• 한국수정란이식학회지
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• 제29권2호
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• pp.163-169
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• 2014

The objective of this study was to evaluate the characteristics of Korean Native Cattle sperm frozen-thawed with L-cysteine and/or catalase. The semen from bulls was collected by the artificial vagina method, and Triladyl containing 20% egg-yolk and/or L-cysteine (L), catalase (C) and L-cysteine + catalase was added to the diluted semen for cryopreservation. The results showed that sperm viability was significantly higher in the L-cysteine + catalase ($69.49{\pm}3.16%$) group than in the control ($60.5{\pm}3.94%$) group (p<0.05). Acrosome damage was significantly lower in the L-cysteine ($17.12{\pm}1.08%$) group than in the control ($21.46{\pm}1.14%$), catalase ($20.54{\pm}0.76%$), and L-cysteine + catalase ($19.29{\pm}0.65%$) groups (p<0.05). In addition, the level of intact mitochondria in the spermatozoa was significantly higher in the L-cysteine ($58.65{\pm}1.39%$) group than in the control ($50.63{\pm}2.37%$) group (p<0.05). The hydrogen peroxide level in the frozen-thawed sperm was significantly lower in the L-cysteine ($3.74{\pm}1.66%$), catalase ($4.65{\pm}1.87%$), and L-cysteine + catalase ($8.11{\pm}2.15%$) groups than in the control ($13.22{\pm}1.6%$) group (p<0.05). The glutathione level was significantly higher in the L-cysteine ($1.33{\pm}0.03%$) group than in the control ($1.08{\pm}0.06%$), catalase ($1.05{\pm}0.02%$) and L-cysteine + catalase ($1.11{\pm}0.03%$) groups (p<0.05). In conclusion, L-cysteine and catalase could protect the membrane of Korean Native Cattle sperm from damage during sperm cryopreservation. Especially, L-cysteine was more effective for keeping acrosomes and mitochondria intactness during sperm cryopreservation.

• 한국수정란이식학회지
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• 제21권1호
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• pp.69-75
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• 2006

본 연구는 제주도 축산진흥원과 농촌진흥청 난지농업연구소에서 사육되고 있는 제주마 5두를 사용하여 인공질을 이용, 정액을 채취하여 정액의 일반성상 및 CASA를 이용하여 정자의 운동성과 생사 염색으로 기형률을 조사하였다. 제주마 정액의 일반 특성으로 총 정액량은 평균 42.5ml이었으며 평균 pH는 7.3, 평균 정자농도는 평균 $198.5X10^6/m1$ 이었다. 정자의 운동성은 $64.3{\pm}23.2%$이며, motility parameters 별로 VAP $70.4{\pm}28.7{\mu}m/s,\;VSL\;69.6{\pm}28.9{\mu}m/s,\;VCL\;94.1{\pm}30.0{\mu}m/s,\;ALH\;2.3{\pm}0.7{\mu}m/s,\;BCF\;7.6{\pm}1.1Hz,\;STR\;99.1{\pm}1.2%,\;LIN\;77.1{\pm}12.7%$로 나타났다. 정자 부위별 기형률은 두부가 평균 4 %, 경부가 평균 20 %, 미부가 평균 4 %로 나타났다. 이 연구를 통해 천연기념물로 지정되어 있는 제주마의 유전자원 보존과 증식을 위한 인공수정기술의 실용화를 위한 기초 자료를 제공하고자 한다.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.121-126
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• 2007

