Effects of cryoprotective agents and treatment methods on sperm cryopreservation of stone flounder, Kareius bicoloratus

Lee, Yun Ho; Park, Jung Yeol; Lee, Il Young; Zidni, Irfan; Lim, Han Kyu

doi:10.1016/j.aquaculture.2020.735969

Cited by 14 publications

(9 citation statements)

References 30 publications

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“…Furthermore, these are natural strategies that organisms and plants adopt to survive to adverse conditions (desiccation and freezing) ( Lencioni et al, 2015 ; Gertrudes et al, 2017 ). Trehalose and sucrose are two disaccharides that have a positive effect during sperm cryopreservation when used as single or combined cryoprotectants in several species such as ram ( Öztürk et al, 2020 ), stallions ( Pérez-Marín et al, 2018 ), boar ( Pezo et al, 2020 ), stone flounder ( Lee et al, 2021 ), Greenshell™ mussel ( Smith et al, 2012a ), and Australian flat oyster ( Hassan et al, 2017a , b ). A previous work developed by our group established several steps for cryopreservation protocol of C. angulata sperm (type of extender, type and concentration of permeant cryoprotectant, type and sperm concentration in package and freezing rate) ( Riesco et al, 2017a ).…”

Section: Introductionmentioning

confidence: 99%

Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm

et al. 2021

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Sperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a successful strategy to reduce cryodamage in sperm of several species, once allowing to stabilize the plasma membrane constituents. Therefore, this study intends to understand the effects of sugars in the plasma membrane, DNA integrity, and oxidative response during Portuguese oyster sperm cryopreservation. Three cryoprotectants solutions with an initial concentration of 20% dimethyl sulfoxide (DMSO) and 20% DMSO complemented with 0.9 M trehalose or sucrose in artificial seawater were employed. Sperm samples of mature males were individually collected and diluted 1:10 (v/v) in artificial seawater followed by addition of cryoprotectants [1:1 (v/v)]. Thereafter, sperm was loaded into 0.5 ml straws, maintained at 4°C for 10 min, frozen in a programmable biofreezer at −6°C/min from 0 to −70°C, and stored in liquid nitrogen. Samples were thawed in a 37°C bath for 10 s. Several techniques were performed to evaluate post-thaw quality. Sperm motility and DNA integrity were analyzed by using computer-assisted sperm analysis (CASA) software and comet assay. Flow cytometry was employed to determine membrane and acrosome integrity and to detect intracellular reactive oxygen species (ROS) and apoptosis activity. Lipid peroxidation was determined by malondialdehyde (MDA) detection by using spectrophotometry. Sperm antioxidant capacity was evaluated through glutathione peroxidase, glutathione reductase, and superoxide dismutase. Motility was not affected by the extenders containing sugars; these compounds did not reduce the DNA damage. However, both the trehalose and sucrose protected plasma membrane of cells by increasing cell viability and significantly reducing MDA content. The same finding was observed for the ROS, where live cells registered significantly lower levels of ROS in samples cryopreserved with sugars. The activity of antioxidant enzymes was higher in treatments supplemented with sugars, although not significant. In conclusion, the addition of sugars seems to play an important role in protecting the Crassostrea angulata sperm membrane during cryopreservation, showing potential to improve the post-thaw sperm quality and protect the cells from cryoinjuries.

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Section: Introductionmentioning

confidence: 99%

Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm

et al. 2021

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“…The most effective CPA for fish spermatozoa has been identified to be DMSO [ 27 ], mainly because of its small molecular structure, which allows it to easily enter and exit the sperm cell [ 28 ]. The DMSO provided good frozen–thawed sperm quality in starry flounder [ 29 , 30 , 31 ]. For some other flatfish species, DMSO has been considered to be reliable, including Brazilian flounder (10% DMSO), summer flounder (10% to 20% DMSO), and stone flounder (7.5% to 10% DMSO), but not for others including winter flounder and yellowtail flounder [ 5 , 27 , 28 , 29 ].…”

Section: Discussionmentioning

confidence: 99%

Effects of Cryoprotective Medium Composition, Dilution Ratio, and Freezing Rates on Spotted Halibut (Verasper variegatus) Sperm Cryopreservation

