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Motility grade and duration of motility (s) of M. cephalus sperm under different concentrations of glucose. Different letters denote significant differences (P < 0.05).
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The effects of osmolality and pH of the seawater and non-ionic media (glucose) on sperm activation in Mugil cephalus was evaluated. The effect of cryopreservation was documented by cryopreserving the sperm diluted with a cryomedium (V2 extender + 10% dimethylsulfoxide) in a programmable freezer. The highest motility grade (4) or (3) was recorded wh...
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... s) was recorded at 800 mM of glucose followed by 1000 mM of glucose (140 ± 24 s). However, the lowest motility grade of 0.5 ± 0.0 and sperm motility duration of 106 ± 6 s (P < 0.05) was observed when sperm were activated with 600 mM of glucose. On the other hand, there was no motility when the sperm were activated with 200 and 400 mM of glucose (Fig. ...
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The present study aimed to evaluate cryo-injury during the cryopreservation process in sterlet (Acipenser ruthenus) sperm, focusing on ultrastructural characteristics. Post-thaw sperm quality parameters, including total motility rate, curvilinear velocity (VCL), linearity (LIN), plasma membrane integrity, antioxidant status, DNA damage, and fine ul...
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... The cryopreservation of fish sperm includes various species, such as sturgeons, salmon, mullet, bream, grouper, cod, snapper, carp, and eel. In these species, cryoinjury manifests as swollen, ruptured, or dehydrated heads, flagella, midpieces, and tails, along with swollen or absent mitochondria and damaged or missing plasma membranes (Dadras et al. 2022;Díaz et al. 2019;Balamurugan et al. 2019;Taddei et al. 2001;Tian et al. 2015;Ottesen et al. 2012;Liu et al. 2007Liu et al. , 2010Tsai et al. 2010;Yao et al. 2000). Ninhaus-Silveira et al. (2009) attempted to cryopreserve the embryos of Prochilodus lineatus using propylene glycol. ...
Climate change has profoundly impacted coral reefs worldwide, resulting in a concerning and irreversible deterioration. Preserving the biomaterials of coral species is essential, and cryopreservation presents a viable method for achieving this goal. This study aimed to evaluate cryoinjury via ultrastructural data. The planulae of Stylophora pistillata were subjected to vitrification and nanolaser warming utilizing species-specific vitrification solutions with gold nanoparticles. Ultrastructural analysis of the planulae using a transmission electron microscope to assess the cryoinjury incurred during cryopreservation. The results indicated that swimming planulae sustained greater cryoinjuries compared to their immobile counterparts. These cryoinjuries included swelling of the microvilli, breakage of the flagella in the epidermis, tissue fragmentation, disintegration, and cell loosening. Symbiodiniaceae exhibited abnormalities ranging from mild (chloroplast swelling and degraded chromosomes) to severe (nucleus margination and stroma darkening), with membrane blebbing being a common moderate abnormality. Cryoinjuries also resulted in shrunken Symbiodiniaceae and disintegrated lipid bodies. Despite the observed cryoinjuries, both swimming and immobile planulae can be used for cryopreservation. The ultrastructural evidence gathered in this study can help to improve cryopreservation protocols for S. pistillata and other scleractinian corals by enhancing settlement and post-settlement survival.
... This result was in agreement with the sperm cryopreservation studies in Dicentrarchus labrax (Zilli et al., 2003), Oncorhynchus mykiss (Cabrita et al., 2005), Epinephelus lanceolatus (Park et al., 2022) and Pacific oysters (Gwo et al., 2003). On the contrary, Balamurugan et al. (2019) found that the sperm DNA integrity in the grey mullets Mugil cephalus was not affected by the cryopreservation process. As these integrities are critical for sperm to perform their biological functions, methods that could maintain their states should be evaluated to improve the sperm cryopreservation technique if they are practical (Peris-Frau et al., 2020;Xin et al., 2020). ...