The objective of this study was to establish the optimal conditions for hypoosmotic swelling (HOS) test to assess the functional integrity of the membranes of boar fresh or frozen/thawed spermatozoa. When pooled semen sample was incubated for 30 min at $37^{\circ}C$ with different test solution of varied osmolarity, the highest percentage of HOS positive spermatozoa was observed in a 150 mOsmol fructose/Na-citrate solution (33.6%). Incubation time did not affect significantly the score of HOS positive spermatozoa observed in a 150 mOsmol fructose/Na-citrate solution at $37^{\circ}C$, but the osmolarity affected the score of HOS positive spermatozoa under the same condition above. Fresh semen was significantly better than frozen/thawed semen in semen parameters evaluated such as motility, viability, membrane integrity and lipid peroxidation (p<005). In the relationships of sperm parameters, motility vs viability, motility vs membrane integrity and viability vs membrane integrity were positively correlated ($0.82{\sim}0.94$) but lipid peroxidation vs other estimated factors was negatively correlated ($- 0.90{\sim}- 0.98$). Among the evaluation methods, motility vs Viability, motility vs membrane integrity and lipid peroxidation vs other estimated factors were significantly correlated (p<0.05). These results of this. study indicate that the optimal condition of HOST in boar spermatozoa is a 150 mOsmol fructose/Na-citrate solution for 30 min incubation at $37^{\circ}C$ and HOST can substitute the examination of motility, viability and lipid peroxidation.

• 한국수정란이식학회지
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• 제32권3호
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• pp.221-226
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• 2017

Spermatozoa viability can be assessed by microscopy, flow cytometry, and other methods using fluorescent stain. Flow cytometry can be used to examine the morphological and functional characteristics of spermatozoa in a short time. The purpose of this study was to compare the viability of cryopreserved spermatozoa in Jeju black cattle by two dual fluorescent stain methods. Semen of Jeju black cattle raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA were collected with artificial vaginal technique. Sperm was diluted with $Triladyl^{(R)}$-egg yolk diluent and then was performed cryopreservation. There was no significant difference in viability of spermatozoa according to the two dual fluorescent stain methods. However, when the distribution of spermatozoa according to the staining method was compared, the spermatozoa group stained with 6-CFDA/PI was more clearly distinguished than the spermatozoa group stained with calcein AM/PI.

• Clinical and Experimental Reproductive Medicine
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• 제43권4호
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• pp.193-198
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• 2016

Objective: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect. G6PD plays a key role in the pentose phosphate pathway, which is a major source of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH provides the reducing equivalents for oxidation-reduction reductions involved in protecting against the toxicity of reactive oxygen species such as $H_2O_2$. We hypothesized that G6PD deficiency may reduce the amount of NADPH in sperms, thereby inhibiting the detoxification of $H_2O_2$, which could potentially affect their motility and viability, resulting in an increased susceptibility to infertility. Methods: Semen samples were obtained from four males with G6PD deficiency and eight healthy males as a control. In both groups, motile sperms were isolated from the seminal fluid and incubated with 0, 10, 20, 40, 60, 80, and $120{\mu}M$ concentrations of $H_2O_2$. After 1 hour incubation at $37^{\circ}C$, sperms were evaluated for motility and viability. Results: Incubation of sperms with 10 and $20{\mu}M\;H_2O_2$ led to very little decrease in motility and viability, but motility decreased notably in both groups in 40, 60, and $80{\mu}M\;H_2O_2$, and viability decreased in both groups in 40, 60, 80, and $120{\mu}M\;H_2O_2$. However, no statistically significant differences were found between the G6PD-deficient group and controls. Conclusion: G6PD deficiency does not increase the susceptibility of sperm to oxidative stress induced by $H_2O_2$, and the reducing equivalents necessary for protection against $H_2O_2$ are most likely produced by other pathways. Therefore, G6PD deficiency cannot be considered as major risk factor for male infertility.

• 한국수정란이식학회지
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• 제26권4호
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• pp.245-250
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• 2011

The aim of this study was to evaluate the post-thawed characteristics of leopard cat semen. In this experiment, semen was collected from two leopard cats (A and B) at wild animal center in Seoul Grand Park in Korea. After collection, the sperms were washed with D-PBS and diluted by the freezing medium (Irvine science, USA) and stored in liquid nitrogen. The post-thawed concentration was $357{\times}10^6sperms/ml$ for A and $97{\times}10^6sperms/ml$ for B. The viability of post-thawed sperm from A and B individual was 24.0% and 19.0%, respectively. Pre-freezing motility of A and B individual semen was 68.54% and 56.65. Leopard cat A had more normal sperm than that of B (69.5% vs. 54.5%). Acrosome integrity analysis detected live (14.5% vs. 9.0%), damage (39.0% vs. 44.0%) and dead (46.0% vs. 47.0%) in leopard cat A and B, respectively. The present results concluded that leopard cat semen can be collected successfully by electro-ejaculation method and cryopreserved successfullyfor future use in different assisted reproductive technologies. The cryopreservation protocol needs to be modified for increasing post-thawed viability of leopard cat spermatozoa.

