• Reproductive and Developmental Biology
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• 제35권3호
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• pp.363-367
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• 2011

The purpose of this study was to improve of frozen-thawed sperm using magnetized water in Korean native cattle. Before cryopreservation, without egg-yolk Triladyl$^{(R)}$ solution was flowed though magnet [0, 2000, 4000 and 6000 Gauss(G)] for S min. The freezing of dilluted semen added with Triladyl containing 20% egg-yolk. Analysis of frozen-thawed sperm was estimated viability with SYBR14/PI double stain, membrane intact with hypoosmotic swelling test (HOST), acrosome reaction with FITC-PNA, mitochondria membrane function with Rhodamin 123 by flow- cytometry. Sperm viability was significantly higher in 4000G group than other groups (p<0.05). However, the Hypoosmotic Swelling Test(HOST) was significantly higher in fresh, 4000 and 6000G than 0 and 2000G (p<0.05). In addition, mitochondria membrane damage and acrosome damage were significantly lower in 6000G group than other groups (p<0.05). in conclusion we suggest that magnetized water could be improve ability of sperm on cryopreservation in Korean native cattle.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.385-391
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• 1997

The purpose of this study was to examine the effects of anti-sperm antibody (ASA) on the fertilization processes using conventional IVF and ICSI procedure in human and hamster oocytes. In human IVF, we have observed restricted fertilization with sperm testing positive for ASA. ($23{\sim}90%$ IgA, 60-97 % IgG). However, if ICSI was perform in the next IVF cycle with the same patients, we could successfully fertilize the oocytes (37%; p<0.001), thus achieving pregnancy and delivery. When the sperm were cocultured in medium containing ASA, there were binding of ASA to sperm surface. In addition, the mean rate of the acrosomal reaction in an in vitro acrosome reaction test was lower for Ab-bound sperm (43.5%) than for Ab-free sperm group (51.3%, p<0.05). We used human sperm and hamster oocytes to confirm the negative effects of the ASA on fertilization. The sperm and/or oocytes have been expose to medium containing ASA before IVF and ICSI. In this experiment, the ASA was bound to the oocyte and sperm surface. The following results were obtain by using various combinations of ASA free or ASA bound sperm with ASA free or ASA bound oocytes for IVF. When ASA free sperm were inseminate with ASA free and ASA bound hamster oocytes, the fertilization rates are 89.6% and 74.3% respectively. However, when ASA bound human sperm were use the results were 62.5% and 55.6% respectively. These shows the fertilization rate was significantly decreased in both ASA bound and ASA free oocytes when using ASA bound sperm. No difference found when ASA are present on the oocyte surface. When the hamster oocytes was treated by ICSI with ASA free or ASA bound human spermatozoa, no significant difference was found. These results showed that ICSI is the most promising method for couples who fertilization was not possible by conventional IVF because of ASA.

• Clinical and Experimental Reproductive Medicine
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• 제25권3호
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• pp.251-260
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• 1998

It is well known that the clinical test for responsibility of accurate fertilization capacity in male partners is very important to diagnose and treat the infertility. However, it has been reported that the traditional semen analysis cannot accurately predict fertilization and pregnancy potential. The present study was performed to evaluate the acrosomal reaction to ionophore challenge (ARIC) test as a prognostic indicator for fertilization of sperm and oocyte in an in vitro fertilization and embryo transfer (IVF-ET) program. From March 1996 to Februry 1997, 30 couples undergoing IVF program were allocated to this study group. All female partners in the study group were 35 years old or less and their serum level of basal follicle stimulating hormone (FSH) and estradiol $(E_2)$ were normal. All the male partners have normal parameters of semen analysis. The ARIC tests were performed on the day of ovum pick up and in vitro insemination in all the male partners. The controlled ovarian hyperstimulation (COH) using luteal long protocol of gonadotropin releasing hormone (GnRH) agonist was used in all couples for IVF-ET. The acrosomal reaction with $10{\mu}l$ of 10% DMSO was induced spontaneously in $10.1{\pm}9.8%$, and acrosomal reaction with calcium ionophore A 23187 was induced in $27.4{\pm}18.1%$, and the ARIC value was $17.4{\pm}16.2%$. There were no significant correlation between the ARIC value and the fertilization rate ($r^2$=0.044, p=0.268). There were also no significant correlation between the ARIC value and the percentage of the grade I, II embryos ($r^2$=0.046, p=0.261). On the basis of above results, it was suggested that ARIC test might not be a useful prognostic indicator for fertilization in IVF-ET in male partners with normal parameters of conventional semen analysis. We guessed that IVF-ET could be performed to the patients primarily without universal appilcation of ARIC test to all male partenrs, and if fertilization failure occurs, the micro assisted fertilization (MAF) such as intracytoplsmic sperm injection (ICSI) might be used as an alternative mode of treatment with acceptable success rate.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.43-43
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• 2001