The germplasm of farmed Yesso scallop Patinopecten yessoensis in China has deteriorated since its introduction more than 40 years ago. The main aim of this study is to develop a sperm cryopreservation technique to assist the newly established program to manage this issue in China. This study investigated the factors important to the development of a non-programmable sperm cryopreservation technique in this species, including cryoprotectant agent [CPA; dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycine, sucrose, glucose and trehalose], equilibration time, rack height and thawing temperature. A low post-thaw sperm fertilization rate of 27.67 ± 2.52% was produced with the parameters optimized with the permeable CPAs (DMSO, PG and EG) only: 6% DMSO, 10 min equilibration time, 5 cm rack height and 30 • C thawing temperature. This rate was further improved to 44.00 ± 2.00% by adding 3% glucose into 6% DMSO. This addition had also improved the integrities of post-thaw sperm DNA, plasma membrane and acrosome, mitochondrial membrane potential and activities of some enzymes. Results from this study also showed that the post-thaw sperm morphology and ul-trastructure were intensively compromised. In addition, the sperm agglutination found in the newly spawned sperm might be one of the phenomena resulted from the so-called germplasm deterioration, especially in the stock used in this study. The further exacerbation of agglutination after cryopreservation would also compromise the post-thaw sperm performance. The sperm cryopreservation technique established in this study would provide a better option to assist in managing the genetic diversity of farmed Yesso scallops in China.
... In present study, the Kurokura-2 + DMSO (KD), Kurokura-2 + Methanol (KM), and Alsever's solution + Methanol (AM) extenders were found to maintain DNA integrity of C. mrigala sperm compared to Alsever's solution + DMSO (AD) extender. Different species show different effects of cryoinjury owing to specific chromatin structure and the composition of extender along with cryoprotectants (Balamurugan et al., 2019). ...
This study aimed to evaluate the performance of different extenders for the cryopreservation of Cirrhinus mrigala milt. The extenders under investigation included Kurokura-2 + DMSO (KD), Alsever`s solution + DMSO (AD), Kurokura-2 + Methanol (KM), and Alsever`s solution + Methanol (AM). Sperm quality assessments encompassed critical parameters such as sperm motility, motility duration, viability, and DNA integrity. The initial analysis of fresh milt revealed motility rate of 88.33±1.66%, pH of 7.52±0.76, a volume of 7.83±0.44 mL, a sperm count of 3.55±0.40 x 1010/mL. Upon thawing, it became evident that the KD extender yielded significantly higher post-thaw sperm motility (%) at 68±1.67, compared to the KM (40.00±2.89), AD (38±1.67), and AM (41.67±4.40). Additionally, sperm motility duration (seconds) exhibited a notable increase in the KD extender (78.67±10.413) in comparison to the KM (52.00±5.291), AD (46.66±1.66), and AM (41.67±1.667) extenders. The KD extender also contributed to higher sperm viability (%) at 66.00±1.00, whereas KM and AM exhibited similar values at 59.00±3.00 and 55.33±6.38, respectively. Furthermore, DNA integrity (%) remained consistently high for KD, KM, and AM (98.67±0.66, 98.00±1.52%, and 97.33±1.20%), while a lower percentage was observed for AD (88.00±1.15). In conclusion, the modified Kurokura extender, supplemented with 10% DMSO, showcased a pronounced positive impact on the cryopreservation of Cirrhinus mrigala milt.
... It is well known that both cold storage and freezing/thawing of spermatozoa may affect factors such as sperm plasma membrane integrity, motility, ATP content, and DNA integrity (Cabrita et al., 2005;Balamurugan et al., 2019;Kommisrud et al., 2020). Furthermore, these parameters are recognized as important factors for sperm cells to reach and fertilize the eggs and for proper embryo development (Liu et al., 2007;Cabrita et al., 2008;Pérez-Cerezales et al., 2010;Dzyuba et al., 2017). ...