• Animal Bioscience
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• 제34권2호
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• pp.198-204
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• 2021

Objective: The aim of this study was to ascertain the effects of adding green tea extract (GTE) to skim milk-egg yolk (SM-EY) extender on both the quality of post-thawed bull semen and the pregnancy rates of the recipient cows. Methods: Twelve ejaculates from four Simmental bulls, aged 3 to 5 years and weighing 900 to 950 kg, were diluted SM-EY extender, added with 0, 0.05, 0.1, and 0.15 mg GTE/100 mL extender and then frozen. After four weeks storage in liquid nitrogen, the sperm were thawed and evaluated for viability, motility, intact plasma membrane (IPM), and DNA fragmentation. Meanwhile, the estrus cycles of 48 recipient cows were synchronized by intramuscular administration of a single injection of 5 mg prostaglandin F2α. Estrus cows were divided into four equal groups and inseminated artificially 18 to 20 h after the onset of estrus by using semen from each extender group. Pregnancy was diagnosed by measuring serum progesterone levels at 21 days, followed by transrectal palpation 90 days after insemination. Results: The findings revealed that adding 0.1 mg of GTE/100 mL extender produced the highest percentages of sperm viability (70.67%±1.75%), motility (69.17%±1.47%), and IPM (69.23%±1.21%) and the lowest percentage of DNA fragmentation (3.00%±0.50%). The pregnancy diagnosis revealed that all cows (36/36) inseminated using frozen semen in GTE addition extender were pregnant (pregnancy rate 100%), whereas the pregnancy rate of the control group was 83.33% (10/12). Conclusion: It may be concluded that 0.1 mg GTE/100 mL extender yields the best quality of spermatozoa and that all variants doses of GTE in extender produce a higher pregnancy rate among recipient cows.

• Journal of Animal Science and Technology
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• 제49권6호
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• pp.729-736
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• 2007

본 연구는 돼지 정액의 체외보존에 있어 pyruvate, taurine 및 melatonin의 항산화 효과와 생존력의 평가방법간의 상관관계를 검토하고자 실시하였다. 돼지 정액의 체외보존시 항산화 효과를 평가하기 위하여 체외보존 신선정액에 pyruvate(1mM), taurine(50 mM) 및 melatonin(100nM)에 H2O2(100uM)을 혼합첨가 및 항산화제 단독처리하여 체외에서 3~12시간까지 배양하면서 3시간 간격으로 정자의 운동성, 정자의 원형질막의 완전성 및 지질 산화도를 측정하였다. 정자의 운동성은 항산화제 첨가구가 다른 처리구에 비해 높은 결과를 나타내었고, Hypoosmotic swelling test(HOST) 방법을 이용한 정자의 원형질막 완전성에서도 운동성과 유사한 결과를 나타내었다. 정자의 원형질막의 지질산화도 검사에서는 항산화제 첨가구와 무첨가구가 H2O2 첨가구와 H2O2와 항산화제의 혼합 첨가구에 비해 낮은 결과를 나타내었다. 또한 평가방법들 간의 상관관계에서는 운동성과 정자의 원형질막의 완전성에는 정의 상관관계를 나타내었고, 정액의 성상검사 방법과 지질산화검사 방법에서는 부의 상관관계를 나타내었다. 본 실험의 결과는 정액희석액에 항산화제의 첨가는 정자의 운동성 및 정자의 원형질막 완전성을 증가시키고, 정자의 원형질막의 지질산화도를 감소시키는 것으로 나타났다. 또한 정자의 원형질막의 완전성 검사 (HOST)는 정자의 원형질막의 지질산화도 검사 및 운동성 검사를 대신하여 정자의 질적 평가에 유용하게 이용될 수 있을 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제22권12호
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• pp.1614-1619
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• 2009

