• 한국패류학회지
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• 제20권1호
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• pp.17-26
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• 2004

In present study, attempts were made to preserve abalone (Haliotis discus hannai) sperm in liquid form at low temperature, to evaluate the effect of various diluents in short-term storage on sperm, and cryopreservation procedures were optimized for the cryoprotectants and freezing rates, as well as the motility, survival rate, and the ultrastructural changes of sperm after short-term storage and cryopreservation were observed. The abalone sperm reached maximum motility until about 4 min after activation. The motility was constant for about 16 min, after which it dropped gradually, and about 50 min later all motility ceased. In Hanks' balanced salt solution (HBSS, 300 and 400 mOsmol/kg) and 150, 250 and 350 mOsmol/k artificial seawater (ASW), the sperm was immotile. After 100% ASW was added, motility of those sperm, which are in 300, 400 mOsmol/kg HBSS, 250, 350 mOsmol/kg ASW, could be again restored incompletely. Sperm motility can be maintained for 20 days of cold storage only in ASW of 850 and 1200 mOsmol/kg. A high motility index of 3.5-4.5 was observed for the first 8 days in 850 and 950 mOsmol/kg ASW. In other diluents sperm motility was constant less than 10 days, and the motility index was obviously lower than that of sperm in 850 and 1200 mOsmol/kg ASW. After 20 days of cold storage, survival rates of 10.2%-20.7% were obtained in ASW and 300 mOsmol/kg HBSS, and that in 400 HBSS (65.3%) was significantly higher than others. The constant period of sperm motility stored in 850 mOsmol/kg ASW was obviously longer than that in 1200 mOsmol/kg ASW after 6 days of storage. The sperm plunged into liquid nitrogen all died except that sperm using 15% glycerol as cryoprotectant restored 10.4% of motility. The highest motility index (3.4) was obtained with 5% glycerol and freezing procedure: $-50^{\circ}C$/min from $20^{\circ}C$ to $-80^{\circ}C$.

• 한국수정란이식학회지
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• 제26권1호
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• pp.71-78
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• 2011

This study was undertaken to find out the effect of cholesterol and serum albumin on sperm ability and lipid peroxidation levels period to the liquid storage of miniature pig sperm. Ejaculated semen from miniature pigs was collected by gloved-hand method into a pre-warmed ($37^{\circ}C$) thermos bottle, and extended with Modena solution {with and without BSA, methyl-beta-cyclodextrin (-cholesterol) and cholesterol loaded cyclodextrin (+cholesterol)}. Each semen was assessed for viability (SYBR-14/PI staining) and acrosome intactness, intensity and capacitation status by chlorotetracycline (CTC) staining at 1, 3, 5, 7 and 10 days of storage. At for the effects of cholesterol and serum albumin on lipid peroxidation, semen were incubated with $H_2O_2$ ($10\;{\mu}M$), and lipid peroxidation level were measured by flow cytometry using the lipid peroxidation reporter probe $C_{11}-BODIPY^{581/591}$. The result, lipid peroxidation level in sperm added with cholesterol were lower in $10\;{\mu}M$ $H_2O_2$ compared to the added sperm with serum albumin. Also, added cholesterol to sperm had significant (p<0.05) higher viability when storage for 7 and 10 days and lower when 10 days of storage percentage of acrosome-reacted sperm (AR pattern) in acrosome state as say result compared to other treated groups. In conclusion, role of cholesterol during lipid storage in miniature pig spermatozoa was protected boar spermatozoa from lipid peroxidation prior to lipid storage. Addition serum albumin during lipid storage in sperm may be induce sperm membrane damage by lipid peroxidation. Therefore, addition of cholesterol to miniature pig sperm will be lead to extension of liquid storage periods.

• 생명과학회지
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• 제33권7호
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• pp.586-592
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• 2023

