• 한국수정란이식학회지
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• 제31권4호
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• pp.367-374
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• 2016

This study was conducted to evaluate the effect of transfer temperature of epididymis on survival rate of semen and development ability of B6D2F1 mice embryos. No significant differences were noted in the survival rate of semen ($59.0%{\pm}0.1$ vs. $47.6%{\pm}0.1$), in vitro fertilization rate ($90.7%{\pm}0.1$ vs. $90.7%{\pm}0.1$), developmental rate ($90.0%{\pm}0.1$ vs. $90.0%{\pm}0.1$), and blastocysts formation rate ($53.1%{\pm}0.2$ vs. $52.3%{\pm}0.2$) between groups. (NS; P>0.05). However, the zona hatched rate was significantly higher in the $4^{\circ}C$ group compared to those of the $37^{\circ}C$ group ($47.8%{\pm}0.1$ vs. $25.6%{\pm}0.2$; p<0.05). When it comes to cell numbers of blastocysts, the % ICM (/total cells) was significantly higher in the group of $4^{\circ}C$ compared to the $37^{\circ}C$ ($27.0%{\pm}0.1$ vs. $18.3%{\pm}0.1$; p<0.05). However there were no differences in total cell numbers ($72.7{\pm}31.6$ vs. $62.0{\pm}36.6$), ICM cell numbers ($17.0{\pm}7.8$ vs. $14.6{\pm}8.6$), TE cell numbers ($55.8{\pm}29.8$ vs. $64.0{\pm}24.4$), the ratio of ICM:TE ($1:4.2{\pm}4.1$ vs. $1:6.4{\pm}7.2$) between two groups (NS; P>0.05). Taken altogether, it is expected to achieve the best developmental ability of B6D2F1 mice embryos in the transfer temperature of epididymis. Also these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice. In future, therefore, it is expected that results herein might be applied for in vitro culture of human embryos.

• 한국가축번식학회지
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• 제20권2호
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• pp.127-134
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• 1996

본 연구는 epidermal growth factor(EGF)가 처리된 돼지 체외성숙란의 수정능력을 조사하기 위하여 실시하였다. 돼지 미성숙란의 성숙을 위해서 배양액내에 EGF(10ng/ml)와 FSH(10$\mu\textrm{g}$/ml) 또는 FBS(10%)를 단독 혹은 공동 첨가함으로서 4처리군으로 처리하였다(1:무처리군, 2:EGF 단독 처리군, 3: FSH와 FBS 공동 처리군, 4: EGF와 FSH 그리고 FBS 공동 처리군). 그 결과 2, 3, 4 처리군의 핵성숙율은 무처리군보다 현저하게 높았다(P<0.001). 수정율에 있어서 EGF 단독 처리군의 경우 3과 4 처리군보다 낮았지만, 무처리군보다는 현저하게 높았다(P<0.05). 더욱이 EGF와 FSH 그리고 FBS의 공동 처리군의 경우 다른 모든 처리군에 비하여 정자 침입뿐만 아니라 웅성 전핵 형성율이 높았다(P<0.05). 따라서 돼지 난포란의 체외성숙시 EGF 단독 처리만으로는 핵성숙에 수반된 세포질 성숙률은 낮았지만, EGF와 FSH 및 FBS를 공동 처리했을 때 돼지 체외성숙란의 세포질 성숙도 효과적으로 유도된다는 것을 알 수 있다.

