• Journal of Animal Science and Technology
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• 제54권2호
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• pp.95-101
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• 2012

본 연구는 희소 한우인 제주흑우의 정액 동결을 위하여 동결보호제로서 glycerol과 amide 계열의 동결보호제인 5%의 DMA, DMF 그리고 7%의 MF를 이용하여 동결 융해 후 정자의 운동성, 생존율, 정자막 온전성 및 정자의 첨체 양상의 변화 조사를 위하여 수행하였다. 제주흑우의 정액을 동결 한 결과 glycerol과 DMF를 사용하였을 때 운동성은 $64.00{\pm}9.62$와 $59.00{\pm}5.48$로 DMA와 MF에 비해 유의적으로 높았으며(p<0.05), 생존율 역시 glycero과 DMF 처리구에서 $58.25{\pm}6.63$과 $53.05{\pm}3.77$로 유의적으로 높게 나타났다(p<0.05). 그러나 glycerol과 DMF 처리구 간의 유의적 차이는 나타나지 않았다. 정자막 온전성 검사(HOST)에 있어서도 역시 glycerol과 DMF 처리에서 정자 미부의 팽창 비율 역시 $45.12{\pm}25.08$과 $44.95{\pm}8.58$로 유의적으로 높았다(p<0.05). 정자의 첨체막 양상 변화에 있어 F pattern의 비율이 MF 처리시 다른 처리구에 비하여 유의적으로 낮았으며(p<0.05), B pattern의 비율은 DMA, DMF 및 MF의 사용시 glycerol에 비하여 유의적으로 증가하였다(p<0.05). AR pattern의 비율에 있어서는 DMF를 제외한 amide의 사용은 F pattern의 감소와 조기 첨체반응에 의한 유의적인 AR pattern의 증가를 볼 수 있었다(p<0.0.5). 그러나 DMF의 사용은 glycerol 보다 AR pattern의 비율이 유의적으로 낮았다(p<0.05). 동결 융해 후 시간에 따른 정자의 생존율과 정자미부 팽창 비율에 있어서 융해 초기에는 glycerol에서 DMF 보다 높은 생존율과 정자 미부 팽창율을 나타냈으나, 1시간 이후부터는 DMF에서 glycerol 보다 높은 생존율과 정자 미부 팽창율을 나타내었다. 이러한 결과는 희소 가축의 생식세포 보존 및 유전자원 확립을 위한 중요한 자료가 될 것이며, 제주흑우 및 희소 가축의 안정적이고 효율적인 동결 정액 제조 연구에 있어 중요한 정보를 제공할 것으로 평가된다.

• 한국수정란이식학회지
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• 제30권1호
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

• Reproductive and Developmental Biology
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• 제37권3호
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• pp.97-102
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• 2013

The objective of this study was to develop of semen transport system for cryopreservation and fertility in bull sperm. The ejaculated semen were diluted with Triladyl containing 20% egg-yolk for transportation. Diluted semen was transported by three methods that there were wrapping tissue (Tissue), sinking under $30^{\circ}C$ water (Water) and sinking between warm water and air (Air) methods. Semen was transported within 2 hours in $0.3^{\circ}C$. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk. And frozen-thawed sperm were estimated with SYBR14/PI double stain for viability, FITC-PNA/PI double stain for acrosome reaction analysis and Rhodamine123 double stain for mitochondrial intact assessment. In results, live sperm (SYBR+/PI-) in Air treatment group ($43.3{\pm}4.7%$) was significantly (p<0.05) higher than other treatment groups (Tissue: $16.3{\pm}2.7%$ and Water: $27.5{\pm}3.1%$), dying sperm (SYBR+/PI+) in Air treatment group ($55.6{\pm}4.7%$) was significantly lower than other treatment groups (Tissue: $77.6{\pm}3.2%$ and Water: $67.6{\pm}3.3%$) (p<0.05). Acrosome reaction in Air treatment group ($0.2{\pm}0.1%$) within live sperm (PI negative region) was significantly (p<0.05) lower than other treatment groups (Tissue: $0.7{\pm}0.2%$ and Water: $0.5{\pm}0.1%$), the acrosome reaction in Air treatment group ($28.6{\pm}2.8%$) within all sperm also was significantly lower than other treatment groups (Tissue: $44.2{\pm}1.8%$ and Water: $36.2{\pm}2.0%$) (p<0.05). And mitochondrial intact in Air treatment group within live ($97.1{\pm}0.4%$) and all ($61.9{\pm}3.3%$) sperm were significantly higher than other treatment groups (Tissue: $85.2{\pm}3.3%$, Water: $87.8{\pm}2.9%$ within live sperm and Tissue: $49.28{\pm}3.7%$, Water: $42.0{\pm}3.1%$ within all sperm) (p<0.05). Therefore, we suggest that transportation by sinking method between warm water and air was beneficial to improvement of fertility in frozen-thawed in bull semen.

