Giant clams (Tridacna spp.) are keystone reef organisms that play important ecological roles in Indo-Pacific coral reef ecosystems. However, their populations have declined substantially because of overexploitation, habitat degradation, and climate change. Cryopreservation offers a potential strategy for preserving genetic resources and supporting future conservation and restoration efforts. In this study, we evaluated the feasibility of cryopreserving D-stage larvae of Tridacna crocea and T. maxima using a two-step freezing protocol. The effects of different cryoprotectants, concentrations, and equilibration times on post-thaw larval integrity and motility were assessed. Larval development was monitored using microscale culture techniques and microscopic observations. The results demonstrated species-specific responses to cryopreservation treatments, with T. maxima generally exhibiting greater cryotolerance than T. crocea. Ethylene glycol produced the highest larval integrity percentage (LIP) in both species. In addition, glycerol and propylene glycol yielded relatively high LIP values in T. crocea, whereas methanol and dimethyl sulfoxide (DMSO) performed more effectively in T. maxima. Post-thaw motility was observed only in a limited number of treatment groups, with the highest motility recorded in larvae treated with 0.75 M glycerol for 30 min and 0.5 M DMSO for 20 min. In contrast, non-cryopreserved control larvae exhibited a progressive decline in motility during culture, decreasing to 12.1% in T. maxima and 4.0% in T. crocea after 24 h. To our knowledge, this study represents the first investigation of cryopreservation in tridacnid D-stage larvae. Although post-thaw motility remained limited, the successful preservation of larval structural integrity demonstrates the potential of cryopreservation as a conservation tool for giant clams. Further optimization of species-specific cryopreservation protocols will be necessary to improve post-thaw recovery and support long-term genetic resource banking.
Freezing-induced ice recrystallization is a major limitation in preserving the quality of high-moisture fruits because of its adverse effects on cellular structure, texture, and nutritional stability. This study investigated vacuum-assisted sucrose impregnation as a cryostabilization approach for improving the quality of frozen pineapple stored at -18 °C for 21 days. Pineapple cubes were subjected to untreated control, atmospheric sucrose impregnation (AI; immersion in 60% (w/w) sucrose solution for 30 min at atmospheric pressure), and vacuum-assisted impregnation (VI; 31.8 kPa for 20 min followed by 10 min relaxation). Mass transfer, thermal characteristics, physicochemical properties, color, texture, and microstructural changes were evaluated. Vacuum treatment significantly enhanced solute incorporation, increasing cryoprotectant uptake to 11.2%. Differential scanning calorimetry revealed that VI reduced the onset freezing temperature from -1.8 to -3.6 °C and decreased melting enthalpy by approximately 52% compared with untreated samples. Freezable water content decreased from 72.4% to 38.6%, while bound water increased from 27.6% to 61.4%, indicating enhanced water immobilization within the fruit matrix. During frozen storage, VI-treated samples exhibited lower ion leakage (9.1%), greater retention of total phenolic content (67.5 mg GAE/100 g), higher antioxidant activity determined by DPPH scavenging (73.9%) and FRAP (581.3 μmol Fe2+ equivalents/100 g fresh weight), and improved textural stability, maintaining hardness at 21.4 N compared with 10.2 N in control samples after 21 days. SEM observations further confirmed reduced tissue disruption and preservation of cellular architecture. These findings demonstrate that vacuum-assisted sucrose impregnation effectively improves cryostability by regulating water distribution and preserving structural integrity in frozen pineapple.