This study evaluated the effect of fertilization-promoting peptide (FPP) on fertilizing ability and glycosidase activity in vitro of spermatozoa frozen-thawed in pig, Using chlortetracycline fluorescence analysis, the various glycosidase analyses and the oocyte penetration test, we have obtained evidence that FPP can promote the fertilizing ability and glycosidase activity of frozen-thawed spermatozoa in vitro. When frozen-thawed spermatozoa was washed with different concentrations of FPP, there were significantly (P<0.05) more acrosome-reacted in medium with 100 nM than 0, 50, 200 and 400 nM. The penetration rates were also highest in medium containing with 100 nM FPP (P<0.05). On the other hand, the $\beta$-N-acetylglucosaminidase activity was at least twofold higher than other glycosidase. In same glycosidase, however, there were no difference in medium with different concentrations of FPP In another experiment, spermatozoa preincubated in medium with or without FPP for 0, 1, 2, 3 and 4 h were inseminated with oocytes matured in vitro. The percentages of spermatozoa that reached acrosome reaction were affected by preincubation and were higher in medium with that than without FPP. When oocytes were inseminated with spermatozoa preincubated in medium with and without FPP during the different periods, however, penetration rates were decreased with preincubation periods of spermatozoa. On the other hand, when the sperm-oocyte were cultured for 4, 8, 12, 16, 20 and 24 h, the penetration rates were higher in spermatozoa preincubated with that than without FPP and had a tendency to increase as time of culture periods. However, The activities of $\alpha$-fucosidase, $\alpha$ -mannosidase, $\beta$-galactosidase and N-acetyl- $\beta$-D-glucosaminidase were higher in medium with that than without FPP regardless of periods of sperm preincubation and sperm-oocyte culture. These results suggest that FPP may play a positive role in promoting of sperm function and glycosidase activity in vitro in pig.

• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.105-115
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• 2002

Objective : To evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine (X) and xanthine oxidase (XO) system on sperm function, the change of sperm characteristics, lipid peroxidation, and DNA fragmentation in bovine spermatozoa. Materials and Methods: ROS were produced using a combination of 1000 uM X and 50 mU/ml XO. The ROS scavengers: superoxide dismu tase (SOD) (200 U/ml) and catalase (500 U/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without ROS scavengers at $37^{circ}C$ under 5% $CO_2$ incubator. Sperm movement characteristics by CASA (computer-aided sperm analysis), HOST (hypoosmotic swelling test), Caionophore induced acrosome reaction, malondialdehyde formation for the analysis of lipid peroxidation, the percentage of DNA fragmentation using the method of TdT-mediated nick end labelling (TUNEL) by flow cytometry were determined after 2 hours incubation. Results: The action of ROS on bovine spermatozoa resulted in a decreased in capacity for sperm motility, Ca-ionophore induced acrosome reaction and membrane integrity, an increased in malondialdehyde formation and the percentage of sperm with DNA fragmentation. In the effects of antioxidant, catalase completely alleviated the toxic effects induced by the ROS in terms of sperm function and characteristics, however SOD exhibited no capacity to reduce the toxic effects. Conclusion: The ROS can induce significant damages to sperm functions and characteristics. The useful ROS scavengers can minimized the defects of sperm function and various damages of spermatozoa.