• 한국수정란이식학회지
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• 제16권2호
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• pp.117-125
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• 2001

본 연구는 OPS 방법에 의한 돼지미성숙 및 성숙난자의 동결-융해 후 난자의 생존성에 있어서 난구세포의 영향을 검토하였다. 그 결과 미성숙 난자의 동결-융해 후의 성숙율은 난구세포의 부착 (25%) 및 제거 (15%)시 유의적인 차이는 인정되지 않았지만 control group (62%)에 비해서는 유의적으로 낮게 나타났다(P<0.05). 미성숙난자의 동결-융해 후 체외성숙시킨 난자의 체외수정시 난구세포 제거시 (19%), 부착된 (9%) 난자에 비해 높은 정자 침입율을 나타냈으나 유의적인 차이는 인정되지 않았다. 그러나 체외수정후의 난자 성숙율은 난구세포의 부착시 (41%), 제거된 (17%) 난자에 비하여 유의적 (P<0.05)으로 높았으며. 난구세포 제거시 부착된 난자에 비해 유의적 (P<0.05)으로 높은 사멸율을 나타냈다. 한편, 체외에서 성숙시킨 난자의 동결-융해후 체외수정에 이용하였을 때 정자침입율은 난구세포 제거시 (35%, 부착된 (26%)난자에 비해 유의적으로 높았지만 이때 난자의 사멸율은 난구세포 부착 난자에서 오히려 유의성은 높았다. (P<0.05). 또 다른 실험에서 체외수정 후 6시간에서 정자침입율은 난구세포의 유무에 의한 차이는 인정되지 않았으나. 난구세포 제거시 높게 나타났으며, 이때 난자의 사멸율은 난구세포 부착시 높았으나 유의적인 차이는 인정되지 않았다. 그러나 정자침입이 일어난 난자의 경우 다정자 침입난자가 전혀 관할되지 않았다. 본 연구치 결과로부터 OPS방법에 의한 돼지 미성숙, 성숙난자의 동결은 미성숙난자의 경우 난구세포의 부착이 효과적인 것으로 사료되며, 성숙난자의 경우 난구세포 제거시 정자침입율이 높게 나타나 동결-융해시 난구세포가 난자의 성숙과 수정 시 정자의 침입에 관여하는 것으로 사료된다.는 것이 시사되었다.가지고 있는 아동이, 남아의 경우, 경도비만아의 16.3% 중 2등도비만아의 19.0%, 고도비만아의 26.4% 여아의 경우 각각 11.1% 35.0% 및 47.5%인 것으로 나타났으며. 비만도와 심혈관계질환 위험인자 수간의 상관성은 여아에 있어 서 유의 적인 것으로 나타났다(r = 0.331, p = 0.001).7배와 11배의 차이를 보였다.m-coil로 전이 되어짐을 알 수 있었다. 이는 본 연구에서 분리 정제된 curdlan이 정확한 $\beta$-결합을 가진 glucan임이 확인되었으며 생합성된 curdlan의 구조와 분자적인 특성을 확인하였다. 본 연구를 통하여 $\beta$-결합을 가지는 glucan의 신속한 선별법을 구축할 수 있었고 이는 새로운 기능을 가지는 $\beta$-glucan 생합성 효소나 분해효소의 탐색에 이용될 것으로 기대된다. 남학생이 251.4mg, 여학생이 159.3mg이었다. 이상의 결과로 보아 에너지, 단백질, 칼슘, 비타민 A 등 대부분의 영양소 섭취량이 권장량에 미치지 못하여 성장기에 있는 아동들의 영양소 섭취 상태에 문제가 있음을 시사해 주었다. 5. 조사 대상자의 식습관과 영양소 섭취량의 상관관관계는 식사 시간의 규칙성은 단백질, 식이성 섬유소, 비타민 A 및 티아민섭취량과 양의 상관을 나트륨섭취량과는 음의 상관관계가 있었다. 음식의 간(염도)은 인과 콜레스테롤 섭취와 음의 상관관계를 나타냈다. 들깨가루나 들기름 사용 여부는 열량, 탄수화물 및 인의 섭취량과 음의 상관을, 지방, 철분, 리보플라빈 및 나이아신섭취량과는 양의 상관관계를 나타냈었다. 결식여부는 지방, 철분 및 나이아신섭취량과는 양의 상관관계를, 열량, 탄수화물, 인

• Asian-Australasian Journal of Animal Sciences
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• 제17권2호
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• pp.164-167
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• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Clinical and Experimental Reproductive Medicine
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• 제22권3호
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• pp.293-299
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• 1995

