• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.255-262
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• 2000

The aim of this work was to study the effects of ascorbic acid (Asc) and/or ferrous sulfate (Fe$^{2+}$) and spernatozoa preincubation on the in vitro fertilization in porcine. Porcine follicular oocytes matured in culture were inseminated with frozen-thawed boar spermatozoa preincubated for 0, 1, 2, 3, 4 and 5h. The penetration rates (37~51%) were not significantly different between durations of spermatozoa preincubation in medium with 0.1 mM Asc. The addition of 1.0 mM Fe$^{2+}$ during spermatozoa preincubation were not significantly affecting the penetration rates (41~56%). When spermatozoa were preincubated with Asc and Fe$^{2+}$, the penetration rates had a tendency to increase with time of spermatozoa preincubation, and were significantly (P<0.05) higher in spermatozoa preincubated with that than without Asc and Fe$^{2+}$ for 5 h. On the other hand, when spermatozoa were preincubated in fertilization medium without Asc and/or Fe$^{2+}$, the penetration rates were significantly (P<0.05) higher in medium with Fe$^{2+}$ than with Asc or Asc and Fe$^{2+}$ for in vitro fertilization. The rate of polyspermy in penetrated oocytes in medium with Asc and Fe$^{2+}$ decreased with the period of spermatozoa preincubation. Despite different culture conditions for spermatozoa preincubation, no differences were observed in polyspermy rates in the presence of Asc and/or Fe$^{2+}$ These results indicate the advantage of preincubating spermatozoa with Asc and Fe$^{2+}$ and an addition of Fe$^{2+}$ during in vitro fertilization with spermatozoa preincubated maintain penetration potential without increased polyspermy rates on in vitro fertilization in porcine oocytes.on in porcine oocytes.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.93-93
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• 2002

싸움소(투우)는 1999년 문화관광부지정‘한국의 10대 지역문화 관광축제’로 선정되어, 청도군 투우대회가 한국을 대표하는 축제가 되었고, 상설투우장의 건설로 새로운 레져 산업으로 부상하고 있다. 그러나 싸움소 생산에 소요되는 유전인자 보존 및 생산기반은 전무한 실증이며, 본 연구는 청도군내 우수 종모우을 선별하여 정액채취 및 동결정액을 생산하여 군내 한우번식 농가에서 인공수정을 실시하고 계절별 수태율과 송아지의 성비를 조사하였다. 종모우 번개(나이6세, 체중850kg, 97, 98년 우승)와 사자(나이6세, 체중870kg 98, 99년 준우승) 2두로부터 일반적인 방법으로 인공질을 이용하여 1999년 10월에 정액을 채취하였다. 채취된 정액은 35$^{\circ}C$에서 3~5배정도로 희석하여 정자농도와 활력을 평가하였다. 희석정액은 90분간에 걸처 5$^{\circ}C$ 까지 냉각하면서 글리세롤을 첨가한 난황구염산나트륨액으로 여러번 나누어 희석하여 정자의 충격을 피하였다. 글리세롤평형 2시간 후 0.5$m\ell$스트로에 정자수가 3500만/스트로의 분주.봉인하여, 정액의 동결은 액체질소상에서 4~5cm 위에 스트로를 평행으로 놓아 액체질소 가스로 10~15분간 예비동결한 다음, -8$0^{\circ}C$의 초저온 냉장고에서 케니스터에 넣어 -196$^{\circ}C$ 액체질소에 보관하였다. 인공수정을 실시하고 40일 전후에 직장검사를 통해 임신율과 수태율을 조사하고 분만한 송아지의 성비를 기록하였다. 채취한 싸움소의 정액량은 번개와 사자가 각각 평균 4.6$m\ell$와 3.8$m\ell$이고, 동결전의 정자의 활력은 번개와 사자의 정액이 각각 70.3 vs 75.3%, 동결후의 활력은 37.3 vs 40.3%로 유의한 차이는 없었다. 번개와 사자의 동결정액으로 각각 44두와 127두를 인공 수정하였고, 40일 전후의 임신율은 26두 vs 80두(59.1 vs 63.0% )였으며, 수태율은 26두 vs 66두(59.1 vs 52.0% )로 이들간의 유의한 차이는 없었다. 번개와 사자의 수송아지의 성비는 각각 65.3 vs 42.4%로 유의한 차이가 있었다. 싸움소 정액 동결보존과 인공수정으로 싸움소 혈통을 가진 송아지가 생산되었다. 그러나 생산된 송아지가 싸움소의 능력을 가진 것을 선별하여 혈통을 고정시키고, 훈련으로 싸움소의 제질을 발굴하는 것이 앞으로의 연구과제이다.

