• 한국동물생명공학회지
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• 제35권1호
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• pp.58-64
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• 2020

This present study was conducted to investigate protective effect of discontinuous Percoll gradient containing alpha-linolenic acid (ALA) before freezing process on viability, acrosome damage, mitochondrial activity, and oxidative stress of frozen-thawed boar spermatozoa. The separation of spermatozoa by discontinuous Percoll gradient was performed by different concentration of Percoll solution (45/90%) containing ALA combined with bovine serum albumin (BSA), and collected sperm in each Percoll layer was cryopreserved. To evaluate viability, acrosome damage, mitochondrial activity, and reactive oxygen species (ROS) level of frozen-thawed sperm, flow cytometry was used. Morphological abnormalities were observed under light microscope. In results, viability of sperm from 90% Percoll layer was higher than control and 45% Percoll group (p < 0.05). Separated sperm in 90% Percoll layer had lower acrosome damage and morphological abnormalities than control as well as viability, whereas 45% Percoll group was higher (p < 0.05). Similar with acrosome damage and abnormalities, mitochondrial activity was slightly enhanced and the population of live sperm with high ROS level was decreased by 90% Percoll separation, however, there was no significant difference. Supplementation of 3 ng/mL ALA into Percoll solution increased sperm viability and decreased population of live sperm with high ROS compared to control (p < 0.05). In conclusion, discontinuous Percoll gradient before freezing process could improve efficiency of cryopreservation of boar sperm through selection of sperm with high freezing resistance, and supplement of ALA during Percoll gradient might contribute suppression of ROS generation via stabilizing of plasma membrane during cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• 제49권2호
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• pp.117-126
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• 2022

Objective: This study examined whether the addition of triple antioxidants (3A)-10 µM acetyl-L-carnitine, 10 µM N-acetyl-L-cysteine, and 5 µM α-lipoic acid-in freezing-thawing medium during human sperm cryopreservation using the sucrose vitrification (SuV) and liquid nitrogen vapor (Vapor) techniques could improve post-thaw survival of spermatozoa. Methods: We analyzed 30 samples from healthy human sperm donors. Each sample was allocated into one of five groups: fresh control, SuV, SuV+3A, Vapor, and Vapor+3A. The sperm motility, morphology, viability, intracellular and extracellular reactive oxygen species (ROS) levels, and sperm DNA fragmentation (SDF) were evaluated. Results: The cryopreserved spermatozoa had significantly reduced percentages of motility (p<0.05) and viability (p<0.05). Antioxidant supplementation non-significantly improved these parameters (p>0.05). No significant differences were found in sperm morphology between the fresh and frozen-thawed groups (p>0.05). After freezing, the extracellular ROS levels in the frozen-thawed groups were significantly higher (p<0.05) than in the fresh group. However, we did not find any differences in intracellular ROS parameters among these groups (p>0.05). The SDF was higher in the SuV and Vapor groups than in the fresh group, but without statistical significance (p=0.075 and p=0.077, respectively). Conclusion: Cryopreservation had detrimental effects on sperm motility, viability, and extracellular ROS levels, without changing the morphology or intracellular ROS levels. Antioxidant supplementation was slightly effective in preventing SDF in frozen-thawed spermatozoa.

• Clinical and Experimental Reproductive Medicine
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• 제48권4호
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• pp.316-321
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• 2021

Objective: Amino acids can protect sperm structure in cryopreservation due to their antioxidant properties. Therefore, the present study aimed to investigate the protective effect of L-carnitine (LC) and N-acetyl cysteine (NAC) on motility parameters, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA damage, and human sperm intracellular reactive oxygen species (ROS) during vitrification. Methods: Twenty normal human sperm samples were examined. Each sample was divided into six equal groups: LC (1 and 10 mM), NAC (5 and 10 mM), and cryopreserved and fresh control groups. Results: The groups treated with LC and NAC showed favorable findings in terms of motility parameters, DNA damage, and MMP. Significantly higher levels of intracellular ROS were observed in all cryopreserved groups than in the fresh group (p≤0.05). The presence of LC and NAC at both concentrations caused an increase in PMI, MMP, and progressive motility parameters, as well as a significant reduction in intracellular ROS compared to the control group (p≤0.05). The concentrations of the amino acids did not show any significant effect. Conclusion: LC and NAC are promising as potential additives in sperm cryopreservation.

