• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.85-85
• /
• 2001

본 연구는 돼지 난자의 체외 수정시 sucrose 첨가가 돼지 난자의 다정자 침입율에 미치는 영향을 조사하기 위하여 실시하였다. 돼지 난포란은 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 $\mu\textrm{g}$/$m\ell$ FSH, 0.5 $\mu\textrm{g}$/$m\ell$ LH, 및 10 ng/$m\ell$ EGF가 첨가된 TCM199 배양액에서 42-44 시간 동안 배양하여 체외 성숙을 유도하였으며, 2 mM caffeine과 0.1% BSA가 첨가된 체외수정용 mTBM 배양액에 3% sucorse를 첨가하여 6시간동안 수정을 유도하였다. 수정된 난자의 다정자 침입율을 조사하기 위해 체외수정 후 12, 13 및 14 시간대에 각각 1% orcein으로 염색하여 전핵 형성율을 조사하였다. 돼지 난자의 체외수정시 3% sucorse를 첨가하였을 때, 전핵 형성율을 조사한 결과 대조구에서 미수정, 1, 2, 및 3 PN 이상의 전핵 형성율은 각각 57.6 7.6, 9.7, 및 25%로, 처리구 74.2, 7.6, 13.6, 및 4.5% 에 비교하여 다정자 침입율이 유의하게 높았다(p<0.05). 그리고 3% sucorse를 첨가한 체외수정용 배양액에서 수정을 유도한 후 12, 13, 및 14 시간대별로 나누어 전핵 형성율을 조사한 결과, 수정 후 시간이 경과되면서 다정자 침입율이 증가되는 것을 볼 수 있었다 그러나 대조구 60.7%에 비해서는 각각 32.5, 36.0, 및 33.3%로 현저히 낮은 것을 볼 수 있었다.(Table Omitted). 또한, 3% sucorse가 첨가된 배양액에서 수정된 수정란의 발달율을 조사한 결과, 배반포 생산율은 처리구 21.8%로 대조구 6.9%에 비해 유의하게 높은 것을 확인 하였다. 따라서, 돼지 난자의 체외수정시 3% sucorse첨가는 체외수정란 생산에서 문제가 되고 있는 다정자 침입율을 저하시키고 배발달율을 향상시킴으로서 수정란의 체외 대량생산 효율성은 물론 수정란 이식시 수태율을 증가시킬 수 있을 것으로 사료된다.

• Korean Journal of Animal Reproduction
• /
• v.20 no.1
• /
• pp.1-8
• /
• 1996

The objective of this study was to determine effects of oviductal fluid on the sperm penetration and subsequent in vitro development of porcine oocytes. The addition of oviductal fluid to the fertilization medium decreased sperm pen etration and the mean number of spermatozoa in penetrated eggs. The number of spermatozoa firmly bound to zona pellucida was also decreased in the presence of oviductal fluid. Chlortetracy cline (CTC) fluorescence patterns were used to determine incidence of capacitation and acrosome reaction. The proportion of capacitated a and acrosome free spermatozoa increased when spermatozoa were exposed for 1.5 and 3 h to oviductal fluid. These results suggest that the factor(s) in secretion from the oviduct reduces polyspermic fertilization and the number of spermatozoa that will penetrate porcine oocytes. The reduction of polyspermic penetration by oviductal secretions may be due to a reduced number of spermatozoa in the fertilization me-dium into an intact acrosome.

• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.73-73
• /
• 2002

돼지 난포란의 체외성숙, 수정 및 배양에 관한 연구는 생명공학 기술을 도입하기 위한 기반 기술로 최근까지 계속 진행되고 있으나, 돼지 난포란을 이용한 효율적인 수정란 생산은 불규칙적인 웅성전핵 형성율과 높은 다정자 침입율 그리고 체내에서 발달된 배반포에 비하여 적은 세포수는 돼지 체외수정 체계에 있어서 끊임없는 문제점으로 제기되고 있다. 따라서 본 연구에서는 난포란을 이용한 보다 효율적인 수정란 생산을 위하여 돼지난자의 체외수정시 정자의 adenylate cyclase 활성을 조절하여 수정능획득 및 자발적인 첨체반응을 조절하여 정상적인 수정을 돕는 것으로 알려진 ADP를 체외수정 배양액에 첨가했을 때 수정율, 다정자 침입율, 배발달율 및 배반포의 세포수에 미치는 영향을 조사하였다. (중략)

