• Journal of Embryo Transfer
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• v.32 no.3
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• pp.201-207
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• 2017

To increase the productivity of in vitro development, the antioxidants have been used for culture system of bovine oocytes and embryos. However, comparative studies on these molecules are rare and direct beneficial effects on blastocyst production cannot be discriminated for best results. The study was conducted to determine the influence of N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime (CYS) on maturation competence of COCs from GV to MII stage and productivity of blastocyst formation during in vitro fertilization and culture. There was no difference among maturation rates of oocytes to metaphase II with polar body with antioxidants for any of the treatment groups (p>0.05). However, the significant improvement on the rate of blastocysts ($32.3{\pm}5.0%$) was found in 0.1 mM CYS treatment than 0.3 mM NAC, 0.2 mM NACA or 0.5mM GSH (p<0.05). The addition of NAC ($18.8{\pm}3.7%$) or NACA ($21.2{\pm}3.9%$) did not improve development competence to morula and blastocysts than control ($24.4{\pm}4.1%$) and GSH ($26.5{\pm}5.0%$) (p>0.05). Our study showed that medium supplementation with CYS during IVM and IVC improved the rate of bovine embryo development but not with NAC, NACA and GSH addition.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.241-251
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• 1997

체외성숙과 수정된 돼지 난자의 체외발달능이 체외배발생 배양액인 NCSU 배양액에 0.4% BSA, 10% 혈청 혹은 아미노산 (2% BME 아미노산 용액과 1% MEM 아미노산 용액)을 첨가함으로서 조사되었다. 본 실험에 공시된 난자는 체외수정 추 30시간 (2-세포기)혹은 48 시간 ($2{\sim}4$-세포기)에 회수하였다. 실험I에서 0.4% BSA가 첨가된 NCSU 배양액에서 2-세포기 난자들의 배양경과시간에 따른 발달능을 조사한 결과, 배양 후 72 시간 (체외수정 후 102 시간)에 상실배기와 배반포기 배가 나타났으며, 배양 후 120 시간째 (체외수정 후 150 시간)에도 팽창된 배반포기 배까지만 발달하였다. 실험II는 체외수정 후 48 시간의 분할된 ($2{\sim}8$-세포기) 난자들의 핵과 외관적 분할구와의 수적 차이를 조사한 결과, $2{\sim}4$-세포기보다는 5-세포기 이상에서 핵과 분할구의 조화에 차이가 많았다. 실험III에서는 $2{\sim}4$-세포기 난자들을 배양후 5일째의 배반포들의 투명대의 두께, 난자 크기 그리고 inner cell mass (ICM)과 trophectoderm (TE)의 세포 배열을 조사한 결과, 난자의 크기가 커짐에 따라서 투명대가 얇아지고 전체 세포수가 증가하였지만, ICM의 비율은 차이가 없었다. 실험IV에서는 BSA, 혈청 혹은 아미노산이 첨가 혹은 무첨가된 배양액내에서 $2{\sim}4$-세포기 난자들의 배반포 후 부화능력을 조사한 결과, 모든 군에 있는 난자들은 팽창된 배반포기 배까지 발달할 수 있었던 반면, 난자의 부화는 아미노산 혹은 혈청이 포함된 배양액에서만 일어났다. 더우기 상실배기와 배반포기 시기에 혈청의 첨가는 부화 배반포기 배의 발달을 현저히 증가시켰다. 또한 아미노산과 혈청의 영향을 받은 팽창 배반포기 배는 얇은 투명대, 팽창된 난자의 크기 그리고 ICM과 전체 세포수의 증가를 보였다. 이상의 결과로 미루어 볼때, 배양액내에 대한 아미노산과 혈청의 첨가는 돼지 배반포기 배의 부화를 유도할 수 있다고 보며, 더우기 이들 요소들은 투명대의 두께, 난자의 크기 그리고 ICM과 전체 세포수에 영향을 미친다.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.185-189
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• 2004

The purpose of this study was to determine the possibility of in vitro maturation of tiger oocytes. Immature oocytes were recovered from a pair of ovaries. A total of 78 oocytes was collected, of which forty three were classified as good oocytes with compact cumulus cells and uniform cytoplasm. Forty three COCs were in vitro matured at $39^{\circ}C$, 5% CO2 in air atmosphere for 48 h in a IVM medium (TCM-199 supplement with 10% FBS, 0.6 mM cysteine, 0.2 mM pyruvic acid and 10 IU/mL HMG). Experiment I: the morphologic evaluation was conducted by measuring the diameter of oocytes with or without ZP, the thickness of ZP and the diameter of cytoplasm by microeyepiece at the same magnification (${\times}$100). Experiment II: the evaluation of meiotic development was conducted of the nuclear development stage of tiger oocytes. The results were summarized as follows: 1. The diameter of tiger oocytes $(176.5\pm6.1{\mu}m)$ with ZP was significantly (p<0.05) bigger than that of bovine oocytes $(150.7\pm4.9{\mu}m).$ The ZP thickness of tiger oocytes $(20.4\pm2.9{\mu}m)$ was significantly (p<0.05) bigger than that of bovine oocytes $(12.0\pm2.6{\mu}m;$ p<0.05). However, there was no significant difference in the diameter of cytoplasm (without ZP) between tiger $(122.1\pm9.7{\mu}m)$ and bovine oocytes $(118.7\pm7.5{\mu}m).$ 2. The rates of meiotic development of tiger oocytes were achieved GV (12.5 %) and MII (50.0%), respectively. These results indicated that tiger oocytes could be developed to MII in in vitro culture system.

