• 한국수정란이식학회지
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• 제27권1호
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• pp.51-55
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• 2012

The objective of this study was to investigate the motility and kinematics of boar sperm that while stored at 4C. The samples of fresh boar semen were place into an extender, Androhep, and stored at $4^{\circ}C$. In three of these samples, cryoprotectants were added. The sperm's motilities and kinematics were evaluated by using microscope (${\times}400$) and the viability status was evaluated by using with eosin staining method. The 5 sample groups are; Goup A:Androhep (extender), stored at $17^{\circ}C$. Group B:Androhep (extender), stored at $4^{\circ}C$. Group C:Androhep (extender), + 3% glycerol (cryoprotectant), stored at $4^{\circ}C$. Group D:Androhep (extender), + 3% DMSO (cryoprotectant), stored at $4^{\circ}C$. Group E:Androhep (extender), + 3% ethylene glycol (cryoprotectant), stored at $4^{\circ}C$. In group A, the sperm's motility was reduced. On day one the sperm's motility was ($85.7{\pm}2.3$) and day 5 the motility was ($43.9{\pm}3.3$). In group B, C and D the sperm's motility were reduced to 0 on day 5. In group E the sperm's percentage of motility decreased. On day one the sperm's motility was ($42.0{\pm}0.5$) and day 5 the motility was ($2.3{\pm}0.3$). When comparing cryoprotectant in samples of boar sperm there is a slight improvement in the results when the use of Androhep Lite (extender), + 3% ethylene glycol (cryoprotectant), stored at $4^{\circ}C$ are used. Based on these results, ethylene glycol can protect sperm from heat shock at $4^{\circ}C$, but not satisfactory level. However, it showed the possibilities of liquid semen preservation at $4^{\circ}C$ by using cryoprotectant.

• 한국양식학회지
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• 제10권3호
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• pp.273-279
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• 1997

자주복(Takifugu rubripes) 정자의 액상보존을 위한 기초자료를 얻고자, 수조사육한 전장 43.5-45.1 cm, 체중 1.7-2.0 kg의 2-3년생 자주복 수컷을 재료로 하여, 정자의 액상보존 효과를 연구하였다. 희석액으로 marine fish ringer solution과 1% NaCl을 사용하여 7일간 보존하였을 때, SAI와 정자의 생존율이 각각 1.7과 $59.0\pm1.5%,\;1.6$과$\;56.7\pm6.2%$로 가장 높았다. 정자를 3-5배로 희석하여 보존하였을 때, 가장 높은 SAI와 정자의 생존율을 보였으며, 보존온도로는 0- 5$^{\circ}C$가 적절하였다. SAI와 정자의 생존율을 높이기 위한 항생제로는 neomycin (800 ppm)이 효과적이었다.

• Clinical and Experimental Reproductive Medicine
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• 제44권1호
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• pp.52-55
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• 2017

The aim of this study was to report a successful pregnancy using completely immotile frozen-thawed spermatozoa selected by laser. A single laser shot was used to detect the presence of viable immotile spermatozoa in fresh and frozen-thawed testicular spermatozoa. The viability rate was 55.8% after the laser detection, and cryopreservation was carried out immediately. The thawing test was performed on the day of oocyte pick-up, and no motile sperm were observed after extending the culture for another 4 hours, while a survival rate of 39.8% was detected using the laser. In all, five mature oocytes were injected, resulting in four cases of normal fertilization (80%) on day 1. Further, two high-quality day 3 embryos were transferred, which resulted in a singleton pregnancy. Our study demonstrates that completely immotile spermatozoa are worth cryopreserving for further intracytoplasmic sperm injection, which provides a new insight into male fertility preservation in cases of completely immotile spermatozoa.

• 한국가축번식학회지
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• 제9권2호
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• pp.153-157
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• 1985

Ejaculated and epidiymal goat spermatozoa were preserved for 0, 6, 12 adn 18 h, and 0 and 18 h in a semi-aerobic condition at 20-$25^{\circ}C$, and preincubated for 5-6 h in a CO2 incubator in m-KRB solution. Then they were preincubated at different concentrations (3-5, 25-48 and 105-190$\times$107/ml), and ability of penetration into zona-free hamster eggs in vitro was examined. When ejaculated spermatozoa were preincubated in m-KRB solution after presservation for 12 and 18 h, 12 and 29% of zona-free eggs were penetrated, and only 4% of eggs were penetrated by epididymal spermatozoa which were preincubated after preservation for 18 h. When spermatozoa were preincubated at a low concentration, the penetration rates were very low. But when the sperm concentration during preincubation was 25-48 and 105-190$\times$107/ml, the penetration rates increased to about 30%.