• 한국동물생명공학회지
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• 제36권2호
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• pp.106-115
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• 2021

Sperm cryopreservation is an important method of assisted reproductive techniques and storing genetic resources. It plays a vital role in genetic improvement, livestock industrial preservation of endangered species, and clinical practice. Consequently, the cryopreservation technique is well organized through various studies, especially on Korean native cattle (Hanwoo). However, the cryopreservation technique of Korean native brindled cattle, which is one of the native cattle species in Korea, is not well organized. Therefore, it is necessary to develop a Supplementary Table technique for the cryopreservation of Korean native brindled cattle. For this purpose, it is important to first evaluate the quality of the currently produced cryopreserved sperm of Korean native brindled cattle. In this study, we randomly selected 72 individual Korean native brindled cattle semen samples collected from 8 different region research centers and used them to evaluate sperm functions. We focused on the quality evaluation of cryopreserved Korean native brindled cattle semen following the measurement of motion kinematics, capacitation status, intracellular ATP level, sperm motility, and cell viability. Then, the values of each of the eight groups were derived from various sperm parameters of nine individual samples, including sperm motility, kinematics, cellular motility, and intracellular ATP levels, which were used to compare and evaluate sperm function. Overall, differences in various sperm parameters were observed between most of the research centers. Particularly, the deviations of motility and motion kinematics were high according to the sample. Therefore, we suggest that it is necessary to develop a standard method for the cryopreservation of Korean native brindled cattle semen. We also suggest the need for sperm quality evaluation of the cryopreserved semen of Korean native brindled cattle before using artificial insemination to attain a high fertility rate.

• Asian-Australasian Journal of Animal Sciences
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• 제23권10호
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• pp.1276-1281
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• 2010

The present study was conducted to investigate the effects of butylated hydroxytoluene (BHT) supplementation on diluted, cooled and frozen-thawed ram spermatozoa. After primary evaluation of collected ejaculates, only semen samples with motility of more than 70% and sperm concentration higher than $3{\times}10^3$ sperm/ml were used for cryopreservation. The selected semen samples were then pooled and diluted 1:4 with Tris Citrate Fructose Yolk (TCFY) extender supplemented with different concentrations of BHT (0.5, 10, 2.0 and 3.0 mM). As the control, semen was diluted and frozen in the diluent without BHT. Motility, progressive motility, viability, membranes and acrosome integrity were evaluated after dilution (part 1), cooling (part 2) and freezing and thawing (part 3). The results of the first part of the experiment showed that there were no significant difference between treatments in the motility, progressive motility, viability, membranes and acrosome integrity of spermatozoa, but the results with 2.0 mM BHT were slightly better than obtained with other levels of BHT and control extender. Significantly better results (p<0.05) were observed in the second part of the experiment for cooled spermatozoa characteristics, when extender was supplemented with 2.0 and 3.0 mM BHT. Furthermore, the results obtained in the third part of the experiment indicated that, after freezing and thawing, all evaluated semen characteristics were improved significantly (p<0.05) by increasing BHT levels, with the best results obtained for extender containing 2 mM BHT. Comparison of these results with those of control diluent, the effects of supplementation were significantly (p<0.01) better. However, the higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of spermatozoa compared to extender containing 2.0 mM BHT. In conclusion, the results obtained in this study showed that the semen quality of rams was improved when BHT was added to extender used before the freezing process.

• Asian-Australasian Journal of Animal Sciences
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• 제11권4호
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• pp.445-449
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• 1998