• 한국가축번식학회지
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• 제26권3호
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• pp.275-289
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• 2002

본 연구는 종모우의 정자수정능력 평가방법의 개발과 정자 기능 및 성상 변화에 영향을 미치는 요인을 알아보기 위하여 시행하였다. 동결-융해된 종모우 정액을 대상으로 정자의 운동성과 정자의 형태를 분석하였고, 정자의 기능 검 사 항목으로서 체외수정(IVF), HOST, Ca-ionophore에 의한 첨체반응율, 정자의 ROS 측정을 위한 luminol, lucigenin-dependent chemiluminescence, LPO 분석을 위한 malondialdehyde의 측정 및 TUNEL (terminal deoxynucleotidyl transferase(TdT) dUTP nick end labelling) 기법을 이용한 정자의 DNA fragmentation를 측정하였으며 이들 각각의 조사 항목들의 분석치들과 체외수정율 및 배발생율과의 상관관계를 조사하여 다음과 같은 결과를 얻었다. 1. 고수정군과 저수정군의 체외수정율과 배반포 발생율의 평균은 각각 64.4%와 34.3%, 18.50%와 6.2%였으며 두 군간에 통계학적으로 유의한 차이를 보였다(P<0.05). 고수정군과 저수정군의 정자운동성과 첨체반응률은 각각 평균79.0 %와 66.2%, 40.7%와 22.9%로 두 군간에 통계학적으로 유의한 차이를 보였으나(P<0.05), 정상형태 정자의 비율과 HOST는 각각 평균 94.6%와 92.7%, 69.4%와 59.8%로 두 군간 유의한 차이를 보이지 않았다. 2. Luminol dependent chemiluminescence, LPO 및 DNA fragmentation의 평균은 고수정군과 저수정군에 있어서 각각 6.4와 6.5, 2.Onmol와 3.Inmol 및 2.6%와 7.4%로 두 군간 통계학적으로 유의한 차이를 보였으나(P<0.05), lucigenin dependent chemiluminescence는 4.7와 4.6로 두 군간 유의한 차이를 보이지 않았다. 3. 체외 수정율은 정자의 운동성 및 첨체반응율과 통계학적으로 유의한 정(positive)의 상관관계(r=0.87, p<0.01; r=0.81, p<0.05)를 나타내었으며, luminol dependent chemiluminescence, lipid peroxldation 및 DNA fragmentation과는 통계학적으로 유의한 부(negative)의 상관관계 (r= -0.81, p<0.05; r: -0.74, p<0.05; r : 0.81, p<0.05)를 나타내었다. 그러나 체외수정율은 정상형태 정자의 비율, HOST 및 lucigenin dependent chemiluminescence와는 유의한 상관 관계를 나타내지 않았다. 4. 배반포 발생율은 첨체반응율과 통계학적으로 유의한 정의 상관관계(r=0.71, p<0.05)를 나타내었으며, luminol dependent chemiluminescence, lipid peroxidation 및 DNA fragmentation과는 통계학적으로 유의한 부의 상관관계(r= -0.71, p<0.05; r= -0.89, p<0.01; r= -0.71, P<0.05)를 나타내었다. 배반포 발생율은 정자의 운동성, 정상형태 정자의 비율 및 HOST, lucigenin dependent chemilumihescence와는 유의한 상관관계를 나타내지 않았다. 이상의 결과를 종합해 보면 정액질의 저하에 ROS의 영향이 밀접히 연관되어 있음을 알 수 있으며, 또한 본 연구에서 적용된 기법들은 정액질의 평가 및 정자 수정능력 향상을 위한 기술개발에 있어서 유용한 평가 방법으로 이용될 수 있을 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제35권3호
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• pp.203-212
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• 2008