Oocyte vitrification is widely used in fertility preservation and assisted reproductive technologies (ART), but current protocols rely on sequential cryoprotectant exposure steps that are labor-intensive and operator-dependent. This study evaluated the feasibility of a simplified one-step vitrification and warming protocol using mouse metaphase II oocytes. An initial optimization phase using 716 oocytes identified the optimal exposure time to the newly developed vitrification and warming solutions. Subsequently, 876 oocytes were allocated to fresh controls, standard multi-step vitrification, or the optimized one-step protocol. Outcomes included post-warming survival, meiotic spindle integrity, chromosome alignment, reactive oxygen species (ROS) levels, mitochondrial distribution, intracytoplasmic sperm injection (ICSI) outcomes, blastocyst development, total blastocyst cell counts, and full-term development after embryo transfer. An one-step exposure time of 2 min achieved the highest post-warming survival (100%), comparable to the standard protocol (98.9%; p = 0.080). Immunofluorescence analysis demonstrated normal spindle morphology, chromosome alignment, oxidative stress levels, and mitochondrial distribution across groups. Survival after warming and ICSI, as well as blastocyst formation rates, did not differ significantly between vitrification methods and fresh controls (p > 0.05). Although blastocyst total cell numbers were slightly reduced in vitrified groups, full-term development rates were comparable among all groups, and offspring developed normally to adulthood. These findings demonstrate that one-step vitrification and warming can preserve oocyte developmental competence while substantially simplifying cryopreservation procedures, supporting its potential as a practical alternative to conventional vitrification protocols in ART.
We evaluated the use of alpaca oocytes vitrified using the Microdrop or Cryotech technique for in vitro embryo production. Grade I-II COCs (n = 1094) were recovered from slaughterhouse ovaries, and in vitro matured for 36 h. Then, MII oocytes were randomly distributed in groups: a) vitrified using two techniques [Microdrop (VM group; n = 250) or Cryotech (VC group; n = 244)], b) just exposed to the corresponding vitrification/thawing solutions (EM group; n = 200 or EC group; n = 200) or c) non-exposed nor vitrified (i.e. fresh oocytes; control group; n = 200). After treatment administration oocytes from all groups were IVF using epididymal sperm and in vitro cultured for up to 8 days. The vitrification technique and the exposure to vitrification solutions of MII oocytes negatively affected (p < 0.001) the cleavage rate compared to control group. On the other hand, exposure to Microdrop vitrification solution (EM group) and vitrification using the Microdrop technique significantly (p < 0.001) reduced the embryo developmental rate at all stages evaluated. Early blastocysts rate was affected (p < 0.001) by treatment, and it was greatest (p < 0.001) for the EC and VC, intermediate (p < 0.05) for the control, and lowest (p < 0.001) for VM and EM groups. Expanded and hatched blastocyst were not observed in the groups where oocytes were subjected to either Cryotech or Microdrop vitrification technique. We conclude that Cryotech is a more suitable vitrification technique for MII alpaca oocytes than Microdrop, under the conditions used in this study (timing and cryoprotectants concentration), allowing the development of early blastocyst at a rate comparable to control group.
Extending the storage of donated livers beyond the current 4-6 h via freezing could significantly improve transplant availability. However, mitigating freezing injury in complex organs remains challenging because conventional cryoprotective agents (CPAs) are difficult to perfuse uniformly and several biologically important injury mechanisms are not well controlled. Ice recrystallization, where larger ice crystals grow at the expense of smaller ones, is one such mechanism. To address this, we evaluated two ice recrystallization inhibitors (IRIs), N-(2-fluorophenyl)-C6-azido-D-gluconamide (2FA) and N-(2-chlorophenyl)-D-gluconamide (4CLA), for hepatocompatibility and tissue permeation during a 4 h portal vein perfusion of rat livers. Hepatic function assessments showed no general toxicity for either IRI. However, 4CLA-perfused livers exhibited biliary stasis and lacked bile production. Conversely, 2FA maintained bile production and, in post-perfusion biopsies subjected to freezing, successfully reduced ice crystal growth within 200 μm of portal venules. These findings demonstrate that 2FA is a highly promising candidate for supporting long-term liver preservation in the frozen state.
