Bivalves represent an important aquaculture supply, which has led to a great demand for this resource and consequently an increase in its production (Wijsman et al., 2019). Therefore, aquaculture needs to diversify the species produced for a greater offer. Portuguese oyster [Crassostrea angulata (C. angulata)] is a species with high potential for aquaculture production and market acceptance. It was a key species for the European shellfish industry in the 1970s; however, due to a disease and poor remediation and management measures, the natural populations decreased (Boudry et al., 1998). Nowadays, there are only three places with pure populations of Portuguese oyster, namely, in the Sado and Mira estuary (Fabioux et al., 2002) and the Guadalquivir River (Michinina and Rebordinos, 1997). Therefore, it is important to develop techniques to ensure the conservation and recovery of native populations of the Portuguese oyster, but also to enhance and support their aquaculture production (Anjos et al., 2017; Riesco et al., 2017a; Pogoda et al., 2019).

Cryopreservation methodologies are advantageous tools that may enhance conservation strategies and reproductive technologies. The applicability of sperm cryopreservation involves the maintenance of improved genetic lines or endangered species (Martínez-Páramo et al., 2017; Riesco et al., 2017a). In addition, it allows to extend the breeding season and synchronize spawning between males and females (Hassan et al., 2015). This procedure stores reproductive cells at low temperatures. Thus, one of the major challenges of cryopreservation is to prevent the associated cryodamage involving biological structures (plasma membrane, mitochondria, and chromatin) and functions of the cell in a molecular and biochemical point of view (Cabrita et al., 2010; Sieme et al., 2016). Spermatozoa characteristics make this type of cells very prone to suffer cryodamage, mostly due to the high content of polyunsaturated fatty acids of their membranes (Cabrita et al., 2014). Osmotic and thermal stress, intracellular ice crystallization, high level of reactive oxygen species (ROS), and imbalance in antioxidant defense system are some examples of consequences that may compromise the quality of the cryopreserved material (Amidi et al., 2016). These bring up the necessity to evaluate the lethal and sublethal cryodamage. The analysis of sperm quality in invertebrates has been performed by using several techniques such as motility, membrane integrity, morphology, DNA integrity, lipid peroxidation, detection of intracellular ROS, mitochondrial membrane potential, and acrosome integrity (Akcha et al., 2012; Le Goïc et al., 2013; Vignier et al., 2017; Gallo et al., 2018, 2020). However, in cryopreservation studies, most research in bivalves applies only motility, plasma membrane integrity, and fertilization as tools to assess post-thaw sperm quality. Only a few works apply specific techniques such as malondialdehyde (MDA) content determination, DNA fragmentation, and acrosome integrity (Smith et al., 2012b; Liu et al., 2016; Riesco et al., 2019). Therefore, it is crucial to establish quality assessment methodologies that are already widely applied to mammals (Gangwar et al., 2020; Öztürk et al., 2020) and marine vertebrates (Riesco et al., 2017b; Sandoval-Vargas et al., 2021a). This will allow to understand more specifically which organelles and sperm functions are affected by cryodamage. By identifying the type of damage, it is possible to outline the strategy to prevent it. One common and successful strategy to prevent cryodamage is the supplementation of freezing media with compounds that protect cells against freezing injuries (Elliott et al., 2017). Antioxidants, proteins, and sugars are some examples of non-permeant cryoprotectants used in supplementation (Martínez-Páramo et al., 2012; Riesco et al., 2017c; Diogo et al., 2019). Sugars are large molecules that act outside of the cells, interacting with the plasma membrane. Due to their high viscosity, they interfere with the physical and chemical properties of the extender solution (Fuller, 2004). 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). Nevertheless, supplementation of permeant cryoprotectant has not been yet evaluated for C. angulata.

This study aims to explore the effect of trehalose and sucrose, when combined with one permeant cryoprotectant as dimethyl sulfoxide (DMSO), on the structure and functions of cryopreserved spermatozoa. Simultaneously, the different cryodamage levels that compromise the quality of the spermatozoa during cryopreservation were evaluated through motility, DNA and plasma membrane integrity, content of ROS, apoptosis activity, acrosome integrity, lipid peroxidation, and antioxidant activity.