• The Korean Journal of Zoology
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• v.22 no.4
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• pp.175-183
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• 1979

This simple culture device is based on the microtube culture method (Cho, 1974). The microtube culture system was placed in a plastic tube (Falcon, 12 $\\times$ 75 mm) and the transporting system used in the present study were Trans-Temp 310 (Royal Industries, 209 D 135-1). The simple culture device in the Trans-Temp 310 seems to be an available method for short term culture and some distance transportation of oocytes and embryos.

• 대한생식의학회:학술대회논문집
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• 2007.11a
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• pp.104.2-105
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• 2007

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.91-91
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• 2003

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.75-80
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• 2003

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.303-309
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• 2005

This study was conducted to examine the effects of OA on metaphase of meiosis II and the mitochondrial activity of cytoplasm in bovine cumulus oocytes complexes(COCs) during in vitro maturation. Hanwoo COCs were collected from the slaughterhouse cow ovaries and matured in TCM199 supplemented with $0.1\%$ PVA, 0.2 uM, 2 uM, 20 uM OA for the maturation rate of OA concentration. For the maturation effects between OA and cycloheximide(CX), COCs were matured in TCM199 with 25 ug/mL CX, 25 ug/mL CX (6 hrs culture) plus 2 uM OA or 2 uM OA only at a atmosphere $5\%\;CO_2,\;95\%$ air $39^{\circ}C$ for 6, 12, 24 hrs. To evaluate the nuclear types of matured COCs, cumulus cells were removedby $0.5\%$ hyaluronidase sol. and oocytes were fixed in 1:3 acetic acid ethyl alcohol for 30 sec. and then stained with $0.1\%$ basic Fuchsin sol. For the detection of fluoriscent intensity (FI) of matures oocytes, cumulus cells were removed same as performed above and were stained with 20 nM mite tracker for 20 min. at $39^{\circ}C$. Mitochondrial activity of FI in matured oocytes was imaged by laser conforcal microscopy (Fluoview, Olympus, Japan) and were measured scanned face on 5 um from median to endpoint of oocytes. Statical analysis of nuclear types observed the three replicates was carried out with ANOVA and Fisher's protected least significant difference test using the STATVIEW program. FI of matures oocytes was compared the multiples of the least intensity among the measured oocytes. Maturing in TCM199 supplemented with $0.1\%$ PVA, 0.2 uM, 2 uM, 20 uM OA, metaphase B were showed 72.0, 50.0, 70.0, $68.8\%$, respectively and there were different significant(p<0.05). In the case of treatment with OA and CX, metaphase were $73.8\%,\;8.2\%,\;45.5\%,\;73.7\%$ in $0.1\%$ PVA-TCM199, 25 ug/mL CX, 25 ug/mL CX plus OA or 2uM OA only, respeclively. FI was revealed the increasing tendency during the process of maturation. Whereas FI in CX was decreased about 3 times compared to the other treatments of 6 hrs maturation. We conclude that OA regulates bovine COCs maturation and induces the mitochondrial activity during the process of maturation.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.413-421
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• 1997

In vitro culture has provided new information on the mechanisms involved in fertilization how sperm and oocyte fuse together. At the same time, results obtained in vitro have led to new questions. Techniques for In vitro maturation of porcine oocytes have progressed such that the problem of the low rate of pronucleus formation with in vitro matured oocytes after in vitro fertilization has been nearly improved. On the other hand, porcine spermatozoa have been shown to be capacitated if the fertilization medium contains caffeine and Ca$^2+$, but the incidence of polyspermy in IVM-IVF oocytes is still high. To prevent polyspermy, co-culture with oviductal cells, sperm preincubation with porcine follicular fluid or control of sperm concentration, have been examined with significant effects but still remarkably high rates of polyspermy. The under standing of these influences is a prerequisite to enhancing in vitro production of porcine em bryos.

• Journal of Embryo Transfer
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• v.7 no.2
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• pp.97-110
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• 1992

돼지난포란의 안정된 체외배양 체계를 위하여 도살장에서 재취된 난소로부터 난포 및 난자난구세포체의 혀태적인 선정기준을 설정하고 이의 이론적 배경을 확립하였다. 난소를 난포상태 및 분포나 황체 존재 여부에 따라 A,B 및 C 의 세 가지 형태로 분류하고 각 type의 난소에서 직경 3-5mm인 난포로부터 난포란을 회수하였다. 회수된 난포란을 난구세포 부착상태에 따라 Good, Fair 및 Poor 의 세 가지 형태로 분류하여 각각을 호르몬이 첨가된 M16+FCS 배양액으로 35시간 동안 체외배양하여 성숙율을 비교 검토 하였다. 난소의 형태에 따라 회수된 난포란 중 Good 또는 Fair 형태는 Type A 및 C 난소에서 85%를 차지한 반면, Type B 난소에서는 53%에 불과하였다. 또한 이들 난포란을 체외배양한 결과 Type A 및 C 에서 회수된 난포란은 90 및 85%의 높은 성숙율을 보인 반면, Type C 의 난포란은 33%의 저조한 성숙을 나타냈다. 한편 핵형 분석 및 조직학적 분석에서도 Type C 난소의 경우 난포란의 핵형이 대부분 GVBD 및 퇴화 형태를 보였으며, 폐쇄포난포의 비율도 Type A 난소의 53%에 비해 월등히 높은 85%를 나타내어 성숙율 비교실험의 결과와 일치되는 경향을 나타내었다. 따라서 본 실험의 선별기준에 의한 돼지 난소 및 난포란의 형태적 분류 작업에 의해 난포란의 체외배양 성적 향상 및 안정된 배양체계의 확립이 가능하였다.