Seafood supplied by fishery and aquaculture has been generally recognized as the key alternative protein source for the increasing global population (Zhang et al., 2022). Currently, the global fishery has reached the roof for sustainable production, whereas the aquaculture is in constant growth (Zhang et al., 2022). However, the acceleration of this growth will be needed to keep the pace of population growth in the world which is anticipated to be achieved through systemic approaches to address key issues identified in different species. So far, the application of many research findings has been compromised due to lack of information on late developmental stages or subsequent generations, especially in species with a long generation interval. In finfish, this issue has been alleviated by the introduction of model species. For example, zebrafish Danio rerio has been used as a model species to investigate issues with broader implications in aquaculture and fishery as this species has a short life cycle and could be easily maintained in laboratory conditions (Aleström and Winther-Larsen, 2016). In addition, zebrafish has also long been the model species for oocyte cryopreservation in fish and has recently been recommended as a model to study generation effects of sperm cryopreservation (Dong et al., 2008).

Molluscs represent the second largest farmed group worldwide, accounting for 20.2% of total world aquaculture production in 2020 (FAO, 2022). The main species currently farmed are oysters, clams, cockles, scallops and mussels, which all belong to bivalve (FAO, 2022). Although sperm cryopreservation techniques have been published in many of these species (Liu et al., 2015), their application in commercial productions and/or cryobanking has been restricted due to the lack of information on their potential long-term impacts (Fernández-Díez and Herráez, 2018; Bøe et al., 2021). These concerns have been further stressed recently as negative effects of sperm cryopreservation on progeny fitness have been reported in fish and mammalian species, such as brown trout Salmo trutta (Nusbaumer et al., 2019), Japanese eels Anguilla japonica (Müller et al., 2018), horses (Ortiz-Rodriguez et al., 2019) and mice (Jia et al., 2015), and a urgent study to address these knowledge gaps was recommended at the development of Atlantic salmon gene banking (Bøe et al., 2021). However, investigations on performance of economic important traits and generations are time-consuming and challenging in farmed marine bivalve species because of (1) their long generation interval, from one year in Pacific oysters Crassostrea gigas (Yang et al., 2021) to multiple years in scallop Patinopecten yessoensis (Silina, 2018); and/or (2) high costs and challenges to manage the experiments in the open marine environments (Heres et al., 2022). All these obstacles could be addressed if a model species could be applied.

The dwarf surfclam Mulinia lateralis is a dioecious marine bivalve species and has all the characters as a model species: a short generation interval (approximately three months), well established laboratory cultivation technique, and accessibility of genomic information (Yang and Guo, 2006; Li et al., 2022; Guo et al., 2023). In fact, this species has already been used as a model species in some studies, such as the optimization of tetraploid production methods (Yang and Guo, 2006) and the elucidation of shell formation mechanisms for bivalves (Guo et al., 2023). In order to use M. lateralis as a model species to investigate the effects of bivalve sperm cryopreservation on the performance of resultant progeny and/or subsequent generations, the first step is to develop a non-programmable sperm cryopreservation technique commonly used in this group. In this study, the parameters (type and concentration of cryoprotectant agent, equilibration period, rack height, thawing temperature and sperm to egg ratio) key to this technique have been optimized.

Maintaining sperm capability to fertilize eggs is the most important goal in the development of sperm cryopreservation techniques (Martínez-Páramo et al., 2017). In bivalves, although sperm motility is the common method to assess the post-thaw sperm quality, the published papers have suggested that the post-thaw D-stage larval rate would be more reliable to predict the further development of progeny produced by the cryopreserved sperm (Gwo et al., 2003; Ieropoli et al., 2004; Adams et al., 2008; Hassan et al., 2015). In addition, data on performance of D-stage larvae are easy to collect as the period required for fertilized eggs to develop into this stage is short (< one day) and they have not started feeding.

This study has developed a non-programmable sperm cryopreservation technique in the dwarf surfclam M. lateralis, resulting in a post-thaw D-stage larval rate similar to the fresh control with the optimized cryopreservation parameters. This result suggested that the technique commonly used in the bivalve sperm cryopreservation studies is suitable for M. lateralis. Therefore, this species could be used as a model to accelerate the investigation of sperm cryo-impact on the performance of resultant progeny at late developmental stages and/or subsequent generations, as these questions are the key concerns limiting the application of this technique in marine bivalve aquaculture and germplasm cryobanking.