• The Korean Journal of Zoology
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• v.31 no.1
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• pp.21-28
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• 1988

The present study was carried out to examine the fertilizability of the mouse oocytes pre-ex-posed to dbcAMP which is a well-known inhibitor of the oocyte maturation. The oocytes once cultured in the dbcMP-containing medium for a certain length of, time were cultivated in the dbcMp-free medium to induced the maturation, then mixed with sperms, and observed following culture for 24 hours. The fertilization rate of cocytes was judged by the index of the number of 2-cell embryo developed 24hr following insemination. The fertilization rate of the oocyte previously incubated with dbcAMP (100 g/ml) for 2, 4, 8 16 hours was 32.3, 14.5, 4.7 and 8.8%, respectively, while that of the control was 53.3% indicating that the fertilizability was decreased as a function of time exposed to dbcAMP. The pretreatment of dbcMP, however, didn't affect the process of sperm penetration to egg. In addition, there is no prominent changes in the morphological architecture of fertielized eggs which has been exposed to dbcAMP as revealed by electron microscopic observation. Consequendy, it can be concluded that the mouse cocytes once inhibited their maturation by dbcMP may retain, in some extent, the fertilizability, although most of the fertilized egg may not proceed to further development because of the failure of pronucleus formation.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.59-64
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• 2008

The objective of this study was to estimate modification of semen quality during storage. Liquid boar semen samples extended in Beltsville Thawing Solution were stored at $17^{\circ}C$ up to 5 days. While % motility and linearity significantly decreased eon day 3 in extender, the qualitative motility patterns were maintained satisfactorily. Also the storage of boar semen up to 5 days before insemination did not significantly changed the acrosome intactness. However, acrosome changed sperm significantly increased and capacitated sperm significantly decreased from day 4. No significant modifications in acrosome integrity were showed during sperm storage; these results suggest that liquid boar semen may keep the quality in extender for 3 days.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.263-273
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• 2002

This study was undertaken to evaluate the effects of $\beta$-mercaptoethanol ($\beta$-ME) on lipid peroxidation and fertilization ability in vitro by xanthine (X) - xanthine oxidase (XO) system in boar spermatozoa frozen-thawed. The boar spermatozoa were treated with X and/or XO, and the spermatozoa viability were measured by the eosin-nigrosin stain method. In control group, level of vitality in boar spermatozoa were higher than in medium with X, XO and X+XO groups. No significant differences, however, were observed under the all conditions. The percentage of spermatozoa that reached acrosome reaction were significantly (P<0.05) higher in sperm treated without that than with $\beta$-ME under the all conditions. On the other hand, when spermatozoa were inseminated in medium with X and/or XO, the penetration rates in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. The lipid peroxidation of sperm was evaluated on the basis of malondialdehyde (MDA) production. The MDA were higher in sperm treated without that than with $\beta$-ME under the above all conditions. However, significant differences were not observed between medium with and without $\beta$-ME. Sperm-SH group were higher detected in medium with that than without $\beta$-ME under the all conditions. The activity of sperm binding to Bona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were significantly (P<0.05) higher than in medium with X+XO groups. The sperm binding in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. These results suggest that addition of $\beta$-ME in X-XO system may play a positive role in improving of fertilization ability in vitro.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.163-169
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• 2001