Oysters are low trophic filter feeders that constitute an important resource for fisheries and aquaculture. However, due to bad management of seed production and anthropogenic impacts, some species such as the C. angulata, which shows potential for aquaculture diversification, are at risk of disappearing. Cryopreservation is a helpful method for genetic resources preservation. This method is a useful tool that may support the management of C. angulata pure natural banks and, at the same time, aquaculture production activities. However, the cryopreservation process produces cellular stress that generates damage in cells (Xin et al., 2020). Due to the complex dynamic structure of spermatozoa, it is necessary to establish different protocols for each species, toward the reduction of cryodamage. The supplementation with non-permeant cryoprotectants is a successful strategy to reduce cryodamage in cells through the use of high viscosity, high-molecular weight, and non-toxic compounds (Elliott et al., 2017). These strategies have been used in sea bass (Dicentrarchus labrax), dusky grouper (Epinephelus marginatus) and sea bream (Sparus aurata) improving either motility or cell viability or enhancing the antioxidant system by the addition of antioxidants (ascorbic acid, α-tocopherol), amino acids (taurine, hypotaurine), antifreeze proteins (AFPI, AFPIII), or sugars (glucose, sucrose) (Martínez-Páramo et al., 2012; Zilli et al., 2014; Riesco et al., 2017c).

In this study, sugars incorporated into the freezing media improved sperm plasma membrane integrity and reduced ROS levels, acrosome damage (only trehalose), and lipid peroxidation. This was probably due to high-molecular weight of sugars, which promoted cellular dehydration by replacing the water in the membrane that improved permeant cryoprotectant incorporation (De Leeuw et al., 1993). At the same time, due to a high viscous environment, the surrounding of the cells is stabilized, protecting the plasma membrane from cold damage (Nicolajsen and Hvidt, 1994; Woelders et al., 1997). This protective effect of sugars in post-thaw sperm was also reported for several species such as boar, Salmonidae fish, and Australian flat oyster (Gómez-Fernández et al., 2012; Nynca et al., 2016; Hassan et al., 2017a). Trehalose and sucrose have shown an important role as supplement of the freezing medium during cryopreservation of bivalves, being mainly applied in larvae (Choi et al., 2008; Labbé et al., 2018) and less in sperm (Hassan et al., 2017a; Demoy-Schneider et al., 2018). Moreover, studies in bivalve sperm were performed with a low number of quality analyses both in the fresh and post-thaw samples, being these analyses focused mainly on sperm motility, plasma membrane integrity, and fertilization success (Vitiello et al., 2011; Horváth et al., 2012; Suquet et al., 2016; Hassan et al., 2017a,b), thus lacking important information on sublethal damage. The use of a wide range of quality analyses in the establishment of a cryopreservation protocol is useful to investigate the specific structure and functions of spermatozoa that are being damaged (Cabrita et al., 2014). This information will support the establishment of strategies to mitigate cryodamage (Diogo et al., 2019; Riesco et al., 2019; Kim et al., 2020; Hossen et al., 2021). Therefore, in this study, the analysis performed on post-thaw sperm revealed the effects that trehalose and sucrose supplementation had on motility, DNA and plasma membrane integrity, ROS and apoptosis (caspases pathway) detection, acrosome integrity, lipid peroxidation, and antioxidant enzymes activities (SOD, GPx, and GR). This allowed an exhaustive evaluation of sperm quality and cell cryodamage. Sperm motility is a widely used quality indicator, since its activation is necessary for sperm to reach oocytes for successful fertilization (Cabrita et al., 2008; Boulais et al., 2019). In this study, post-thaw sperm motility did not show significant differences between cryoprotectant solutions; therefore, sugars supplementation did not have an effect on this parameter. In opposition, Hassan et al. (2017a) reported an improvement of post-thaw sperm motility of Australian flat oyster (Ostrea angasi) when using 10% DMSO supplemented with 0.45 M trehalose. In previous studies, our group showed that sperm motility did not seem so relevant when compared with other parameters that can jeopardize cells, due to the fact, that even with few motile cells, the long sperm motility duration of this species would allow oocytes to be fertilized (Riesco et al., 2017a). This fact is also supported in Crassostrea virginica by Yang et al. (2012), who showed that even low sperm motility after thawing did not compromise the ability of sperm to fertilize the oocytes and further develop into D-larvae. According to Figueroa et al. (2016), one structural damage that can compromise post-thaw sperm motility of Salmo salar was the damage inflicted to the mitochondria. Mitochondria is an organelle also present in bivalve sperm that participates in the synthesis of ATP, producing the majority of the energy necessary for motility (Boulais et al., 2015). For this reason, some changes or damage in this structure or metabolic pathways can compromise sperm performance. In this way, in further studies, the evaluation of mitochondrial functionality such as plasma membrane potential and ATP content should be applied, since it could help to explain the motility results.