Semen quality was compared between the local Katjang and the cross-bred (local Katjang ♀ ${\times}$ German Fawn ♂) bucks. There were on significant genotypic differences in semen characteristics of concentration (first ejaculate : $6.19{\pm}1.30$ -versus $6.33{\pm}1.40{\times}10^9/ml;$second ejaculate: $5.82{\pm}1.10$ - versus $5.68{\pm}1.45{\times}10^9/ml$, for Katjang and the cross-breds, respectively), percentage live (first ejaculate: $77.61{\pm}1.33%$ versus $77.81{\pm}0.53%$; second ejaculate: $81.97{\pm}1.59%$ versus $82.74{\pm}0.96%$, for Katjang and cross-breds, respectively) and percentage of normal sperms (first ejaculate: $12.54{\pm}3.88%$ versus $26.45{\pm}3.83%$; second ejaculate: $38.68{\pm}3.65%$ versus $28.54{\pm}4.38%$, for Katjang and cross-breds, respectively), with the exception of seminal volume and sperm motility. Means of all variables were within the values reported for other goat breeds, In contrast, the differences in semen characteristics between the first and second ejaculations of both genotypes were more distinct, the second ejaculations always had more volume, more normal sperms and better sperm motility but less sperm concentrations. Removing the seminal plasma and replacing it with tris-citrate buffer greatly prolonged the viability of sperms of both genotypes when stored at $5{^{\circ}C}$. Sperm motility seens to be a good indicator of sperm viability. However, the sperms of the corss-bred bucks withstood the washing process better and their swimming abilities were superior ($8.12{\pm}0.46mm/min$) when compared to those of the local Katjang breed ($5.42{\pm}0.49mm/min$). The higher content of calcium ions in their seminal plasma (first ejaculate: $10.5{\pm}0.8$ versus $10.6{\pm}0.8mg/100ml$;second ejaculate: $15.3{\pm}0.8$ versus $16.1{\pm}0.8mg/100ml$, for Katjang and cross-breds, respectively) means that in natural matings the sperms of the cross-breds would be at an advantage compared to those of the local Katjang, since calcium ions reportedly initiate acrosomal reactions.

• 한국수정란이식학회지
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• 제28권3호
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• pp.297-302
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• 2013

Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of genetically elite populations. Castration, catastrophic injury, sudden death or any other event that makes semen collection or mating impossible may prematurely terminate a stallion reproduction. Stallion epididymal spermatozoa vary widely in the loss of progressive motility, acrosomal integrity, and viability during freezing and thawing. The objective of this work was to investigate the effect of (1) freezing package types on cryopreservation efficiency, (2) thawing temperatures (37, 56 or $70^{\circ}C$) on Computer Assisted Sperm Analysis (CASA) parameters and (3) post-thawing incubation time (0, 1, 2 or 4h) on castrated stallion epididymis. Post-thawed sperm motility ranged between 59.69% and 64.28% ($56^{\circ}C$ and $37^{\circ}C$) in various thawing temperatures. When stallion epididymis sperm was frozen, straw was better than freezing tube on VCL (Velocity of Curvilinear Line) and VAP (Velocity of Average Path) parameter. Higher percentage of motility was observed at $37^{\circ}C$ thawing temperature even though no significant difference was observed among various temperatures. The motility, VCL, ALH (Amplitude of Lateral Head displacement), VAP, BCF (Beat-Cross Frequency) and STR (Straightness index) parameter of post-thawed sperm were significantly decreased by increasing the incubation time for all thawing temperatures. The present study showed that type of freezing package (Straw vs. Freezing tube) was not significantly different on cryopreservation efficiency. Furthermore, stallion epididymal spermatozoa frozen-thawed at $37^{\circ}C$ for 1 min resulted the highest proportion of motility and velocity movement. In addition, motility and viability of frozen-thawed stallion epididymal spermatozoa were also decreased by incubation.

• 한국수정란이식학회지
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• 제29권4호
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• pp.385-391
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• 2014

Objective of this study was to investigate the effect of nicotinic acid (NA) on the characteristics in fresh semen of miniature pig. We evaluated viability, acrosome reaction and mitochondrial integrity of sperm on 0, 3, 7 and 10 days during storage period with nicotinic acid. As results, the survival rate of sperm in 15 mM NA (day 3, $87.8{\pm}1.2%$; day 5, $84.0{\pm}2.7%$; day 7, $82.2{\pm}0.9%$) and 30 mM NA (day 3, $87.7{\pm}0.3%$; day 5, $84.4{\pm}2.5%$; day 7, $82.3{\pm}0.7%$) groups were higher than control and 5 mM NA groups in 3, 7 and 10 days of semen storage. The NA-treated sperm on 10 day was used day for observing acrosome integrity. The survival sperm with acrosome reaction was higher in 30 mM NA group (day 3, $2.7{\pm}0.2%$; day 5, $3.3{\pm}0.6%$; day 7, $11.4{\pm}0.3%$) than in the control, significantly (P<0.05). Moreover, the live sperm with mitochondrial integrity was higher in whole treatment groups of NA than control group, significantly (P<0.05). Specially, most mitochondrial integrity on 10 day of semen storage was significantly higher in 30 mM NA group ($90.2{\pm}1.6%$) than other treatment groups (control, $81.8{\pm}3.1%$; 5 mM NA, $83.4{\pm}3.0%$; 15 mM NA, $89.1{\pm}0.7%$, P<0.05). In conclusion, supplement of NA in liquid semen of miniature pig can improve and maintain semen quality, such as viability, acrosome reaction, and mitochondria integrity.