• Korean Journal of Animal Reproduction
• /
• v.22 no.4
• /
• pp.411-417
• /
• 1998

The objective of this study was to dertermine the effects of oviductal fluid and oviductal conditioned medium on polyspermy and in vitro development of porcine oocytes. The addition of oviductal fluid and oviductal conditioned medium in the prefertilization and fertilization medium significantly decreased polyspermy rates and the mean number of spermatozoa in penetrated eggs(P＜0.05). The acrosome reaction rate significantly increased when spematozoa were exposed for 1.5, 3, 4.5h in oviductal fluid and oviductal conditioned medium(P＜0.05). When oocytes cultured for 192h, the percentage of oocytes that developed to the morula and blastocyst stage was higher in culture medium with oviductal fluid and oviductal conditioned medium than without oviductal fluid and oviductal conditioned medium(P＜0.05). These results indicated that the oviductal secreations will effectively reduce both the polyspermy rates and the mean number of spermatwa in penetrated eggs. And the presence of culture with oviductal fluid and oviductal conditioned medium promotes in vitro development of porcine oocytes.

• Korean Journal of Animal Reproduction
• /
• v.21 no.3
• /
• pp.239-246
• /
• 1997

The objective of this study was to evaluate the variation of fertilizing ability following the morphologically normal sperm ratio in porcine IVF using epididymal sperm The results obtained in this experiment were summarized as follows: 1. When the penetration rate (PR), polysper my rate (PSR), pronuclei formation (2PNF) and mean number of sperm (MNS) per oocyte were evaluated according to the percentage of morphologically normal epididyrnal sperm at insemination($\leq$lO%, 10~30% and $\geq$50%). the PR and PSR of $\leq$50% group (82.4, 87.4%) were significantly higher than those of other two groups ($\leq$lO%; 29.7%, 22.6% and 10~30%; 20.3, 37.0%) (p<0.01). Also, the 2PNF per examined oocytes was significantly high in $\geq$ 50% group (p<0.01). 2. When the $\geq$50% group in epididymal sperm was adjusted to 100% (5x1$^5$ cells/ml) , the PSR and 2PNF were not different between epididymal sperm (86.7, 35.1%) and frozen-thawed ejaculated sperm (86.0. 39.4%) although the PR in epididymal sperm (79.7%) was significantly lower than that in frozen-thawed ejaculated sperm (95.5%)(p<0.01). 3. Also. when the PR, PSR, 2PNF and MNS of epididymal sperm were evaluated according to the oocyte: sperm ratio (1:6000, 1:6650. 1:7700 and 1: 10000) at insemination. the PR, PSR and MNS were increased as the oocyte:sperm ratio increases. However, this result indicated that the 2PNF was high in the oocyte:sperm ratio (1:6000 and 1:6650). Therefore. these results suggested that when the percentage of morphologically normal epididymal sperm was more than 50. the fertilizing a ability was very similar to that of frozen-thawed ejaculated sperm and that the detailed evalu¬a ation of morphological normality in porcine IVF using epididymal sperm should be prerequisite to obtain the more effective fertilizing ability.

• Korean Journal of Animal Reproduction
• /
• v.23 no.2
• /
• pp.149-157
• /
• 1999

The functional role of cumulus cells on the penetration and polyspermy during in vitro fertilization in porcine was examined. The penetration rate was significantly higher (P＜0.01) in oocytes with (61%) than without (25%) cumulus cells, but significant differences in polysper-my rates were not observed. When hyaluronidase was added to the fertilization medium with different concentrations, penetration rates in oocytes with cumulus cells were higher than in oocytes without cumulus cells at 0 (61% vs 34% ; P＜0.05), 0.01 (56% vs 35% ; P＜0.05), 0.1 (66% vs 30% ; P＜0.05) and 1.0mg/$m\ell$ (39% vs 27%). The polyspermy rates were lower in oocytes without than with cumulus cells, and had a tendency to decrease with high concentrations of hyaluronidase. In another experiment, the penetration and polyspermy rates had a tendency to increase as time of sperm-oocyte culture was prolonged. At 16 and 20 h after insemination, the penetration rates were significantly higher (P＜0.05) in oocytes with (48 and 62% for 16 and 20 h) than without (25 and 31% for 16 and 20 h) cumulus cells in medium containing hyaluronidase. Polyspermy rates were significantly (P＜0.05) lower in oocytes without (13% and 16%) than with (37% and 48%) cumulus cells at 16 and 20 h after insemination. In cumulus-free oocytes inseminated in medium containing different concentrations of cumulus cells, the penetration rates were significantly (P＜0.05) higher in medium with than without hyaluronidase. The proportion of polyspermy was lower in medium without than with hyaluronidase at 0 (10% vs 0%), 10$^2$(25% vs 0%), 10$^4$(24% vs 14%) and 10$^{6}$ (29% vs 10% ; P＜0.05) cumulus cells/$m\ell$. These results suggest that cumulus cells can have a positive influence on sperm penetration, its action on polyspermy control does appear to function primarily on zona pellucida by co-culture of cumulus cells and oocytes in medium without hyaluronidase.

