1% hyaluronidase. Recovered mature oocytes in study group were cryopreserved by vitrification method using EM grid for $5{\sim}7$ days. In brief, oocytes were exposed in dPBS containing 1.5 M EG and 5.5 M EG+1 M sucrose for 2.5 minutes and 20 seconds each, and then executed vitrification by plunging in LN2 after loading on EM grid. Thawing treated by exposure of 1, 0.5, 0.25 and 0.125 M sucrose solution for 2.5 minutes each in order and used for experiments. Spermatozoa aspirated form the epididymis of 12 weeks old ICR male mice were used for insemination after capacitation. T6 media containing 0.4% BSA were used for fertilization and development. Results: Survival and fertilization rates after thawing were 76.9% and 79.6% respectively. Fertilization rate was lower (p<0.005) than that of control group (92.9%). There was no difference in embryo developmental rates from 2-cell to morula, however, the blastocyst formation rate and mean cell numbers of blastocysts in study group (63.3%, $58.9{\pm}9.2$) were lower compared with those of control group (76.1%, $63.5{\pm}8.9$). Conclusion: Vitrification is an effective method for mouse mature oocyte cryopreservation with high survival and fertilization rate after thawing. And in simple media, fertilization rates and embryo development of frozen-thawed mouse oocytes are satisfactory.

• Asian-Australasian Journal of Animal Sciences
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• 제14권8호
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• pp.1188-1195
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• 2001

A tetraparental chimeric bull was successfully produced by aggregating bovine IVF embryos of F1 (female Holstein${\times}$male Japanese Black) and F1(female Japanese Brown${\times}$male Limousin) and culturing in vitro without the zona pellucida at Yamaguchi Research Station in Japan. In the microsatellite genotyping, 12% (28/228) microsatellite primer sets ware potentially useful for this parentage analysis in the chimeric bull, 78.6% (22/28) of microsatellite present in the chimeric bull were uniquely contributed from the Japanese Black and 21.4% (6/28) from Limousin. This chimeric bull semen was used in producing IVF embryos. The chromosome preparations were made from peripheral lymphocytes. Based on chromosome analysis the Chimera had apparently normal chromosomes (29 acrocentric pairs, one large sub metacentric X chromosome and one small sub metacentric Y chromosome). The proportion of acrosome reacted spermatozoa after 1 h of incubation was higher (p<0.01) with the Chimera than with the Holstein and in Japanese Brown bulls. But did not differ from Japanese Black and Limousin bull sperm. Fertilization rates observed after 5 h of sperm-oocyte incubation with Chimera sperm were higher (p<0.05) than with Japanese Brown and (p<0.01) than with Holstein sperm, but did not differ from Japanese Black and Limousin sperm. The cleavage rates of IVF oocytes inseminated with Chimera sperm were also higher (p<0.001) compared with Holstein, (p<0.01) Japanese Brown and (p<0.05) Limousin, but did not differ from Japanese Black sperm. The blastocyst rates of IVM oocytes inseminated with sperm were higher (p<0.05) than in Limousin, Japanese Brown and Holstein, but did not differ from Japanese Black. Chimeric cattles were produced by aggregation of parthenogenetic (Japanese Brown) and in vitro fertilized (Holstein) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus) and in vitro fertilized (Holstein) embryos at the St. Gabriel Research Station in Louisiana. The aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding. The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derieved embryos cultured without agar than when cultured with agar. The development rate to blastocysts, however, was not different among the treatment. To verify parthenogenetic and the cells derieved from the male IVF embryos in blastocyst formation, 51 embryos were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zonafree chimeric embryos at 24 h following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP. Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transfer of 40 chimeric embryos at the Louisiana station. Two pregnancies were Jost prior to 4 months and one phenotypically chimeric viable male born.