• 한국임상수의학회지
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• 제24권4호
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• pp.577-583
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• 2007

It is important to obtain semen with good quality for efficient fertilization and pregnancy. To obtain these semen, various methods have been developed but most of these methods are time consuming and require costly equipment. Therefore, the objective of this research is to investigate the usability of column filtration system as quick and simple method to get sperm with better quality. Ejaculates were obtained from 5 dogs and analyzed with basic quality parameters before each filtration. Sperm concentration was adjusted to $5{\times}10^7/ml$ after dilution. The experimental groups were divided into non-filtered group(control) and filtered groups(glass wool, Sephadex 5% and Sephadex 20%). Ejaculates were filtered through each filter system and assessed by recovery rate of sperm, motility, normal morphology, CFDA/PI stain and plasma membrane integrity(hypo-osmotic swelling test, HOST). The lowest recovery rate of spermatozoa was recorded in glass wool filtration group, followed by 20% Sephadex filtration group(p<0.05). There was no significant difference between control(non-filtered) and 5% Sephadex filtration poop. Also, there was no significant difference of sperm motility assessed under light microscope among experimental groups. Morphological normality of canine spermatozoa was the highest in the glass wool filtration group and the lowest in the 5% Sephadex filtration group with no significant differences versus 20% Sephadex filtration and control group, respectively(p<0.05). Viability of canine sperm assessed by CFCA/PI staining was the highest in the glass wool filtration poop with no significant difference versus the control group, and the lowest in the 20% Sephadex filtration group with no significant difference versus 5% Sephadex filtration group, respectively(p<0.05). HOS values of canine sperm was the highest in the 20% Sephadex filtration group with no significant difference versus 5% Sephadex filtration group, and the lowest in the control poop with no significant difference versus glass wool filtration group, respectively(p<0.05). Therefore, these results indicated that filtration treatment for extended canine sperm would be useful method to get sperm with better quality by trapping the damaged sperm, consequently filter would be physical barrier against injured or immotile sperm.

• Animal Bioscience
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• 제37권6호
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• pp.1001-1006
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• 2024

Objective: This study aimed to investigate the effect of Codonopsis pilosula polysaccharide (CPP) on the motility, mitochondrial integrity, acrosome integrity rate, and antioxidant ability of sheep sperm after preservation at 4℃. Methods: Semen from healthy adult rams were collected and divided into four groups with separate addition of 0, 200, 400, and 1,000 mg/L CPP. Sperm motility was analyzed using the Computer-Assisted Semen Analysis software after preservation at 4℃ for 24, 72, 120, and 168 h. Sperm acrosome integrity rate was analyzed by Giemsa staining at 24, 72, and 120 h, and mitochondrial membrane integrity was analyzed by Mito-Tracker Red CMXRos. The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content of spermatozoa were measured after 120 h of preservation. Results: The sperm viability and forward-moving sperm under 200 mg/L CPP were significantly higher than that in the control group at 72 h (61.28%±3.89% vs 52.83%±0.70%, 51.53%±4.06% vs 42.84%±1.14%), and 168 h (47.21%±0.85% vs 41.43%±0.37%, 38.68%±0.87% vs 31.68%±0.89%). The percentage of fast-moving sperm (15.03%±1.10% vs 11.39%±1.03%) and slow-moving sperm (23.63%±0.76% vs 20.29%±1.11%) in the 200 mg/L group was significantly higher than control group at 168 h. The mitochondrial membrane integrity of the sperm in the group with 200 mg/L CPP was significantly higher than those in the control group after storage at 4℃ for 120 h (74.76%±2.54% vs 65.67%±4.51%, p<0.05). The acrosome integrity rate in the group with 200 mg/L (87.66%±1.26%) and 400 mg/L (84.00%±2.95%) was significantly higher than those in the control group (80.65%±0.16%) after storage for 24 h (p<0.05). CPP also increased T-AOC and decreased the MDA concentration after preservation at 4℃ (p<0.05). Conclusion: Adding CPP could improve the T-AOC of sperm, inhibit lipid peroxidation, and facilitate semen preservation.