• Journal of Embryo Transfer
• /
• v.21 no.2
• /
• pp.129-135
• /
• 2006

This study was conducted to investigate the effect of in vitro maturation (IVM) duration of porcine follicular oocytes on maturation rate, polyspermic rate, and subsequent embryo development. The nuclear maturation rates of oocytes matured for 36, 38, 40, 42 and 44 hr were similar between 68.0, 78.0, 79.5, 73.8 and 81.8% respectively. There was no significant difference in the rates of polyspermy after in vitro feritilization (IVF). The cleavage rate in the group of 36 hr was significantly higher in than that of 40, 44 hr (p<0.05) but not to 38 and 42 hr. The development rate to blastocyst stage was significantly higher in the group of 38 hr (23.1%) than that in the group of 44 hr (15.6%) (p<0.05) but not to 36, 40 and 42 hr. These results suggest that the aged oocytes for 44 hr is not required for the production of bias to cysts derived from porcine IVF embryos.

• Korean Journal of Animal Reproduction
• /
• v.18 no.3
• /
• pp.199-206
• /
• 1994

We present here a new PCR-based cloning technique that allows the different PCR products during mouse embryogenesis. Recently, mRNA differential display described by Liang & Pardee (Science 257, 1992) and re-confirmed by Zimermann & Schultz (PNAS 91,1994). This method will detect the appropriate changes in the temporal patterns of expression or in the transition from maternal control to zygotic control as well as the functional difference of embryo with polyspermy or monospermy, the difference of expression between successfully hatched blastocyst and blastocyst failed to hatching, response to agents, and cell cycle regulation. By this methods, we have cloned an eDNA, which showed mouse 2 cell specific expression. Genomic DNA digested with EcoRI showed approximately 15 kb and then showed higher expression in fetal liver rather than adult liver. Furthermore, this gene is likely to have 2 mRNA by alternative splicing.

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

• Clinical and Experimental Reproductive Medicine
• /
• v.25 no.3
• /
• pp.315-322
• /
• 1998

The objective of this study was to test the effect of catalase on penetration in vitro by spermatozoa preincubated with xanthine and/or xanthine oxidase. The penetration rates were, significantly (p<0.05) higher in spermatozoa preincubated without (66 and 38%) than with (40 and 15%) catalase for 0 and 30 min. When spermatozoa were preincubated and inseminated in medium with xanthine, the penetration rates were significantly higher (p<0.05) in medium with (68, 70 and 49% for 0, 30 and 60 min) than without (33, 41 and 19% for 0, 30 and 60 min) catalase. However, in oocytes were' inseminated with spermatozoa pre incubated with or without catalase in the presence of xanthine oxidase, no decrease in penetrations rates were observed for up to 60 min of preincubation. In another experiment, the penetration rates were significantly (p<0.00l) higher in medium with (75, 55 and 52%) than without (14, 4 and 8%) catalase when oocytes were inseminated with spermatozoa preincubated for 0, 30 and 60 min in the presence of xanthine plus xanthine oxidase. On the other hand, The rate of polyspermy in oocytes penetrated in medium without catalase in the presence of xanthine or xanthine plus xanthine oxidase decreased with time of spermatozoa preincubation. However, no differences were observed in polyspermy rates in the medium with xanthine oxidase alone despite presence of catalase. These results indicate the advantages of spermatozoa pre incubated with xanthine plus xanthine oxidase in the presence of catalase to increase penetration potential and with suppressed polyspermy in porcine.