목 적: 정자의 동결 과정에서 생길 수 있는 급격한 온도 차에 의한 동결 충격이나 동결 상해등에 의한 세포막의 손상, 세포의 기능 장애 등은 정자의 수정능에 영향을 미칠 수 있다. 본 연구에서는 인간 정자를 동결 보존하는 과정에서 콜레스테롤 전처리가 정자의 운동성 및 기능보존에 미치는 영향을 알아보고자 하였다. 연구방법: 본원을 내원한 14명 남성의 정자를 대상으로 콜레스테롤을 첨가하지 않은 대조군 (control)과 여러 농도의 콜레스테롤을 동결보존액에 첨가한 실험군에서 정자의 동결-융해 후 상태를 다음 3가지 방법으로 비교, 분석하였다. 1) 정자 분석, 2) calcium ionophore로 유도된 첨체 반응 검사, 3) 정자 염색질 구조 분석 (sperm chromatin structure assay). 결 과: 첫째로 인간 정자의 운동성은 $0.5{\mu}g$ 농도의 콜레스테롤을 첨가한 동결보존액에서 동결-해동하였을 경우, 콜레스테롤을 첨가하지 않은 군에 비해 유의적 차이를 보이며 증가하는 것을 확인하였다 ($33.46{\pm}1.48%$ vs. $30.10{\pm}1.07%$, p<0.05). 다음으로 동된 정자의 첨체 반응 검사에서도 콜레스테롤을 첨가한 동결보존액에서의 첨체 반응이 일어나는 정자의 비율이 첨가하지 않은 군에 비해 유의하게 높게 관찰되었다 ($53.60{\pm}1.60%$ vs. $47.40{\pm}1.86%$, p<0.05). 마지막으로 정자 염색질 구조 분석에서는 콜레스테롤을 첨가한 군이 첨가하지 않은 군에 비해 정자의 DNA손상이 적게 나타남을 확인하였다. 결 론: 본 실험은 동결보존액을 통한 정자 원형질막 내 콜레스테롤 함유량의 증가가 동결-융해 후 정자의 운동성과 수정능(capacitation status)을 증가시키고 DNA 손상을 방지하는 역할을 한다는 결과를 보여주었다. 이러한 결과를 통해 동결보존액 내 콜레스테롤의 첨가는 인간 정자의 동결보존 동안 발생할 수 있는 동결 상해를 줄여줄 수 있는 유용한 방법으로 사료된다.

• Reproductive and Developmental Biology
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• 제36권1호
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• pp.1-6
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• 2012

This study evaluated a sexed sperm ability to produce embryos by flow cytometer. Hanwoo bulls sperm were separated to X and Y sperm via Hoechst 33342 stained with near UV laser or performed the pre-sorted without near UV laser beam in flow cytometry. Pre-sorted sperm had significantly higher viability ($84{\pm}1.15%$, $p$<0.05) compared to other sorted groups in frozen-thawed semen. For fresh semen, pre-sorted sperm had the higher viability ($79{\pm}3%$, $p$<0.05) than those of the X and Y sperm ($44.7{\pm}1.67$ and $41.7{\pm}1.2%$) separated by differences of DNA content. On the other hand, pre-sorted and X sperm sorted according to differences in DNA content had significantly higher viabilities ($24.3{\pm}1.2$ and $25.7{\pm}0.9%$, $p$<0.05) compared to that of the sorted Y sperm ($13.7{\pm}1.2%$) in the hypoosmotic swelling test. The proportion acrosome reaction in the sorted X sperm was higher ($55.0{\pm}1.7$ and $45.0{\pm}1.5%$) than those of the sorted Y-sperm ($32.3{\pm}0.9%$, $p$<0.05). However, the sperm morphologies of the sorted groups were not significantly differences. In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm. Further study is needed to determine the optimal procedures to enhance male and female embryos and sorting accuracy.