돼지 정액을 저장하는 동안 산화스트레스의 발생은 정자의 질과 생존에 영향을 미치는 중요한 인자이다. 정액의 저장은 온도 변화, 동결보호제 등의 다양한 스트레스 인자에 노출되어 있다. 이러한 정자 내에서의 산화스트레스는 활성산소종의 생성에 의해 발생되며, 이는 지질, 단백질, DNA와 같은 세포를 구성하는 물질에 산화적으로 손상을 일으킨다. 활성산소종과 항산화물질의 균형있는 체계는 정자의 생존과 그 기능을 유지하는 데 중요한 역할을 한다. 정액을 장기간 보존하게 되면 활성산소종의 수준이 증가하여 정자의 운동성, 막 온전성, DNA 온전성에 영향을 미치게 된다. 또한, 활성산소종에 의해 유도된 지질과산화 반응은 정자막의 유동성과 안정성에 영향을 미쳐 정자의 운동성을 감소시킨다. 그리고, DNA의 산화적 손상은 DNA 단편화를 일으켜 정자의 DNA 온전성을 손상시킬 수 있다. 결론적으로, 정액을 보관하는 동안 발생되는 산화스트레스는 정자의 질과 기능을 유지하는 데 중요하다. 따라서, 산화스트레스의 기본적인 메커니즘과 정자의 기능에 미치는 영향을 이해하는 것은 산화스트레스로부터의 손상을 최소화하고, 효율적이고 기능적인 정자의 저장 방법을 개선하기 위한 효과적인 전략과 연구 개발을 위해 중요할 것으로 판단된다.

• 한국발생생물학회지:발생과생식
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• 제7권1호
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• pp.9-13
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• 2003

말똥성게 정자의 냉장보존을 위한 여러 가지 희석액의 보존 효과를 비교하였다. 다양한 농도의 포도당 용액, 인공해수(ASW)를 희석액으로 하여 정자를 4$^{\circ}C$에서 보존하였다 보존효과는 정자 활성지수 (SAI), 생존율 및 말똥성 게 난에 대 한 수정률로 평가하였다. ASW와 1.2 M glucose액을 희석 액으로 하여 30일간 냉장보존하였을 때 가장 높은 운동성 및 생존율을 유지하였다. 정자의 냉장보존을 위한 희석액의 적정 pH는 7.0∼8.0 이었다. 정자의 SAI와 생존율을 높이기 위한 항생제로는 neomycin(400 ppm)이 효과적인 것으로 나타났다

• 한국수산과학회지
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• 제38권4호
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• pp.239-244
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• 2005

The effects of diluents composition on cold storage for olive flounder (Paralichthys olivaceus) sperm were examined in terms of the swimming speed of sperm, the percentage of mobile sperm, and fertilization rates. The following results indicated that cold storage methods with fresh conditions could be employed in olive flounder milt preservation. The preserved sperm of olive flounder that was diluted I: 10 with artificial seminal plasma II (ASP II) and Stein's solution (SS) at $0^{\circ}C$ remained motile for 30 days. The most effective condition for cold storage was ASP II and SS at 0 $0^{\circ}C$ for the sperm, although there is no significant difference statistically. No difference in the fertilization rate was found between fresh sperm and the preserved ones with ASP I, II and SS at 10 days post-storage.

• Applied Microscopy
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• 제19권1호
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• pp.70-88
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• 1989

The storage sac of U. unicinctus can be divided into two parts morphologically as well as functionally ; one is proximal and the other distal storage sac. It is because of the seasonal morphological change of the storage sac caused by sperm accumulation in the storage sac. The proximal storage sac contains the mature sperm with the dumbbell-shaped acrosome and well developed one or two mitochondria in the middle piece, whilst the sperm of hemispherical acrosome associate closely with an accessory cell in the distal storage sac. This means that the sperm do not perform the synchronous development in a storage sac, which is not the case of sperm development in the coelomic cytophorus. In addition, the basal membrane of the distal storage sac is different from that of the proximal storage sac in term of tissue formation. Connective tissues, acces-sory cells and small lumen develop on the basal membrane of the distal sto-rage sac, which is well contrasted with the thin basal membrane of the proximal storage sac. The function of the storage sac is discussed in rela-tion of the sperm development and the seasonal change.

• 한국양식학회지
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• 제10권4호
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• pp.381-386
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• 1997

A series of experiments were conducted to compare the effects of various diluents in cold storage on the marbled sole, Limanda yokohamae sperm. Various diluents of glucose, L. yokohamae serum, marine fish Ringer's solution, sodium citrate and Ca2+ free artificial seawater (ASM) (tris-HCI, pH 7.4) containing 3 mM ethylene glycol-bis (2-aminoethyl ether) tetraacetic acid (EGTA) were used to store the sperm at 4℃. The storage effect was evaluated using motility index and survival rate of sperm. Glucose and sodium citrate were found to be better diluents which maintained high motility and survival rate of sperm for a storage period of 10 days. Some morphological changes of spermatozoa were observed during the cold storage with diluents. In particular, a detachment of the nuclear enveloped and of the plasma membrane from the nucleus in spermatozoa was observed. Morphological normality of the stored spermatozoa diluted with 0.3 M glucose was better than that of the stored spermatozoa undiluted or diluted with Ca2+ free ASW containing 3 mM EGTA.