Objective: To investigate the effects of sperm morphology and their co-incubation with oocytes on the outcome of IVF and ICSI. Design: Strict morphology of washed sperm was assessed by Diff-Quick staining method before or after insemination. And the relationships between strict morphology and outcome (fertilization, embryo development and pregnancy) of IVF(with co-incubation) and ICSI (without co-incubation) were determined. Patients: Two-hundreds-and-sixty-three cycles of IVF and ninety-six cycles of ICSI were analyzed in order to clarify the influence of strict sperm morphology of spermatozoa on outcome of IVF and ICSI. These were divided into four groups. according to fertilization method and sperm morphology(Group 1: IVF, ${\geq}$12%, n:227; Group 2: IVF, <12%, n:36; Group 3: ICSI, ${\geq}$ 12%, n=48; Group 4: ICSI, <12%, n=48). Results: The fertilization rates of better morphology groups were higher than those of poor groups: Group 1(68.1%) > Group 2(62.1%), Group 3(78.1%) > Group 4(71.5%). There was no difference in embryo cleavage rates among four groups (>90%), Regarded with the good embryo rates, Group 1(56.8%) was significantly higher than Group 2(42.3%)(P<0.01), but there was no difference between Group 3(64.7%) and Group 4(61.2%). The pregnancy rates were also higher in better morphology groups as well as fertilization rates: Group 1(34.8%)> Group 2(16.7%)(p<0.05), Group 3(40.0%) > Group 4(23.0%)(p=0.08). Conclusion: Co-incubation with poor morphology sperm might adversely affect the quality of embryos. And strict sperm morphology may represent the ability to establish successful pregnancy. In short, the strict sperm morphology can be a good predictor of IVF and ICSI outcome.

• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.193-201
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• 1999

본 연구에서는 소 난자 세포질내 원형정자 및 원형정자 핵을 미세주입 후 수정과정과 체외 배 발달을 조사하였다. 원형정자 주입 3시간 전에 인위적 자극을 주고 원형정자를 주입했을 때 난자들의 수정률이 주입 직후에 자극을 준 것들과 자극을 주지 않은 것들 보다 높았다. 원형정자 주입과 원형 정자핵을 주입한 후 전핵 형성률과 전핵 이동률을 조사하였으나 유의차를 발견할 수 없었다. 전핵의 형성과 이동중에 미세소관의 움직임을 간접형광면역법 및 공촛점 현미경을 사용하여 조사해 본 결과, 난활성 직후 난자의 표층에서 미세소관이 발생해서 이것에 의해 웅성 및 자성전핵이 난자 중심부로 이전됨을 볼 수 있었다. 수정 후 원형정자의 세포질내 Mitochondria의 분포상태를 알아보기 위해 MitoTracker에 노출시킨 원형정자를 주입한 후 형광현미경에서 관찰해 보았다. 그 결과 원형정자의 Mitochondria가 주입 후 4-cell 이 전까지 는 관찰되었으나 그 이 후에는 발견 할 수 없었다. 원형정자를 미세주입한 후 소 난자를 체외에서 배양했을 때 2.5%의 난자가 배반포로 발달하였다. 이상의 결과는 체외 배양한 소 난자 내에 원형정자 혹은 원형정자 핵을 미세 주입하여 정상적인 수정과 배발달이 가능한 것을 보여 주는 것이다.

• 대한수의학회지
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• 제39권1호
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• pp.220-225
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• 1999