Permeable CPAs could protect the cells intracellularly and extracellularly and are the essential ingredient for sperm cryopreservation in aquatic species (Martínez-Páramo et al., 2017). EG was found to be the most toxic to the sperm of M. lateralis in this study as a lower D-stage larval rate was produced in comparison with other CPAs at each concentration assessed. This result was in agreement with the findings in most marine molluscan, fish and crustacean sperm cryopreservation (Liu et al., 2015; Magnotti et al., 2018; Morales-Ueno and Paniagua-Chávez, 2020). However, it should note that this chemical has often been selected as a suitable CPA for oocyte and larvae cryopreservation in some aquatic and domestic animals (Tervit et al., 2005; Cirino et al., 2019; Tharasanit and Thuwanut, 2021; Zhu et al., 2023).

The equilibration time is the period for CPAs to enter into the sperm without or with limited toxicity prior to the initiation of cryopreservation process. In the current study, the D-stage larval rate was decreased with the increase in the equilibration period in all the CPAs evaluated. Except for 8% DMSO, there was no significant difference in the D-stage larval rate between 10 and 20 min equilibration periods in all other CPAs. This result agreed with the findings in oysters, abalone and mussels, where 10 min was normally identified as the optimal equilibration period (Liu et al., 2015).

Cooling is a key step for cryopreservation. Its rate should be fast enough to reduce the exposure time of sperm to the concentrated CPAs and also slow enough for dehydration to avoid the formation of intracellular ice crystals (Demoy-Schneider et al., 2020). In this study, the highest D-stage larval rate was achieved when the sperm were cryopreserved at 4 cm above the LN surface. This height was lower than those found in abalone H. laevigata and H. rubra (Liu et al., 2015), mussels M. galloprovincialis (Liu et al., 2016), pearl oysters Pinctada fucata martensii ( Kawamoto et al., 2007) and P. margaritifera (Acosta-Salmόn et al., 2007), and great scallops Pecten maximus (Suquet et al., 2014), but higher than those in greenshell mussels Perna canaliculus (Smith et al., 2012) and pearl oysters P. margaritifera (Hui et al., 2011).

Thawing temperature is a critical factor to manage the risk of water recrystallization and its impact on sperm (Demoy-Schneider et al., 2020). Among the thawing temperatures evaluated in this study, 60°C produced the highest D-stage larval rate, although it did not differ significantly from 50°C. This temperature was the same as used in abalone H. laevigata and H. rubra (Liu et al., 2015). However, it was lower than those in oysters C. gigas (Gwo et al., 2003) and C. virginica (Paniagua-Chávez and Terisch, 2001), and abalone H. diversicolor (Gwo et al., 2002), but higher than those in oysters C. gigas ( Adams et al., 2004; Dong et al., 2005) and O. edulis (Vitiello et al., 2011), abalone H. rufescens (Salinas-Flores et al., 2005), pearl oysters (Acosta-Salmόn et al., 2007; Kawamoto et al., 2007; Narita et al., 2008; Hui et al., 2011), mussels (Matteo et al., 2009; Smith et al., 2012) and scallops P. maximus (Suquet et al., 2014). These suggest that the thawing temperature might be species specific and also be related to the cooling methodology used in the same species.

It is generally accepted that cryodamge on sperm is inevitable, resulting in a higher sperm to egg ratio being required for the post-thaw sperm fertilization (Martínez-Páramo et al., 2017; Yánez-Ortiz et al., 2022). In this study, a ratio of 220 times higher than the fresh control was needed to produce a similar D-stage larval rate. This ratio was higher than that in oysters C. gigas (Ieropoli et al., 2004) and scallops P. maximus (Suquet et al., 2014), but lower than studies in most marine molluscan species, such as oysters (Paniagua-Chávez and Terisch, 2001; Gwo et al., 2003; Adams et al., 2004), abalone (Liu et al., 2015), pearl oysters (Hui et al., 2011) and mussels (Smith et al., 2012; Liu et al., 2016).

