• The Korean Journal of Malacology
• /
• v.20 no.1
• /
• pp.17-26
• /
• 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 $-80^{\circ}C$.

• Journal of Embryo Transfer
• /
• v.26 no.1
• /
• pp.71-78
• /
• 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.

• Journal of Life Science
• /
• v.33 no.7
• /
• pp.586-592
• /
• 2023

Oxidative stress is a critical factor affecting the quality and viability of sperm during boar semen storage. Oxidative stress is also a significant concern during the process of freezing semen. The process of semen storage involves exposing the sperm to various stressors, including temperature changes, cryoprotectants, and extended periods of incubation. In addition, oxidative stress can lead to the production of reactive oxygen species (ROS) within the sperm, resulting in oxidative damage to cellular components, such as lipids, proteins, and DNA. Striking a balance between ROS production and the antioxidant defense system is crucial for maintaining sperm viability and functionality during semen storage. Moreover, the prolonged storage of boar semen leads to an increase in ROS levels, which can impair sperm motility, membrane integrity, and DNA integrity. ROS-induced lipid peroxidation affects the fluidity and stability of sperm membranes, leading to decreased sperm motility. Moreover, oxidative damage to the DNA can result in DNA fragmentation, compromising the genetic integrity of the sperm. In conclusion, oxidative stress is a significant challenge in maintaining sperm quality during boar semen storage. Understanding the mechanisms underlying oxidative stress and their impacts on sperm function is crucial for developing effective strategies to minimize oxidative damage and improve sperm storage outcomes.

• Development and Reproduction
• /
• v.7 no.1
• /
• pp.9-13
• /
• 2003

A series of experiments were conducted to compare the effects of various diluents in cold storage on the sea urchin, Hemicentrotus pulcherimus sperm. Various diluents of glucose solutions, artificial sea water(ASW) and 50% ASW were used to store the sperm at 4$^{\circ}C$. The storage effect was evaluated using sperm activity index(SAI), survival rate of sperm and fertilization rate to egg. ASW and 1.2 M glucose were found to be better diluents which maintained high motility and survival rate of sperm f3r a storage period of 30 days. Optimal pH of diluent to store the sperm at 4$^{\circ}C$ is 7.0∼8.0. In order to keep high SAI and survival rate of sperm, addition of 400 ppm neomycin into the diluent revealed the best storage results.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.38 no.4
• /
• pp.239-244
• /
• 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
• /
• v.19 no.1
• /
• pp.70-88
• /
• 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.

• Journal of Aquaculture
• /
• v.10 no.4
• /
• pp.381-386
• /
• 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
• /
• v.37 no.5
• /
• pp.852-861
• /
• 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.

• The Korean Journal of Malacology
• /
• v.24 no.1
• /
• pp.51-57
• /
• 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
• /
• v.30 no.1
• /
• pp.21-44
• /
• 2000

This study was carried out to investigate sperm storage, and the fate of spermatozoa in the female reproductive tract during hibernation in Korean greater horseshoe bat, Rhinolophus ferrumequinum korai. (1) Numerous sperm occurring in uterine lumen and glands were engulfed, and disappeared by the polymorphouclear leucocytes during the hibernation. (2) The stored sperm present in caudal isthmus of oviduct only, the heads of sperm toward the oviductal epithelial cells. Therefore, the projected sperm during the mating season are only alive in the caudal isthmus of oviduct in the long hibernation. The present result suggests that the caudal isthmus of oviduct may play an important role as the principal storage site in capacitation of sperm. (3) In March, the sperm do not occur in the caudal isthmus of oviduct. It suggests that the stored sperm in the caudal isthmus of oviduct should migrate to the ampulla of the site of fertilization to meet ovum in the period of ovulation. The results of this experiment consider that prolonged sperm storage, fate of sperm and sperm migration in the long hibernation have a kind of mechanism for the fertilization.