OSBA(oocytes-sperm binding assay) is a tool developed for rapid test of optimal condition of IVF medium and protein source by binding ability of mouse sperm and egg. Mouse oocyte-cumulus complexes were prepared by removing of the cumulus cells with 0.1% hyaluronidase. 10$\pm$2 oocytes per 30 ${mu}ell$ medium drop were inseminated with 3 ${mu}ell$ sperm suspension and were cultured f3r 3 hours and 24 hours, respectively. And the oocytes were recovered gently and the No. of sperm bound on oocytes were counted. In the Exp. 1, the ratio of oocytes bound with one sperm at least were 60.2%(50/83), 2%(2/77) and 100%(79/79) in the medium with no protein, FBS(15%, v/v) and BSA(0.4%. w/v), respectively, Fetal bovine serum(FBS) seriously inhibited sperm binding on oocyte, although bovine serum albumin(BSA) promoted the binding ability. The inhibiting effect of FBS was dependent on the concentration of FBS. The sperm binding ability according to oocyte maturity was tested in the Exp. 2. There was no significant difference between Met. II (mature) and Met. I (intermediate mature) oocytes in the number of oocytes bound with sperm and the number of sperm bound on oocytes. Finally, in Exp. 3, two batches of Ham's F10 medium with good and poor quality by OSBA were tested (The ratios of embryos developed from PN 1-cell stage to hatched blastocyst; 25% vs. 70%). In the medium with good quality, sperm binding ability was significantly increased (P ＜ 0.05). The ratio of oocytes bound with one sperm at least was 66% and 90% in the medium with poor and good quality, respectively. Conclusively, It was possible to test IVF medium condition rapidly and easily by OSBA.

• Journal of Sericultural and Entomological Science
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• v.15 no.2
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• pp.9-14
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• 1973

In order to find out effects of the generative power of silk worm moth which have been brought up in the high temperature accommodation at their pupa stage. For this specific study, 9 different kinds of male silk worms have been selected as specimen. All those specimen were brought up in the normal temperature at their larvae stage and after the pupation period they have been accommodated in the condition of high temperature for a certain length in accordance with the study programme. Afterwards, those mlae specimen were copulated with Suwon jam 103${\times}$104 which were all brought up in normal conditions. This study was carried out to find the copulation function as well as the ratio of unfertilized eggs(male sterility test). Results of study have been revealed as follows: 1) Although some differences were observed, male pura which have been brought up in the condition of high temperature shown the low rate of unfertilized eggs rather than those were brought up in the normal conpition. 2) In this group the eclosion(emergency) has been found to be poor rather, than those specimen brought up in normal conditions. 3) The copulation function of Moran, Daedong, J124 and C108 specimen were found to be poor than those of Suwon jam. 4) Fertility rate of Moran, Daedong, J124 and C108 was found to be around 65%. This figure is rather lower than what we normally expect. 5) Unfertilized egg ratio of Moran, Daedong and C108 were found to be around 20% if they were brought up in the condition of high temperature for one day from the time of pupation: 40% at 2 days, and 70% at 3 days duration. More than 3 days treatment has shown no progress in the unfertilized egg ratio. 6) One day's treatment for the pupa at the later stage has shown the unfertilized egg ratio of about 10%; 20% at 2 day's treatment, 35% at 3 day's treatment, 40-60% at 4 day's treatment, more than 60% at 5 day's treatment, and the 70% of fertilized egg ratio was only observed when the treatment days come to 7 days. It was understood that the unfertilized egg ratio was high at the antepupa stage rather than that of post-pupa stage. 7) According to the result of observation the sperm in copulatory pouch and seminal receptacle out of the normal female silk worm which have been copulated with the male brought up in the condition of high temperature at their moth stage. The reproduction system found in the control zone has been found to be normal and the sperm is amountful and active in motion while the sperm found in the treatment to be limited in amount and slow in motion. The observation was made within 5 hours from the copulation. 8) According to the result of observation of sperm of seminal receptacles of the female silkworm moth, and according to that observation of sperm in the seminal receptacle in female silkworm moth, the amount of sperm and mobility in the female moth brought up in high temperature were poor comparing that were brought up in normal temperature zone. Some of them are even found to be no trace of such. 9) Appearance and mosle of the copulatory organ of the male silkworm moth was found to be no anatomical change. 10) Testis of the later pupa stage which was brought up in the high temperature was found to be almost net developed to anucleate sperm and they are degenerated at stage of between maturation division and sperm abnormal stage. Mean time at control zone, the formation of anucleate sperm was already observed.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.227-233
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• 1998