• 한국동물생명공학회지
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• 제36권2호
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• pp.69-75
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• 2021

Cryopreservation is a widely-used efficient means of long-term sperm preservation. However, unlike other types of semen, cryopreserved boar semen has reduced fertility and the efforts continue to optimize post-thawing sperm recovery. In this study, we evaluated the effects of various washing solutions (Hulsen solution, lab-made DPBS and commercial DPBS) on post-thawing porcine sperm kinematics (CASA system), viability (SYBR-14/PI) and acrosome integrity (PSA/FITC). We also examined the effect of washing-centrifugation on frozen-thawed semen kinematics. The results indicate that type of washing solution and post-thawing centrifugation alters parameters linked to sperm quality (total motility, progressive motility, viability and acrosome integrity). Significantly higher (p < 0.05) motility and progressive motility were obtained when cryopreserved semen was processed with Hulsen solution. The post-thaw percentage of live and intact acrosomal sperm was significantly higher in group 1 (Hulsen solution) as compared to other groups. Following thawing-centrifugation, the results showed significantly higher motility and progressive motility in group 1 than other groups. However, the latter two DPBS groups did not differ statistically. Taken together, Frozen-thawed spermatozoa motility, acrosome integrity and viability can be affected by the type of washing solution used. Moreover, centrifugation of frozen-thawed semen has an unfavorable effect on total motility and progressive motility.

• 한국양식학회지
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• 제12권1호
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• pp.57-62
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• 1999

숭어 정액의 냉장 및 냉동보존시 적합한 희석액을 선정하고, 알에 대한 수정률을 평가하였다. 비보존 숭어 정자의 머리는 공모양으로 직경 $1.26{\pm}0.08 \{mu}textrm{m}$, 길이 $1.06{\pm}0.07 \{mu}textrm{m}$ 였으며, 과립상의 염색질을 가지고 있었다. 숭어 정자의 냉장보존시($0^{\circ}C$, 10일간) 희석액으로는 동종의 혈정이 가장 높은 정자활성을 나타냈으며, egg-tris, 0.1M, 0.3M 및 0.5M glucos에서는 활성이 서로 비슷하였다. 또한 동해방지제로 10% DMSO, 희석액으로 MFRS를 사용하여 냉동보존한 후 해동시켰을 때 대조구와 유사한 수정률을 보였다. 냉동보존 후 해동시킨 일부 정자 중에서는 세포막이 이탈되거나 소실되는 구조적 변화를 나타냈다.

• 한국수정란이식학회지
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• 제33권4호
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• pp.337-342
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• 2018

Cryopreservation is mainly used for preservation of boar sperm. However, this method stresses the sperm by reactive oxygen species (ROS), and the conception rate and the litter size are not more efficient than the liquid preservation of spermatozoa. Therefore, we use chitosan which is a natural product derived antioxidant compound. We used GnHA (chitosan+hyaluronic acid) and GnHG (chitosan hydrogel) as chitosan complexes to cryopreserve boar sperm for improve sperm metabolism and function. Sperm parameter (sperm motility, progressive motility, path velocity, straight-line velocity, curvilinear velocity) is measured by computer-assisted sperm analysis (CASA) using frozen sperm with GnHA or GnHG (0, 0.25, 0.5, 1 mg/mL), respectively. Also, lipid peroxidation analysis using malondialdehyde (MDA) is performed to confirm the antioxidative effect of chitosan in frozen spermatozoa. CASA analysis showed GnHA and GnHG are effective against cryopreserved boar sperm. And antioxidant effect is measured by lipid peroxidation analysis. GnHA and GnHG, which is chitosan complex are effective for boar sperm cryopreservation by antioxidant effect.