Cold storage and freezing/thawing of milt may affect sperm functionality and the subsequent fertilization ability of milt. This study aimed to investigate sperm quality parameters and fertilization potential of Atlantic salmon milt, stored cold and subsequently cryopreserved, using different storage conditions. The objective was also to assess if analysis of milt metabolites and sperm DNA methylation signatures could be applicable to further elucidate sperm quality and fertilization following preservation. Milt samples were collected from eight mature Atlantic salmon males and stored for 4 days at 2°C and 8°C. Samples were taken on day one of storage at 2°C and on day four of storage at 2°C and 8°C. Storage for 4 days at 8°C is expected to be detrimental to sperm quality, and was included to create contrasts. Correspondingly, aliquots of cold-stored milt were prepared for cryopreservation, resulting in a total of six experimental conditions. Samples from all six experimental conditions were used in fertilization trials and analyzed for sperm viability, motility, ATP content, DNA fragmentation index, and High DNA stainability. In addition, milt samples from four of the males were analyzed for targeted metabolites and DNA methylation signatures by reduced representation bisulfite sequencing. The fertilization trials were performed using sperm:egg ratios of 75 × 10³ and 500 × 10³, respectively. Storage duration, temperature, and cryopreservation of cold-stored milt influenced several sperm quality parameters, metabolites, and DNA methylation signatures. The total motility, progressive motility, ATP, and velocity parameters were the sperm parameters with the strongest correlation to fertilization rates (p < 0.01). Several metabolites were correlated with fertility rates in both cold-stored and cryopreserved samples (p < 0.05). The fertilizing capacity of cold-stored milt was significantly reduced after 4 days of storage at 8°C, while corresponding cryopreserved milt showed reduced fertilization at both storage temperatures (2°C and 8°C) (p < 0.05). The results indicate that cryopreservation of milt stored for 1 day does not compromise either fertilization ability or DNA methylation signatures.
... Its effectiveness on sperm cryopreservation has also been reported for several freshwater fish species, such as naleh Barbonymus sp. (40), Rasbora tawarensis (9), Poropuntius tawarensis (23), Osteochillus vittatus (13), Mugil cephalus (43), Barbus grypsus (44), Cyprinus carpio (45), Tor tambroides and Tor dauronensis (46). It also gave good results on sperm cryopreservation of marine fish, including the Disentrarchus labrax (47), Pagrus major (48), Sillago japonica (49), and Paralichthys olivaceus (50). ...
... Pseudopleuronectes yokohame (63), Mugil cephalus (43), Oncorhynchus mykiss (64), and Epinephelus sp. (65). ...
BACKGRUND: Goldfish Carassius auratus is a popular ornamental fish extensively cultured worldwide. Sperm cryopreservation is a common fish breeding method that ensures sperm availability around the year. Studies on cryopreservation of goldfish sperm, especially on the suitability of cryoprotectant types and pre-freezing time, are scarcely available. OBJECTIVE: To determine the most suitable type of cryoprotectant and pre-freezing for the successful cryopreservation of goldfish sperm. MATERIALS AND METHODS: A completely randomized design with two factors was utilized in this study. The first factor is the type of cryoprotectants, which included methanol, ethanol, ethylene glycol, glycerol, and DMSO. The second is pre-freezing times of 10, 20, 30, and 40 min at each of the pre- freezing temperatures of 4°C, −10°C, and −79°C, meaning that the total times for the ramping down of temperature were 30, 60, 90 and 120 min, respectively. The Ringer solution and 10% egg yolk were used as extender and extracellular cryoprotectant. The sperm was stored at −179°C for 7 days. RESULTS: The ANOVA test showed that cryoprotectants and pre-freezing significantly affected the motility, viability, and fertility of goldfish sperm after freezing in liquid nitrogen for 7 days (ρ<0.05). Furthermore, 10% DMSO combined with 15% egg yolk with an pre-freezing time of 20 min can maintain sperm motility, viability, and fertility higher than other treatments, by 79%, 80%, and 33%, respectively. The agarose gel electrophoresis showed no DNA fragmentation in all samples, including fresh sperm. CONCLUSION: We conclude that 10% DMSO combined with 15% egg yolk and 20 min pre-freezing is the best treatment for goldfish sperm cryopreservation.