The assessment of post-thaw sperm quality could provide useful information for understanding cryodamage to further improve the sperm cryopreservation protocol (Yánez-Ortiz et al., 2022). The cryopreservation process could compromise the cellular structures and organelles, such as plasma membrane, mitochondria, acrosome and flagellum (Partyka et al., 2012; Peris-Frau et al., 2020; Xin et al., 2020), which have been clearly revealed by the electronic microscope images in this study. Sperm membranes are the primary sites of cryoinjury (Partyka et al., 2012). One of the key reasons responsible for altering the membrane integrity is the lipid peroxidation products generated from the oxidative stress, which has been shown in horse (Nelid et al., 2005), fish (Cabrita et al., 2010), fowl (Partyka et al., 2012) and boar (Guo et al., 2021) sperm cryopreservation. In this study, the increased lipid peroxidation level in post-thaw sperm would also contribute the reduced plasma membrane integrity in M. lateralis.

Acrosome reaction is the key event bringing together the fusogenic areas of sperm and egg during fertilization in marine bivalve species (Longo and Scarpa, 1991; Rosati, 1995). It involves in the fusion of plasma membrane of both sex gametes and the release of hydrolytic enzymes from sperm for penetrating the egg envelop (Rosati, 1995). Therefore, maintaining the acrosome integrity is vital during the cryopreservation. In the current study, the acrosome integrity of post-thaw sperm was significantly decreased, which might be due to the acrosomal membrane injury by the increased level of lipid peroxidation after cryopreservation as discussed previously or the formation of ice crystals. This phenomenon has also been reported in the fowl (Partyka et al., 2012) and bull (Jakop et al., 2023) sperm cryopreservation. Mitochondria are highly susceptible to the changes in intracellular reactive oxygen species which could be increased by the electron leakage from the uncoupled mitochondria oxidative phosphorylation during cryopreservation (Partyka et al., 2012). Consequently, as less number of post-thaw sperm could maintain the high MMP, their ATP production and motility rate would be compromised (Partyka et al., 2012). In this study, the post-thaw sperm motility was significantly compromised and this reduction has often been reported in other marine molluscan species (Liu et al., 2015). In this study, the significant decrease in the post-thaw sperm MMP might also be associated with the enhancement of lipid peroxidation level. Similar phenomena have been reported in the sperm cryopreservation in fowls (Partyka et al., 2012) and fishes (Figueroa et al., 2019).

Antioxidants are molecules capable of preventing, delaying or inhibiting the oxidation of other molecules, thus mitigating the oxidative damage [56]. In general, the antioxidant defense of biological systems could be mediated by either enzymatic or non-enzymatic antioxidants (Amidi et al., 2016; Sandoval-Vargas et al., 2021). GSH is a non-enzymatic and the most abundant antioxidant in almost all cell types (Len et al., 2019), participating directly in the neutralization of oxidative damage (Amidi et al., 2016). Both SOD and CAT belong to the enzymatic antioxidant and could quickly neutralize any reactive molecules by different reactions (Amidi et al., 2016; Sandoval-Vargas et al., 2021). In the current study, the activities of these enzymes were all significantly decreased in post-thaw sperm which agreed with the studies in fowls (Partyka et al., 2012), Pacific cods Gadus microcephalus (Wang et al., 2016). These results also indicate that the activities of intracellular antioxidant enzymes might not be enough to neutralize the damages produced by the oxidative stress during the sperm cryopreservation in M. lateralis.

The knowledge gap in cryo-impacts on resultant progeny at late developmental stages and/or next generations has constrained the application of sperm cryopreservation technique in bivalve aquaculture industry for different purposes, such as assistances in commercial production, selective breeding and cross breeding. The gap mainly resulted from the long generation interval, and challenges and high costs of maintaining experiments in the open marine environment where most bivalve species are farmed. These issues could be addressed if a model species, such as M. lateralis, could be used as this species has a very short generation interval (three months) and well established laboratory culture technique. The development of a non-programmable sperm cryopreservation technique in M. lateralis in this study is a cornerstone to bridge the knowledge gap as this technique is commonly used in bivalve species.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The requirement of ethical approval was waived by College of Marine Life Sciences, Ocean University of China for the studies involving animals because Mulinia lateralis belongs to the invertebrate and according to rules and regulations of ethics committee in College of Marine Life Sciences, Ocean University of China, no ethical approval needed. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

ZX: Data curation, Investigation, Methodology, Writing – original draft. ZY: Investigation, Methodology, Writing – original draft. LB: Data curation, Writing – review & editing. XL: Conceptualization, Methodology, Writing – review & editing. XH: Conceptualization, Methodology, Writing – review & editing. YL: Conceptualization, Investigation, Methodology, Writing – review & editing.