The present study was conducted to observe the effect of increased osmolality of basic tris extender supplemented with trehalose and sucrose on post-thawing quality (motility, progressive motility, viability, the rate of acrosome abnormality, total abnormality and membrane integrity) of Markhoz goat spermatozoa. Fresh semen samples were evaluated for motility and sperm concentration. Only semen samples with motility more than 70% and sperm concentration higher than $3{\times}10^{9}$ sperm/ml were used for cryopreservation. In Exp. 1, trehalose (50, 75 or 100 mM) and sucrose (40, 60 or 80 mM) were added to a basic tris diluent. Based on the results of experiment 1, the goal of Exp. 2 was to investigate the combinational effects of the highest and lowest concentrations ($T_{100}+S_{80}$ or $T_{50}+S_{40}$) of trehalose and sucrose. As the control, semen was diluted and frozen in the tris diluent without trehalose or sucrose. The results in Exp. 1 showed that all evaluated spermatozoa characteristics improved significantly after freezing and thawing (p<0.05) and at the same time the increase of trehalose and sucrose concentrations in basic extenders was seen, with the best results obtained for extenders containing 70 and 100 mM trehalose and 80 mM sucrose. Comparing these results with those of control diluents, the effects of supplementation were significantly (p<0.05) better. In Exp. 2, the results showed no significant differences (p>0.05) between $T_{100}+S_{80}$ and $T_{50}+S_{40}$ extenders, but the results of $T_{50}+S_{40}$were slightly better than obtained with $T_{100}+S_{80}$ diluents. Furthermore, the results of this experiment indicated that the sperm characteristics in the isotonic control extender were significantly (p<0.05) lower than examined extenders. In conclusion, the results of this study indicated that goat sperm can tolerate hypertonic trehalose and sucrose solutions better than isotonic control diluents in the freezing period. In particular, these positive effects have been shown for acrosome integrity, which is very important for the fertilization capacity of sperm. The data indicated that addition of trehalose plus sucrose to the freezing extender can be recommended for cryopreservation of goat spermatozoa, but more data is needed on pregnancy rate, acrosome reaction and IVF to ascertain the real effect.

• Reproductive and Developmental Biology
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• 제38권4호
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• pp.159-163
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• 2014

The objective of this study was to determine the effects of E. coli on boar sperm quality and reproductive performance in sows after artificial insemination. Three different levels of E. coli were artificially inoculated to semen with following concentrations; Control, 500, 5,000 and 50,000 colony forming unit (cfu)/ml. Semen samples were preserved at $17^{\circ}C$ for 5 days. Sperm motility was significantly decreased (p<0.05) on day 3 in the group inoculated with 5,000 cfu/ml compared to control groups. In all treatment groups, sperm motility was gradually decreased as storage time increased, but the decline pattern was more drastic in the groups inoculated with 5,000 and 50,000 cfu/ml groups from day 3 (p<0.05) compared to control group. After 3 day of storage at $17^{\circ}C$, sperm viability in sample inoculated with the highest concentration (50,000 cfu/ml) of bacteria was less (p<0.05) than that of control group. The pH of semen sample pH was maintained 7.2~7.5 in all groups during the experimental period. No differences (p>0.05) were found for both storage time and bacterial concentration. The pregnancy rate and live born piglets tend to decrease by increasing the concentration of E. coli in semen. In particular, the rate of pregnancy was lower in the group inoculated with 50,000 cfu/ml (58.3%) compare to the other groups (81.8, 75.0, 76.5%). These results suggest that the contamination of E. coli in boar semen negatively affects fertilizing ability of boar sperm and the reproductive performance obtained from sows after artificial insemination.