Deoxyribonucleic acid integrity evaluation is essential, since gametes need to provide secured genetic contribution to the offspring to avoid embryo abortion (Cabrita et al., 2010). Sperm freezing with sucrose addition showed significant higher DNA damage when compared to trehalose treatment and control. In equine sperm, DNA was not affected by cryopreservation by using different trehalose and sucrose concentrations (Pérez-Marín et al., 2018). Also, El-Sheshtawy et al. (2015) tested the effect of different concentrations of trehalose and sucrose (0.05, 0.1, and 0.2 M) on post-thaw quality of bull sperm and showed that freezing media supplemented with 0.05 M trehalose and 0.05 and 0.1 M sucrose produced low levels of sperm DNA fragmentation, while higher concentrations of these sugars make the sperm more prone to DNA damage. In this study, trehalose and sucrose were only tested at 0.45 M, taking into consideration the literature in similar species (Adams et al., 2004; Hassan et al., 2017a), being necessary in further studies to evaluate the effect of other concentrations. This will allow inferring about their possible effect on DNA protection and may justify our results with sucrose treatment, as demonstrated by El-Sheshtawy et al. (2015).

The membrane stabilization promoted by trehalose and sucrose allowed an improvement of sperm plasma membrane integrity. Apart from that, the high viscosity of sugars and the reduction in ice crystal formation may have contributed to the results obtained, as demonstrated by Nicolajsen and Hvidt (1994) and Woelders et al. (1997). Hassan et al. (2017a) showed that post-thaw sperm viability of Australian flat oyster (Ostrea angasi) was improved when a solution with 10% DMSO complemented with 0.45 M trehalose was applied. The membrane protection through sugars supplementation also reduced lipid peroxidation. Zhu et al. (2017) showed that trehalose supplementation improved plasma membrane integrity and reduced lipid peroxidation in rabbit post-thawed sperm. Sperm plasma membrane possesses high levels of lipids, which are prone to oxidation during stress events such as cold exposure (Cabrita et al., 2014). Additionally, sugars can create bonds with the polar head of phospholipids in the plasma membrane (Anchordoguy et al., 1987), thus improving membrane protection and preventing lipid peroxidation. Altogether, these factors allowed a higher maintenance of the sperm plasma membrane integrity and stabilization during cryopreservation. Although this effect was not consistent for all the tested sugars, acrosome-reacted cells were lower in trehalose treatment when compared with control. This stabilization could be the responsible factor for acrosome protection, which is an essential structure for fertilization. It has enzymes engaged in lysing the oocyte membrane and their release allow the penetration of spermatozoa into the egg (Boulais et al., 2019). This study allowed understanding the effects of cryopreservation in different sperm structures essential to achieve successful fertilization. However, future studies are necessary to investigate fertilization success and offspring quality generated from C. angulata cryopreserved sperm.

Reactive oxygen species are molecules produced naturally to protect the cell and regulate signal pathways (Ighodaro and Akinloye, 2018). However, when an imbalance of ROS occurs, several cell structures containing lipids, proteins or enzymes, and chromatin may be affected by oxidative stress (Sandoval-Vargas et al., 2021b). In this study, there was a reduction in the formation of ROS in post-thaw sperm supplemented with sugars when compared to DMSO treatment. Concomitantly, low levels of lipid peroxidation were also detected in sperm treated with sugars, improving in these treatments the stabilization of the plasma membrane. Low levels of ROS and MDA associated with an improvement in rabbit sperm membrane integrity were reported by Zhu et al. (2017) when sperm was supplemented with trehalose. Trehalose and sucrose also showed a high protective effect against oxidative stress in boar post-thawed sperm by reducing the ROS levels and improving the cell viability when compared with lactose (Pezo et al., 2020). The relationship between ROS levels and lipid peroxidation was also reported by Sandoval-Vargas et al. (2021a) for coho salmon (Oncorhynchus kisutch) sperm, which demonstrated that post-thawed samples with high levels of superoxide anions showed high MDA content and low levels of viable sperm. In this study, the sugars seemed to reduce the damage associated with oxidative stress by protecting the lipids present in oyster sperm plasma membrane, thus avoiding their peroxidation by maintaining a balance of ROS.