... However, cryogenic storage for 6 and 12 months affected DNA quality significantly, resulting in a loss of integrity of approximately 31% and 58%, respectively. Some previous studies have shown that short periods of cryostorage (one week and two months) do not affect significantly the sperm DNA integrity of grey mullet (Mugil cephalus) (Balamurugan et al., 2019) and Atlantic salmon (Salmo salar) (Figueroa et al., 2016). On the other hand, human sperm stored for more than 15 weeks in liquid nitrogen showed a statistically significant increase in α-tubulin detection (Desrosiers et al., 2006). ...
... Previous studies have also reported that cryopreservation procedures cause substantial morphological alterations in fish spermatozoa (He and Woods, 2004;Liu et al., 2007;Balamurugan et al., 2019;Figueroa et al., 2019). In fact, this damage may occur after the dilution of the milt in the cryopreservation medium, and increase with deep-freezing (Billard, 1983). ...
To date, little attention has been paid to identifying the effects of long-term cryopreservation on sperm quality for Piaractus orinoquensis. The object of this study was therefore to evaluate the effect of long-term cryopreser-vation (24 h, 1, 6 and 12 months) on sperm motility, viability, DNA integrity, ATP content, total antioxidant capacity (TAC), morphology and sperm ultrastructure in this species. Milt samples from six males were cry-opreserved in a medium containing final concentrations of 7.5% Me 2 SO, 4.1% glucose and 9.0% egg yolk. The samples were frozen in liquid nitrogen (LN) vapor and stored in LN for periods of 24 h and 1, 6 and 12 months. After thawing, both the motility rate and the viability decreased significantly compared with fresh sperm; however, these parameters did not differ among the four cryopreservation times. The DNA integrity and ATP content decreased significantly after 6 months of cryopreservation. There were no significant differences in TAC values between fresh and cryopreserved sperm. The total sperm abnormalities in cryopreserved samples were about 5-fold higher than in fresh sperm; short tail was the most common defect occurring after cryostorage. The ultrastructural analysis reveals that P. orinoquensis spermatozoa consist of an ovoid head without acrosome, a cylindrical mid-piece, and a single flagellum. The nuclear fossa is located at the base of the nucleus and contains the centriolar complex. There are 1-2 ring-shaped mitochondria located in the mid-piece. The flagellum shows a 9 + 2 organization of microtubules in the axoneme. Post-thaw spermatozoa presented damage such as swelling and rupture of the plasma membrane, mitochondrial damage, loss of the electron-dense chromatin of the nucleus , and degeneration in the middle region.
... Damage to the mitochondria of a derivative during freezing and thawing can lead to a decrease in the mitochondrial membrane potential [39]. The changes caused by cryopreservation and found in the present study were similar to changes in some ultrastructures of fish spermatozoa [40] and different mammalian species such as llamas [39], horses [18], and rams [41]. In the study by Atroshchenko et al. (2017) [18] the authors found many spermatozoa with acrosome fragmentation due to false acrosome reaction, with acrosome hyperplasia (noncompact acrosome content), and nuclear changes and vacuolization of chromatin. ...
... In the study by Atroshchenko et al. (2017) [18] the authors found many spermatozoa with acrosome fragmentation due to false acrosome reaction, with acrosome hyperplasia (noncompact acrosome content), and nuclear changes and vacuolization of chromatin. In the study by Balamuru-gan et al. (2019) [40], it is showed that freezing and thawing caused various ultrastructural changes, such as damage to the plasma membrane around the sperm head, flagellum fragmentation, and complete flagellum loss in fish sperm. In the study by Zampini et al. (2020) [39], acrosome damage, loss of mitochondria, disorganization of the axoneme and periaxonemal structures, and invagination in the nucleus of llama spermatozoa were observed with the help of TEM. ...