• Reproductive and Developmental Biology
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• 제39권3호
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• pp.69-76
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• 2015

In a conventional sense, dried-spermatozoa are all dead and motionless due to the lost of their natural ability to penetrate oocytes both in vivo and in vitro. However, their nuclei are completely able to contribute to normal embryonic development even after long-term preservation in a dried state when the dried-spermatozoa are microinjected into the oocytes. In this sense, dried spermatozoa must still be alive. Thus, defining spermatozoa as alive or dead seems rather arbitrary. Several drying method of sperm including freeze-drying, evaporative/convective-drying and heat-drying were represented in this review. Although the drying protocol reported here will need further improvement, the results suggest that it may be possible to store the male genetic resources.

• 한국수정란이식학회지
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• 제21권4호
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• pp.345-351
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• 2006

본 연구는 항생제가 첨가되지 않은 돼지 혼합액상 정액을 17$^{\circ}C$ 정액 보관고에 보관하면서 보관일수의 증가가 따라 정자의 운동성, 정액 내 세균의 증식 여부 및 체외 수정란 생산 효율에 미치는 영향을 조사하고자 하였다. 정자의 운동성은 1일 (78.7$\pm$2.4%)에 비하여 3일(78.7$\pm$2.4%)과 5일째(64.8$\pm$2.4%)는 유의적으로(p<0.05) 낮은 운동성을 나타내었다. 보관일수에 따른 정액 내 세균수의 변화는 보관 5일이 $57.8\pm105.2\times10^4$ Cfu로 0일과 3일의 $32.1\pm76.8\times10^4$ Cfu와 $26.9\pm46.6\times10^4$ Cfu에 비하여 유의적인(p<0.05)증가를 나타내었다. 보관된 정액을 이용하여 체외 수정한 결과, 정상적인 수정(2PN)은 1일과 3일째의 66.0$\pm$2.7%와 64.0$\pm$2.7%에 비하여 5일째에는 56.0$\pm$2.6%로 유의적인(p<0.05) 차이를 나타내었다. 체외 수정란의 발달율에서 난할율은 1일째의 75.0$\pm$l.4%에 비하여 3일과 5일째는 70.0$\pm$0.3%와 71.0$\pm$0.3%로 유의적으로(p<0.05) 낮게 나타났으며, 상실배로의 발달율에 있어서 1일째는 32.0$\pm$1.4%의 발달율을 보였으나 3일과 5일째에는 28.0$\pm$1.3%와 24.0$\pm$1.3%로 유의적인(p<0.05) 감소치를 나타내었고, 배반포로의 발달율에 있어서도 1일에 15.0$\pm$1.0%에 비하여 3일과 5일은 11.0$\pm$0.9%와 8.0$\pm$0.9%로 유의적으로(p<0.05) 낮은 발달율을 나타내었다. 이상의 결과를 종합할 때 항생제가 첨가되지 않은 돼지 혼합 정액은 보관일수 3일째부터 정자의 운동성이 감소하고 세균수는 증가하였다. 또한 보존된 정액을 이용하여 체외 수정을 실시할 경우, 보관일수가 증가할수록 정상 수정율과 체외 발달율이 감소함으로 항생제를 첨가하지 않는 경우 3일 이상 정액을 보관하여 사용하지 않는 것이 바람직 하다고 사료된다.

• 한국수정란이식학회지
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• 제28권1호
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• pp.31-39
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• 2013

Alpha-tocopherol as an antioxidant acts in preservation of chilled semen by preserving cell membrane damage from lipid peroxidation. Optimum concentrations of ${\alpha}$-tocopherol in egg yolk-citrate (EYC) extender need to be studied in crossbred bull's semen. Different concentrations of ${\alpha}$-tocopherol viz. 0, 1, 2, 4 and 6mg per ml of extender were used. Semen was collected once a week from four bulls used to regular collection, aged 4 to 7 years, weighing 320 to 450 kg, and with body condition score 4 to 4.5 and scrotal circumference 23 to 32 cm. Semen was evaluated routinely and sperm morphology was viewed under light microscope at ${\times}1,000$ magnification after fixing with buffered formal saline. Over 90% had normal head, acrosome, mid-piece and tail. Semen was diluted with egg-yolk-citrate extender to produce $15{\times}10^6$ spermatozoa/ml and 0, 1, 2, 4 and 6 mg/ml ${\alpha}$-tocopherol were added. The semens amples were kept at $8^{\circ}C$. Sperm motility and viability were examined daily up to 5 days under light microscopy at ${\times}200$ magnification. Sperm viability was acceptable (${\geq}40%$) up to the $4^{th}$ day with all concentrations of ${\alpha}$-tocopherol and up to the $5^{th}$ day with 2 mg/ml ${\alpha}$-tocopherol. Sperm motility was acceptable (${\geq}40%$) up to the $3^{rd}$ day irrespective of ${\alpha}$-tocopherol concentration, and up to the $4^{th}$ day with 2 mg/ml ${\alpha}$-tocopherol. It is suggested that the lifespan of chilled semen may be extended up to 4 days by adding 2mg/ml ${\alpha}$-tocopherol.