• Animal Bioscience
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• 제37권5호
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• pp.852-861
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• 2024

Objective: The present study aimed to investigate the effect of β-nicotinamide mononucleotide (NMN) supplementation on ram sperm quality during storage at 4℃ in vitro. Methods: Tris-citric acid-glucose solution containing different doses of NMN (0, 30, 60, 90, and 120 µM) was used to dilute semen collected from rams and it was stored at 4℃. Sperm motility, plasma membrane integrity as well as acrosome integrity were evaluated at 0, 24, and 48 h time points after storage at 4℃. In addition, sperm mitochondrial activity, lipid peroxidation (LPO), malondialdehyde (MDA) content, reactive oxygen species (ROS) content, glutathione (GSH) content, superoxide dismutase (SOD) activity, and apoptosis were measured at 48 h time point after storage at 4℃. Results: Results demonstrate that the values obtained for sperm motility, acrosome integrity, and plasma membrane integrity in the NMN treatments were significantly higher than control (p<0.05). The addition of 60 µM NMN significantly improved ram sperm mitochondrial activity and reduced LPO, MDA content, and ROS content compared to control (p<0.05). Interestingly, sperm GSH content and SOD activity for the 60 µM NMN treatment were much higher than those observed for control. NMN treatment also decreased the level of Cleaved-Caspase 3, Cleaved-Caspase 9, and Bax while increasing Bcl-2 level in sperm at 48 h time point after storage at 4℃. Conclusion: Ram sperm quality can be maintained during storage at 4℃ with the addition of NMN at 60 µM to the semen extender. NMN also reduces oxidative stress and apoptosis. Overall, these findings suggest that NMN is efficient in improving the viability of ram sperm during storage at 4℃ in vitro.

• 한국패류학회지
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• 제24권1호
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• pp.51-57
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• 2008

With the purpose to estimate the possibility of short-term storage and cryopreservation for sperm of Charonia sauliae, which is a potential preparation for its artificial reproduction and further research, in this study, protocols for short-term storage and cryopreservation of trumpet shell sperm was optimized. The effects of different immobilizing solutions, dilution ratios were estimated for short-term storage. And the effects of different cryoprotectant extenders and freezing rates were estimated for cryopreservation in terms of motility and survival of sperm. The results indicated that the artificial sea water of 350 mOsmol/kg is a better immobilizing solution and sperm which was diluted at a ratio of 1:1 (v/v) had higher motility and survival rate during short-term storage. The effect of 5% dimethyl sulfoxide was significantly better than those of other cryoprotectant extenders. And a freezing rate of $-20^{\circ}C\;min^{-1}$ showed better effect than other freezing rates. In conclusion, this study optimized some key factors of the short-term and cryopreservation of C. sauliae sperm, which can provide valuable data for germ-plasm conservation and artificial propagation of C. sauliae.

• Applied Microscopy
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• 제30권1호
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• pp.21-44
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• 2000

동면기 (11월부터 3월까지) 동안의 한국산 관박쥐(Rhinolophus ferrumequinum korai) 자성 생식도관 내의 정자저장, 정자 생존 여부 및 정자이동을 알아보기 위하여 전자현미경으로 관찰한 결과는 다음과 같았다. (1) 자궁내강, 자궁선내의 정자들은 다수의 백혈구들에 의해 포식되고 소멸되었다. (2)정자들은 수란관 미측 협부에서만 저장되었고, 정자들의 두부는 상피세포쪽을 향하고 있었다. 이는 교미기(10월 초-중순경)에 사출된 정자들이 긴 동면기 동안에 수란관 미측 협부에서만 생존 가능함을 의미하며, 수란관의 미측 협부가 정자의 수정능 획득(capacitation)에 필요한 최적의 장소임을 의미한다. (3) 동면후기인 3월의 수란관 미측협부에는 정자들이 관찰되지 않았다. 이는 배란기인 4월에 난자를 만나기 위해 정자들이 수정부위인 팽대부 쪽으로 이행하였음을 의미한다. 이상의 결과로 미루어 보아, 긴 동면기 동안의 자성 생식도관 내에서의 정자의 장기저장과 생존여부 및 정자이동은 수정을 위한 일종의 mechanism이라 여겨진다.