The aim of these experiments was to investigate the effects of bovine oviduct epithelial cells (OEC) derived from different segments to bind sperm binding and maintain their motility in vitro. In experiment 1, the number of sperm attached to OEC derived from isthmus or ampulla, the motility of unattached sperm during co-culture and fertilizing ability were assessed. In experiment 2, heparin treated sperm (hsp) or no treated sperm (nsp) were used to evaluate OEC binding ability of capacitated sperm. In experiment 1, regardless of their origin, approximately 65% of the sperm were attached to OEC within 2h. From 6h of co-culture, the numbers of unattached sperm on ampullary OEC were significantly higher than those on isthmic OEC (p<0.005). From 12h of co-culture, the motility of unattached sperm on isthmic OEC were significantly higher than those on ampullary OEC(p<0.05). The cleavage rate of oocytes inseminated on OEC derived from isthmic segment was also significantly higher than those from ampullary segment (p<0.01). In experiment 2, the numbers of unattached hsp on OEC were significantly higher than those of controls (p<0.01), between 2-24h examination. From 12h of co-culture, the motility of unattached nsp were significantly greater than those of hsp (p<0.01). These results show that bovine OEC derived from the isthmus play more important role(s) for sperm binding, maintaining motility and fertilization in vitro than those from the ampulla, and heparin induced capacitation may change sperm binding ability on OEC in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제16권1호
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• pp.15-21
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• 1989

An in vitro fertilization assay employing zona-free hamster embryos was used to investigate human spermatozoal fertilitzing ability. Yanaghimarchi et al.(1976) first introduced this cross species fertilzation technique, with its application as a diagnostic tool for male infertility. Human spermatozoa were preincubated for 3 to 4 hrs in B W W medium at concentration of $4{\times}10^6$ sperm/ml prior to the addition to zona-free hamster embryos. After 3 hrs, human sperm was evaluated for fertilizing potential by the presence of swelling or decondencing sperm head in the cytoplasm. The results of penetration rates for sperm were as follow : 1. The average penetration rate of a 7 fertile donor group was $47.8{\pm}27.67%$(Range 14.3-98.0%) 2. The average penetration rate of 12 infertile patients with normal semen analysis was $21.7{\pm}26.9%$(Range 0-38.8%) 3. The average penetration rate of 10 infertile patients with semen abnormalities was $6.1{\pm}8.1%$(Range 0-25%)

• 한국수정란이식학회지
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• 제10권2호
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• pp.171-175
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• 1995

This study was performed to establish the condition and the methods for the techniques of insertion the isolated blastomere cells into cytoplasm, in order to research the develop-mental ability of bovine embryo blastomere cells in vitro produced. After 24h in vitro ovary maturation with the ovaries from a slaughter house, in vitro fertilization was performed to the vital sperms which their mobility were decided by percoll gradient method, with 2~8 cell stage embryos, the blastomeres were isolated in $Ca^2$+. $Mg^2$+-free PBS, and following that embedded into agar and alginate solution, respectively. The rates of in vitro develop-ment are as follows ; in agar embedded 11 among 120(9.2%) 1 /2~1 /3 blastomers cleaved and 6 among 93(6.5%) 1 /4~1 /8 blastomeres cleaved. In sodium alginate-embedded 14 among 84(16.7%) 1 /2~1 /3 blastomeres cleaved and 6 among 85(7.1%) 1 /4~1 /8 blastomeres cleaved. In case of Na-alginate, the rate of the cells were better than those of agar. The results suggest that the techniques for embeeding the isolated blastomeres into gel may help cloning of bovine early embryo without nuclear transplantation.

• The Korean Journal of Ecology
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• 제28권4호
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• pp.193-198
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• 2005

This study investigated the characteristics of the eggs and yolk sac larvae of Korean striped bitterling, Acheilognathus yamatsutae, spawned and grown In mussels. The number of eggs in the ovary was small ($358{\pm}108$ SD). The eggs were oval and large, and the formation of the perivitelline space was narrow. The eggs were hatched at only 41 hours after fertilization but the hatched larvae were underdeveloped. The development of yolk projection and minute tubercles on the skin surface was notable, along with the vividly moving tail in the hatched larvae. The yolk projection and minute tubercles were disappeared upon enhancement of the motor ability of the larvae was enhanced. The formation of eyes and body pigments of the larvae was relatively delayed in comparison with that of other cyprinid larvae. After completely consuming the yolks the larvae escaped from the mussel for free swimming and exogenous feeding.