• 한국임상수의학회지
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• 제34권2호
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• pp.156-160
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• 2017

We investigated the effect of ethylene glycol and antioxidants such as taurine, hypotaurine and trehalose with extenders during cryopreservation of Korean Jeju Black Bull spermatozoa. The cryopreservation of freshly collected spermatozoa was conducted with four different conditions. As a control, spermatozoa were cryopreserved with Tris egg-yolk extenders added 5% ethylene glycol (EG). Taurine (20 mM), hypotaurine (20 mM) and trehalose (20 mM) were individually added into tris egg-yolk extenders with 5% EG. After thawing of frozen spermatozoa with four different conditions, sperm viability, motility, acrosomal integrity, and membrane integrity were investigated. The significant (p < 0.05) improvement of sperm viability showed in all antioxidant treated thawed spermatozoa (taurine; $68.1%{\pm}4.4$, hypotaurine; $69.2%{\pm}6.7$ and trehalose; $68.0%{\pm}4.4$) when compared to control ($63.4%{\pm}5.6$). Neither positive nor detrimental effects of three antioxidants were shown sperm motility after thawing. The results of hypo-osmotic swelling test showed that the membrane integrity of taurine, hypotaurine or trehalose treated thawed spermatozoa ($64.1%{\pm}5.4$, $61.5%{\pm}3.7$ and $59.0%{\pm}4.0$, respectively) had significantly (p < 0.05) higher rate of the swollen sperm compared to control ($53.7%{\pm}9.7$). Hypotaurine treated frozen-thawed spermatozoa had siginificantly higher (p < 0.05) F pattern ratio than taurine, trehalose and control treated frozen-thawed spermatozoa. Trehalose added frozen-thawed spermatozoa had significantly higher (p < 0.05) acrosome reaction pattern ratio than taurine and hypotaurine added frozen-thawd spermatozoa. In this study, we found that antioxidants such taurine, hypotaurine and trehalose treatments during cryopreservation process could reduce damage of spermatozoa of Korean Jeju Black Bull and improved sperm capability of fertilization.

• 한국가축번식학회지
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• 제25권4호
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• pp.317-325
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• 2001

본 연구는 lipid peroxides를 생산하는 것으로 알려진 ascorbic acid (Asc; 0.5 mM)와 ferrous sulfate (Fe$^{2+}$; 1 mM)가 돼지 동결-융해 정자의 체외수정능력에 미치는 영향을 검토하였다. 그 결과, 동결정액의 융해 처리시 배양액내 Asc/Fe$^{2+}$ (38%)의 동시첨가시 대조구 (27%)에 비해 정자의 첨체반응이 유의적으로 높게 유기되었다 (P＜0.05). 정자침입율 또한 Asc/Fe$^{2+}$ (76%)를 동시에 첨가하므로서 대조구 (55%)에 비하여 유의적으로 높게 나타났다 (P＜0.05). 한편 정자의 peroxidation은 상기의 처리구에서 maiondialdehyde (MDA)의 생성에 기초를 두고 평가하였는데, 정자의 처리시 Asc/Fe$^{2+}$의 동시첨가에 의해 MDA의 생성이 증가하였으나 처리구 사이에서 유의적인 차이는 인정되지 않았다. 또한 sulfhydry1(-SH) group의 용량을 측정한 결과 Asc/Fe$^{2+}$를 동시에 첨가한 실험구에서 가장 높게 나타났으나 타처리구와의 사이에서 유의적인 차이는 인정되지 않았다. 또 다른 연구에서 동결-응해정자가 난자의 투명대에 접착하는 정도를 평가한 결과, 대조구와 Asc처리시 Fe$^{2+}$ 처리에 비하여 유의적으로 (P ＜ 0.05) 높은 정자의 접착을 나타냈으나 Asc/Fe$^{2+}$의 동시 처리에 의한 정자접착의 증가는 인정되지 않았다. 결과적으로 lipid peroxidation의 증가를 야기시키는 Asc/Fe$^{2+}$의 동시첨가는 투명대에서 정자의 접착능력을 증가시키지 않았음에도 불구하고 첨체반응과 침입능력을 향상시키는 것으로 나타났다.