... In the study by Keskin et al. (2020) [41] mainly cryodamage to the membrane, as well as mitochondria (electron-lumen matrix) and axoneme (loss of axoneme doublets) cryo-damage were observed in ram sperm STEM. Vesiculation in acrosomal membranes and loss of acrosomal content observed in the study [18,37,[39][40][41] with frozen-thawed semen could be the result of an acrosomal reaction. In the current research, we have not detected the drone sperm acrosome using a transmission electron microscope and have not evaluated the effects of cryopreservation. ...
... Other approaches, such as ultrastructural study using electron microscopy, can provide more specific information, such as damage to the plasma membrane and fragmentation of the flagella. The mentioned approach can provide important information about the morphology and ultrastructural changes in frozen-thawed spermatozoa Balamurugan et al., 2019). Cryo-injury can cause a major challenge for practical application of sperm cryopreservation in aquaculture and fish conservation programs. ...
... In contrast, 10 µg/mL AFPIII combined with 8% DMSO improved DNA integrity in cryopreserved spermatozoa of Pacific abalone (Hossen et al., 2021). In gray mullet, Mugil cephalus, freezing-thawing process had no effect on DNA integrity (Balamurugan et al., 2019). The low DNA damage observed in the present study might be due to the reduced susceptibility of sperm chromatin due to high degree of chromatin packaging (Singh et al., 2003). ...
The present study aimed to evaluate cryo-injury during the cryopreservation process in sterlet (Acipenser ruthenus) sperm, focusing on ultrastructural characteristics. Post-thaw sperm quality parameters, including total motility rate, curvilinear velocity (VCL), linearity (LIN), plasma membrane integrity, antioxidant status, DNA damage, and fine ultrastructure were examined on fresh and cryopreserved sperm with/without addition of a single optimal dose of AFPI (10 μg/mL). A lower motility rate, VCL and plasma membrane integrity, and increased DNA damage (p < 0.05) were observed in frozen-thawed spermatozoa with/without AFPI compared to fresh spermatozoa. The morphology and ultrastructure of spermatozoa were affected during the cryopreservation process with/without supplementation of AFPI. Morphological abnormalities were observed in mitochondria (49–54%) and flagellum (55–57%) of cryopreserved spermatozoa with/without AFPI compared to fresh spermatozoa. In conclusion, the morphology and ultrastructure of spermatozoa were slightly changed after cryopreservation of sterlet spermatozoa with/without 10 μg/mL AFPI.
... A significantly higher percentage of tail DNA in cryopreserved sperm has also been reported in spotted halibut, Verasper variegatus; stone flounder, Kareius bicoloratus; European sea bass, and Dicentrarchus labrax (Zidni et al., 2020;Labbe et al., 2001). By contrast, cryopreservation in gray mullet (Mugil cephalus) and Atlantic croaker (Micropogonias undulatus) has no significant effects on DNA (Balamurugan et al., 2019;Gwo and Arnold, 1992). Variation among fish species is influenced by resistance to cold, cryopreservation medium, chromatin structure, and habitat (Pérez-Cerezales et al., 2009;Figueroa et al., 2016). ...
The giant grouper, Epinephelus lanceolatus, is one of the most valuable tropical commercial fishes. The asynchronous maturity periods between males and females hampers breeding programs. Sperm cryopreservation for gamete conservation is important for fish reproduction. We evaluated the effects of storage for 1, 3, 4, and 5 years on cryopreserved sperm of the giant grouper, E. lanceolatus, in 10% DMSO as a cryoprotectant with artificial seminal plasma and 300 mM sucrose as a diluent. Sperm motility, cell survival rate, DNA damage, fertilization rate, and hatching rate were investigated in this study. Post-thaw sperm motility peaked after 1 year of storage (63.27 ± 2.87%; p < 0.05), followed by 3 and 4 years (54.50 ± 4.33% and 54.40 ± 0.30%, respectively). Post-thaw sperm motility decreased after 5 years of storage. The cell survival rate after 1 year of storage (91.54 ± 3.02%) was similar to that of fresh sperm (98.12 ± 0.44%). The rate of DNA damage was unaffected by storage but significantly higher than that of fresh sperm. The fertility rate was significantly higher after storage for 1 and 3 years (98.06 ± 0.57% and 93.26 ± 1.73%, respectively). Samples stored for 1 and 3 years also resulted in high hatching rates (95.90 ± 1.53% and 88.73 ± 1.15%, respectively), which tended to decrease with increasing storage duration. Sperm cryopreserved for 4 years were of good quality in terms of motility and cell survival rate. However, storage for longer than 3 years decreased the fertility and hatching rates.