Zidni

Lee

Park

et al. 2020

Animals

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The spotted halibut is species that has a high potential market value in Korea, but the supply of seed is unstable because of the limited milt production of males. The objective of this research was to explore different aspects, such as CPAs, diluents, dilution ratio, and freezing rates, to develop an optimal sperm cryopreservation. The parameters assessed were movable sperm ratio, sperm activity index, survival rate, and DNA damage. The CPAs tested in this research were propylene glycol, dimethyl sulfoxide (DMSO), methanol, ethylene glycol, and glycerol. Different diluents, including 300 mM sucrose, 300 mM glucose, Stain’s solution, and Ringer’s solution, were investigated. The previous experiment showed that the optimal CPA for cryopreservation was DMSO with a concentration of 15% with 300 mM as diluent. To determine the effect of the dilution ratio, sperm was diluted to 1:1, 1:2, 1:10, 1:100, and 1:1000 with 300 mM sucrose containing DMSO at a final concentration of 15%. Lastly, the optimal freezing rate of the sperm was evaluated with four different freezing rates (−1, −5, −10, and −20 °C/min). Post-thaw sperm motility was higher with a dilution ratio lower than 1:2, and the freezing rate was less than −5 °C/min. In conclusion, these findings represent the development of a cryopreservation protocol for spotted halibut.

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“…The composition of ASP was based on the biochemical properties of small yellow croaker seminal plasma (Lim et al, 2010). The CPAs and diluents were precooled at 4℃ before use (Lee et al, 2021;Zidni et al, 2020). Sperm was separately mixed with various diluents at 10% EG.…”

Section: Cpas and Diluentsmentioning

confidence: 99%

“…Head intensity (DNAh), tail intensity, and tail migration were assessed using Comet Assay IV lite System (Instem, Staffordshire, UK). The percentage of tail DNA was calculated using the following equation: [100 × (DNAc -DNAh)/DNAc] (Cabrita et al, 2005;Lee et al, 2021;Zidni et al, 2020), where DNAc and DNAh are the sum of the intensities of the pixels in the whole comet area and the head area, respectively (Pérez-Cerezales et al, 2009). The degree of damage was categorized and the percentage of cells within each degree of damage was established.…”

Section: Single Cell Gel Electrophoresis (Scge)mentioning

confidence: 99%

“…Due to the lack of awareness of cryopreservation in Korea thus far, it is necessary to develop basic technologies that can be applied to the actual aquaculture field and conduct physiological research on cryopreserved sperm in aquaculture. In addition, because the types and concentrations of cryoprotective agents (CPAs) and diluents can cause remarkable differences in cryopreservation results, basic information on cryopreservation of fish sperm is necessary (Lee et al, 2021). CPAs play a role in alleviating and regulating conditions unfavorable to cell survival, such as concentration of intracellular electrolytes, increase in osmolality, and formation of ice crystals inside and outside cells, which are generated during the freezing of cells (Ahn et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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Effect of diluent variation on cryopreservation of large yellow croaker Larimichthys crocea

Lim¹,

Zidni²,

Lee³

et al. 2021

Fish Aquat Sci

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The aim of this research was to investigate different factors, including cryoprotective agents (CPAs), diluents, dilution ratios, equilibrium times, freezing rates, and thawing methods to optimize cryopreservation protocols for large yellow croaker (Larimichthys crocea). The parameters evaluated were sperm motility, sperm activity index (SAI), survival rate, and DNA damage. Different types of CPAs, such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), methanol, and glycerol, were tested for sperm preservation. The highest motility, SAI, and survival rate were observed when EG was used. Different diluents such as Stein's solution, Hank's balanced salt solution, marine fish Ringer's solution, artificial seminal plasma (ASP) of small yellow croaker, and Cortland solution were investigated. The highest post-thaw motility was observed upon using ASP as the diluent. Different concentrations of EG were then mixed with ASP to identify the optimal EG concentration. Experimental results showed that the motility (70.33 ± 1.20%), SAI (5), and survival rate (78.30 ± 0.42%) of post-thaw sperm were optimum when 10% EG and ASP were used as the CPA and diluent of cryopreservation, respectively. Post-thaw sperm motility was high at equilibration times below 150 s and at an optimum dilution ratio of 1:1 (sperm: CPA + diluent) and was not significantly different compared with fresh sperm motility. The freezing rate was found to be slow below -10℃/min. The thawing temperature of 45℃ was identified as ideal. The percentage of tail DNA in post-thaw sperm at 10% EG and ASP was also investigated and was found to have more significant DNA damage than that in fresh sperm but significantly lower damage than that in post-thaw sperm at EG concentrations of 5%, 15%, and 20% (p < 0.05). The cryopreservation protocols obtained in this study will be useful in large yellow croaker hatcheries.

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Effects of cryoprotective agents and treatment methods on sperm cryopreservation of stone flounder, Kareius bicoloratus

Cited by 14 publications

References 30 publications

Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm

Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm

Effects of Cryoprotective Medium Composition, Dilution Ratio, and Freezing Rates on Spotted Halibut (Verasper variegatus) Sperm Cryopreservation

Effect of diluent variation on cryopreservation of large yellow croaker Larimichthys crocea

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