• 한국초지조사료학회지
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• 제38권4호
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• pp.320-324
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• 2018

본 연구는 티모시 건초와 농후 사료 위주의 사료를 급여한 한우 씨수소 정소상체 정자 체외수정 효율 조사를 통해 정자의 활용 가능성을 조사하였다. 농후 사료는 체중의 1.8%를 급여하고 양질의 티모시 건초를 자유채식 시킨 14개월령 거세우의 정소에서 분리된 정소상체 미부의 정자를 회수하고 동결 흉해 후 체외수정을 실시한 결과는 다음과 같다. 웅성전핵과 자성전핵이 형성(2PN)된 난자는 정상수정으로, 1개의 전핵(1PN), Expanded Sperm Head (ESH), Polyspermy 형태는 비정상적인 수정의 형태로 평가하였다. 정상적으로 수정된 난자의 비율은 정소상체 정자의 경우 전체 침투율은 49.7% 그리고 정상적인 2PN을 가진 난자는 18.5%를 보였으며, 대조구 정자의 전체 침투율은 54.4%로서 정소상체 정자 보다 높은 결과를 보였으나 유의적인 차이를 보이지는 않았다. 정상적으로 2PN을 형성한 비율은 36.7%로서 정소상체 정자를 이용한 정자 보다 높았으나 유의적인 차이는 없었다. 체외수정 후 발달률 조사에서 정소상체 정자의 분할률은 81.2%, 대조구 정자는 82.7%로 유사한 결과를 보였으나, 배반포 발달률은 정소상체 정자 24.4%와 대조구 정자 12.2%로 정소상체 정자를 사용한 난자의 발달에서는 유의적으로 높았다(p<0.05).

• 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.

• Reproductive and Developmental Biology
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• 제40권3호
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• pp.27-31
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• 2016

The aim of this study was to evaluate effect of ${\alpha}$-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at $37.5^{\circ}C$ for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.

• 한국수정란이식학회지
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• 제18권1호
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• pp.51-59
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• 2003

본 실험은 개의 인공수정에 사용할 정자의 보존에 있어서, 개 정액 동결시 동결속도와 응해온도에 따른 정자의 생존율, 운동성 그리고 intact acrosome의 비율을 조사하였던 바 결과는 다음과 같다. 1. 본 실험에서 실험견의 사출된 평균 신선정액의 농도는 3.44$\times$$10^{8}$ /ml로 정상범위에 들었으며, 정자의 형태학적 판정에서 정상적인 정자의 농도는 평균 59.45$\pm$3.45%로 상대적인 기형율은 약 30~40% 정도 나타났으며, 이는 정상적인 상태의 개 정액이라 할 수 있다. 2 개 정액의 동결속도는 동결하는 높이가 6, 10 및 17 cm 일 때 각각 최저온도는 -11$0^{\circ}C$, 7$0^{\circ}C$, -35$^{\circ}C$로 감소하는 경향을 보였으며 이때 최저온도로 감소하는데 소요되는 시간은 각각 6분, 8분 20초 그리고 12분 50초로 결과적으로 분당 동결속도는 각각 19$^{\circ}C$/min, 8.9$^{\circ}C$/min 그리고 3$^{\circ}C$/min로 나타났다. 3. 정액을 동결속도와 융해온도에 따른 정자의 생존율, 운동성 그리고 intact acrosome의 비율은 동결속도가 3$^{\circ}C$/min일 때 가장 높았으며, 융해 온도는 37$^{\circ}C$일 때 효율성이 가장 높은 것으로 나타났다.4. 동결의 방법에 있어서는 액체질소의 표면으로부터 17 cm 높이에서 동결하는 분당 -3$^{\circ}C$의 동결속도에서 동결하여 37$^{\circ}C$에서 2분간 융해하는 방법이 가장 좋은 결과를 보였으며, 생존성과 운동성은 문제없이 사용할 수 있을 것으로 판단되나, 첨체의 intact한 비율은 저조한 결과를 나타내었다.