Antioxidant enzymes are the first line of defense system of the cell, that keep ROS levels under control (Ighodaro and Akinloye, 2018). High levels of superoxide ions can be metabolized by SOD enzyme, which catalyzes the dismutation of these molecules to form oxygen and hydrogen peroxide, while catalase and GPx enzymes convert the hydrogen peroxide into water and oxygen (Amidi et al., 2016). Therefore, this protective mechanism acts to suppress or prevent the production of harmful molecules. Any changes in the activity of the antioxidant enzymes, such as freezing/thawing events, make the sperm more susceptible to oxidative stress. The detection of the activity of antioxidant enzymes in this study did not show any significant differences between treatments and control, although some authors have shown higher enzymatic activity in rabbit post-thawed sperm when sugars were incorporated in the freezing media (Zhu et al., 2017). This fact may suggest that sugars are not able to protect intracellular enzymes during cryopreservation and, probably, some enzyme denaturation or enzyme cell leakage may have occurred, but their effect is similar to the control.

Caspase-3/7 triggers the beginning of cell death from which the apoptosis process cannot be reversed, leading to cell alterations such as DNA and protein degradation (Elmore, 2007). Several studies have identified apoptosis activation in cryopreserved spermatozoa leading, at latter times, to a decrease in cell viability (Riesco et al., 2017c; Diogo et al., 2018). In this study, there were no significant differences in cellular apoptosis mechanisms identified through caspase-3/7 activities between control and sugar supplemented sperm. These results suggested that supplementation of sugars in the extender did not change cell death type or pathway (early apoptosis, late apoptosis, or necrosis by other mechanisms), according to the subpopulations detected by flow cytometry. The roles of cellular apoptosis and antioxidant enzymes in this study remain unclear. In future studies, these issues should be investigated, as well as, the effect of different freezing and warming rates on antioxidant enzymes activities in C. angulata post-thaw sperm.

This study contributed to the optimization of C. angulata sperm cryopreservation. Through the obtained results, it was possible to confirm selected sugars as successful freezing media, additives to mitigate cryodamage, particularly in reducing ROS production, lipid peroxidation, and improving plasma membrane integrity. In particular, trehalose may have shown a higher protective effect during cryopreservation because its molecular structure creates more connections to phospholipid polar heads than the other tested sugar (sucrose) (Tsai et al., 2018). This effect was evident in reducing acrosome-reacted cells and in the registered levels of DNA fragmentation. This study corroborates the previous findings in several species (Hassan et al., 2017b; Zhu et al., 2017; Öztürk et al., 2020) that freezing media supplementation with trehalose is a good strategy to improve post-thaw sperm quality in C. angulata.

In C. angulata sperm cryopreservation, the supplementation with trehalose and sucrose of the freezing media containing DMSO improved plasma membrane integrity and reduced the oxidative stress and lipid peroxidation caused by ROS.

Sucrose supplementation did not protect chromatin and acrosome structures of C. angulata post-thaw sperm, revealing damage in DNA and acrosome structures.

Sperm cryopreservation with 10% DMSO and 0.45 M trehalose showed a high protective effect in C. angulata post-thaw sperm quality, once improved plasma membrane and acrosome integrity, and reduced lipid peroxidation and superoxide ions levels.

The establishment of a cryopreservation protocol that evaluates several post-thaw sperm quality parameters allowed to establish a strategy to mitigate cryodamage.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

EC designed the experimental work and acquired the funding. EC and DM supervised the experimental work. CA and AS collected the samples. CA, AS, and DD performed the laboratory analyses. CA and EC wrote the manuscript. CA, DM, and EC revised and edited the manuscript. All the authors contributed to the manuscript and approved the submitted manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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