• 한국수정란이식학회지
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• 제32권4호
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• pp.311-317
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• 2017

Cryopreservation of oocytes by vitrification technique may contribute a lot in the field of reproductive biotechnology. The objectives of the present study were to evaluate the effectiveness of two cryo-devices for vitrification of immature oocytes of indigenous zebu cows. Slaughter house derived immature cumulus-oocyte-complexes (COCs) of cows were vitrified using 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA) with 0.5 mol sucrose in TCM 199 supplemented with 20% FBS. Vitrification of COCs was completed after immediate plunging of COCs loaded cryotop or French mini straw into the liquid nitrogen ($LN_2$). Then the COCs containing cryotop or French mini straws were warmed in 0.25 mol sucrose and 20% FBS supplemented TCM 199 followed by in vitro culture in $50{\mu}l$ droplets of bicarbonate buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 hrs at $39^{\circ}C$ with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. Denuded oocytes were also stained by whole mount technique using 1% orcein to examine the maturation by presence of MII chromosomes. The in vitro maturation rate was significantly (p<0.05) higher in oocytes vitrified and warmed using crytop ($47.1{\pm}6.9%$) than that of French mini straw ($15.9{\pm}12.5%$). Moreover, in vitro maturation rate was significantly (p<0.05) highe r in control oocytes (not vitrified) ($84.5{\pm}14.2%$) than that of vitrified oocytes. In conclusion, cryotop is better than French mini straw as cryo-device for vitrification of bovine immature oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.71-76
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• 1998

본 연구는 소 미성숙난자를 electron microscope (EM) grid와 동해제인 EFS30을 이용하여 초급속 동결하였을 때 정상적인 배 발달의 유도가능성 여부를 조사하고자 실시하였다. 동해제는 30% ethylene glycol, 18% ficoll, 0.5 M sucrose와 10% FBS 등이 PBS에 첨가되어 제작된 EFS30을 사용하였다. 난자 생존의 평가기준으로는 성숙, 수정 및 배발달을 조사하였다. 본 연구에서 얻어진 결과는 다음과 같다. 초급속동결-융해 후, 소 미성숙란의 생존율은 43.2%을 나타내었다. 동결-융해군의 체외 성숙 (84.1%)과 정상 자웅전핵 형성율 (57.5%)은 대조군의 결과 (92.5, 65.0%)와 비교하여 볼 때 유의한 차이는 없었다. 또한, 동결군의 체외수정 이후의 $\geq2$-세포기 형성 (65.0%)과 배반포형성율 (30.8%)도 대조군(73.7, 35.7%)의 결과와 유의한 차이를 나타내지 않았다. 따라서 소 미성숙난자는 EM grid와 EFS30 동결액을 이용한 초급속 동결방법에 의해 정상적인 배발달이 유도될 수 있다는 것을 알 수 있었다.

• 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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• 제25권1호
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• pp.1-7
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• 2001

본 연구는 체외에서 성숙된 한우 미수정란이 새로운 초자화 동결 방법인 MVC 방법으로 성공적으로 동결 보존될 수 있는지의 여부를 확인하고자 실시하였다. 초자화 동결을 위해서 미수정 난자는 EG10에서 5~10분간 전처리하고 EG30에서 30초간 노출하였으며 0.25 $m\ell$ 스트로의 내벽에 난자를 각각 적하한 다음, 곧바로 액체 질소에 침지하였다. 응해는 37$^{\circ}C$에서 4단계로 이루어졌다 (1.0M sucrose (S), 0.5 MS, 0.25 MS와 0.125 MS). 한우 미수정 난자를 MVC 방법을 이용하여 초자화 동결하였던 바 체외에서의 발생능이 다른 군과 유의한 차이를 나타내지 않았다 (생존율, 난할율, 배반포 형성율: 동결군 - 91.2, 69.4, 27.9%; 노출군 -100.0, 74.4, 32.3% : 대조군 - 100.0, 78.3, 36.3%). 또한, 체외에서 발달된 동결군의 난자를 6마리의 대리모 소에 이식하였던 바, 4마리가 임신에 성공하여 이중 3마리가 임신중임이 임신 250일에 직장검사를 통하여 확인되었다. 따라서, MVC 동결 방법은 한우 미수정란을 동결하기에 적합한 방법이며 앞으로 이 방법을 통하여 난자은행이 효율적으로 이용될 수 있으리라 사료된다.