• Clinical and Experimental Reproductive Medicine
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• 제50권4호
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• pp.213-222
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• 2023

Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.

• 한국가축번식학회지
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• 제23권2호
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• pp.127-132
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• 1999

본 연구는 소형견 정액의 원정액과 정장제거 정액의 일반성상, 채취분획별 및 단기보존시 정자의 생존성과 아울러 동결보존시의 정자의 생존성에 대해 조사하고자 수행하였다. 1. 원정액과 정장제거 정액에 saline 및 Tris-buffer를 희석 후 각각 일반성상 검사를 실시했을 때 원정액의 경우 정자농도는 5.07$\pm$2.32$\times$$10^{6}$cells/$m\ell$, 정자의 운동성은 95.42$\pm$2.65%, 기행정자수는 4.42$\pm$0.15%로 나타났으며, RSP(saline 및 Tris-buffer)군의 경우 정자농도는 4.69$\pm$3.27~4.25$\pm$3.65$\times$$10^{6}$cells/$m\ell$, 정자의 운동성은 91.17$\pm$3.85~88.52$\pm$3.85%, 기형정자수는 6.57$\pm$0.43~5.54$\pm$0.52%로 나타났다. 2. 분획별 채취 전정액중 제 1 분획에서는 정액량이 0.92$\pm$0.7$m\ell$, 정자농도는 4.57$\pm$0.78$\times$$10^{6}$cells/$m\ell$, 정자활력은 10.72$\pm$3,21%, 기형정자수는 5.50$\pm$0.70%로 나타났으며, 제 2분획에서는 정액량이 2.14$\pm$0.19$m\ell$, 정자농도는 2.01$\pm$0.12$\times$$10^{6}$cells/$m\ell$, 정자활력은 95.44$\pm$4.21%, 기형정자수는 4.31$\pm$0.53%로 나타났다. 또한, 제3 분획에서는 정액량이 2.66$\pm$0.23$m\ell$, 정자농도는 2.35$\pm$0.21$\times$$10^{6}$cells/$m\ell$, 정자활력은 90.71$\pm$2.63%, 기형정자수는 6.33$\pm$0.91%로 나타났다. 3. 원정액과 정장제거 정액을 4$^{\circ}C$ 와 2$0^{\circ}C$ 및 37$^{\circ}C$ 에서 각각 보존했을 때 보존시간별 정자의 활력은 2$0^{\circ}C$ 의 경우 1, 6, 13, 24, 30 및 40 시간에서 각각 98.51%와 98.32%, 86.32%와 92.15%, 83.71% 와 89.20%, 74.29% 와 82.08%, 52.98% 와 72.07%, 15.45% 와 60.02%, 2.41% 와 37.19% 의 정자활력을 나타내어 4$^{\circ}C$와 37$^{\circ}C$에 비해 높은 정자운동성을 나타냈다. 4. 제 2 분획 정액과 정장제거 정액을 각각 제 1차 및 제 2차 희석액으로 희석 평형 시킨 후 동결 융해했을 때 정자의 생존율은 각각 33.3$\pm$8.7, 54.7$\pm$9.5%로서 대조군의 15.4$\pm$5.2% 에 비해 높게 나타났다.

• 한국수정란이식학회지
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• 제28권2호
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• pp.113-119
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• 2013

The study focuses on the quality assessment of Black Bengal buck semen preserved at chilled condition. In this in vitro trial, collected semen from Black Bengal bucks was preserved at chilling temperature ($4{\sim}5^{\circ}C$) in tris-glucosecitrate yolk medium of 1:5 ratios for four days. Artificial Vagina (AV) method was utilized to collect semen from buck. General evaluation of semen includes the color, mass activity and density were measured by direct visual examination. However, computer-assisted sperm analysis (CASA) and phase contrast microscopy were used to figure out the motility (%), hyper-activated (HYP) motility (%) and number of abnormal spermatozoa (%) initially, and at every 24 h intervals. The result revealed that spermatozoa preserved at chilling temperature showed significantly (P<0.05) lower motility and HYP motility with the progression of preservation. The number of phenotypically abnormal spermatozoa significantly (P<0.05) increased following preservation. Although significant positive correlation (r=0.945; P<0.05) was existed between % motile and % HYP motile spermatozoa however, the % of morphologically abnormal spermatozoa was negatively correlated with % motile (r=-0.997; P<0.05) and % HYP motile spermatozoa (r=-0.946; P<0.01). Therefore, we concluded that the quality of chilled semen progressively losses its viability and doesn't remain useable after certain period of preservation with respect to its motility and morphology.