The aim of these experiments was to investigate the effects of oviduct epithelial cells on bovine in vitro fertilization. Oviduct epithelial cell monolayers (OEC) on the 4-well dish were prepared according to general procedures. Monolayers were formed within 5days. The medium for OEC culture (TCM199 with 10% FBS) was replaced with IVF-TALP 2h before each experiment. Macromolecules/proteins from oviductal conditioned medium (OM) were recovered by ultrafiltration, which desalted and concentrated macromolecules greater than 5kDa, and this OM were added to W medium (experiment 1). The cleavage rate in OM+OEC group was significantly higher than in OM group (p〈0.01). In this experiment 2, oocytes were inseminated on OEC with sperm which had been pre-incubated with OEC for 0 or 4h before insemination. In this experiment, oocytes were exposed to sperm only 8 h for clarifying the effect. After insemination, oocytes were cultured in CRlaa. At 42 h post insemination, oocytes were denuded and examined for evidence of cleavage. The cleavage rates of oocytes which were inseminated with OEC treated sperm for 4 h were significantly higher than those of the other group (p〈0.01). In conclusion, sperm released from OEC have more fertilizing ability than those before attachment.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.139-143
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• 2007

This study was performed to investigate the reproductive characteristics of the cloned Hanwoo bulls produced by SCNT. The semen ejaculated from the cloned bulls (C-38 and C-39) and normal Hanwoo bull was properly measured the volume, the number of sperm, and the viability of frozen-thawed sperm. The sperm activity was analyzed using computer assisted sperm analysis (CASA). To analyze fertilizing ability of the cloned bulls, in vitro fertilization and artificial insemination were performed using the frozen-thawed semen. There were no differences in semen volume, sperm concentration, and the viability of frozen-thawed sperm between cloned bulls and normal bull. The difference was statistically significant in total motility, curvilinear velocity (VCL), straight-line velocity (VSL), and average-path velocity (VAP) of both cloned bulls compared to those of normal Hanwoo bull, respectively (p<0.05). The cleavage and blastocyst development rate were not different between the groups. five cloned cows were artificially inseminated using the frozen-thawed semen of C-38, two of them became pregnant. Two second generation calves (one male and one female) were produced. Based on these results, the cloned Hanwoo bulls showed normal reproductive abilities of semen parameters and sperm activity to their comparators and produced cloned calves, although there are some individual differences on the parameters.

• Development and Reproduction
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• v.5 no.1
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• pp.53-57
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• 2001

Sperm capacitation and acrosome reaction (AR) have been known to be Ca$^{2+}$-dependent events. Sperm capacitation accompanies with cholesterol efflux fiom plasma membrane, that eventually stimulates AR. However, whether the AR mediated by cholesterol efflux is Ca$^{2+}$ dependent has not been verified yet. Recently, methyl beta cyclodextrin (MBCD) was found to evoke AR by stimulating the cholesterol efflux fiom sperm membrane. In the present study, we examined the requirement of Ca$^{2+}$ in the MBCD-induced AR. During incubation of sperm in the bicarbonate buffered media MBCD increased AR in a dose-dependent manner regardless of the Ca$^{2+}$ presence. In the presence of low molar concentration of Ca$^{2+}$ (100 ${\mu}$M), MBCD-induced AR was slightly increased compared to Ca$^{2+}$-free condition. In the absence of Ca$^{2+}$ supplement, spontaneous AR was slightly increased during the incubation but inhibited by 100 ${\mu}$M EGTA. MBCD potentiated AR even the presence of EGTA. However, EGTA attenuated MBCD-induced AR, suagesting the functional involvement of intracellular Ca$^{2+}$ in the MBCD-induced AR. Taken together, it was suggested that cholesterol efflux from the sperm plasma membrane was sufficient for induction of AR even in the absence of extracellular Ca$^{2+}$and that a condition permissive for mobilization of intracellular Ca$^{2+}$ is important for MBCD-induced AR.