... Damage to the mitochondria of a derivative during freezing and thawing can lead to a decrease in the mitochondrial membrane potential [39]. The changes caused by cryopreservation and found in the present study were similar to changes in some ultrastructures of fish spermatozoa [40] and different mammalian species such as llamas [39], horses [18], and rams [41]. In the study by Atroshchenko et al. [18] the authors found many spermatozoa with acrosome fragmentation due to false acrosome reaction, with acrosome hyperplasia (non-compact acrosome content), and nuclear changes and vacuolization of chromatin. ...
... In the study by Atroshchenko et al. [18] the authors found many spermatozoa with acrosome fragmentation due to false acrosome reaction, with acrosome hyperplasia (non-compact acrosome content), and nuclear changes and vacuolization of chromatin. In the study by Balamuru-gan et al. [40], it is showed that freezing and thawing caused various ultrastructural changes, such as damage to the plasma membrane around the sperm head, flagellum fragmentation, and complete flagellum loss in fish sperm. In the study by Zampini et al. [39], acrosome damage, loss of mitochondria, disorganization of the axoneme and periaxonemal structures, and invagination in the nucleus of llama spermatozoa were observed with the help of TEM. ...
... In the study by Keskin et al. [41] mainly cryodamage to the membrane, as well as mitochondria (electron-lumen matrix) and axoneme (loss of axoneme doublets) cryo-damage were observed in ram sperm STEM. Vesiculation in acrosomal membranes and loss of acrosomal content observed in the study [18,37,[39][40][41] with frozenthawed semen could be the result of an acrosomal reaction. In the current research, we have not detected the drone sperm acrosome using a transmission electron microscope and have not evaluated the effects of cryopreservation. ...
Instrumental insemination of queen bees using cryopreserved sperm is an important biotechnological tool to address the issues of contemporary beekeeping. Long-term cryopreservation is detrimental to sperm function and drone fertility, killing more than 50% of the sperm during the process. Prediction of cryopreservation damage from freezing drone semen remains elusive. The purpose of this study was to assess the impact of long-term cryopreservation in “Kakpakov diluents” (C46) on morphometric measurements of sperm head of the Apis mellifera L. and ultrastructure. For the experiment, sperm samples from the cryobank of the Federal Beekeeping Research Centre (Russia), frozen since 1993, 2011 and 2013 were used. In the present study, it was found out that cryopreservation had a considerable impact on the drone sperm head morphometry. Sperm head measurements of cryopreserved samples were significantly lower compared with the extended sample for morphometric measurements of the nucleus area 3.55±0.12, 4.07±0.1, 4.31±0.04 (5.57±0.03, respectively); nucleus perimeter 10.57±0.11, 11.11±0.13, 11.59±0.05 (11.72±0.06, respectively); nucleus length 4.66±0.05, 4.82±0.06, 5.07±0.02 (5.22±0.03, respectively); nucleus acrosome length 3.72±0.08, 3.65±0.08, 3.96±0.05 (4.24±0.04, respectively). Cryopreserved and thawed spermatozoa revealed changes in ultrastructure such as vacuolation of the axoneme, intranuclear vacuoles, and loss of the derivative's mitochondria. These details provide valuable data for further experiments on modification of the composition of cryomedium to minimize cryodamage in the semen of honey bee drones.