Vitrification is a fast, simple and cost-effective cryopreservation technique which has emerged as a promising approach for sperm cryopreservation in a variety of animals, including aquaculture species. However, sperm vitrification in bivalves remains undeveloped, although molluscan sperm cryopreservation has been studied for more than 50 years. This investigation aimed to develop a sperm vitrification technique in dwarf surfclam Mulinia lateralia (a model species in bivalve research) and to assess the cryodamage at different levels. The results showed that the synergistic cryoprotective effect of coenzyme Q10 and ficoll was beneficial for the sperm vitrification with the dimethyl sulfoxide (DMSO) as the base cryoprotectant. The optimal cryoprotective solution for sperm vitrification in dwarf surfclam was 8% DMSO + 20 μM coenzyme Q10 + 5% ficoll. Although the D-stage larval rate achieved in vitrified sperm did not significantly differ from the fresh sperm, a sperm to egg ratio of 200 times higher was required in vitrified sperm than the fresh control at fertilization. This increased sperm requirement was associated with the vitrification-induced cryodamage, including reduced sperm motility, plasma membrane integrity, mitochondrial membrane potential, acrosome integrity, enzyme activity and DNA integrity, together with increased lipid peroxidation. In conclusion, the sperm vitrification technique presents a promising and technically achievable alternative for sperm cryopreservation in marine bivalve species.
This study evaluated the long-term cryopreservation efficacy of two previously developed protocols—the Modified Perlite Protocol (MPP) and the Vermiculite Protocol (VP)—using 65 ectomycorrhizal basidiomycete culture strains stored for five years in the vapor phase of a liquid nitrogen tank. Survival rates were assessed based on recovery ratios, with both MPP and VP showing high viability: 64 of 65 strains showed complete recovery under each protocol. Colony morphology remained stable across all methods, showing no visible phenotypic changes due to cryopreservation. Regarding post-thaw mycelial growth potential measured by colony diameter, VP was superior or equivalent to MPP in 49 out of 51 analyzed strains immediately post-thaw, yielding significantly greater growth in 19 of these strains. Further statistical analysis among three protocols (HPP, MPP, and VP) in a subset of 14 strains showed that VP generally produced the largest mean colony diameters immediately post-thaw in most strains and was significantly larger than MPP in six strains, although strain-dependent exceptions were observed. These findings suggest that both MPP and VP are suitable for the long-term preservation of cryosensitive basidiomycete cultures, with VP offering enhanced immediate post-thaw mycelial growth potential.
Extreme cold exposure has emerged as a significant global public health challenge to cognitive function among populations such as military personnel, polar workers, and residents of high-latitude regions. However, the complex mechanisms underlying cold environment-induced cognitive impairment remain to be systematically elucidated. This systematic review synthesizes the effects of cold exposure (CE) on cognitive function and the potential biological mechanisms involved. CE impairs cognition through four interconnected pathways: neuroendocrine dysregulation, oxidative stress, neuroinflammation, and mitochondrial dysfunction. The dual dysregulation of the hypothalamic-pituitary-adrenal axis and the hypothalamic-pituitary-thyroid axis constitutes the core neuroendocrine mechanism, wherein excessive glucocorticoids mediate hippocampal structural damage through mitochondrial reactive oxygen species bursts, autophagic dysregulation, and synaptic plasticity impairment. Oxidative stress is characterized by ROS accumulation and a collapse of antioxidant defenses, with time-dependent decompensation of the Nrf2 pathway exacerbating lipid peroxidation and oxidative modification of synaptic proteins. Neuroinflammation features hippocampal microglial activation and a positive feedback loop involving the HMGB1-NLRP3 inflammasome, driving the cascade release of pro-inflammatory cytokines such as interleukin-1 and tumor necrosis factor-α. Mitochondrial dysfunction manifests as suppressed biogenesis, imbalanced dynamics (Drp1-mediated excessive fission), and decreased oxidative phosphorylation efficiency, leading to neuronal energy crisis and apoptosis. These four pathways amplify each other through positive feedback cycles, ultimately culminating in synaptic plasticity collapse and hippocampal-dependent memory deficits. Based on these mechanisms, this review proposes comprehensive protective strategies, including cold acclimatization training, pharmacological interventions, and targeted therapies. Current research faces challenges such as high heterogeneity in exposure parameters, lack of core body temperature monitoring, and insufficient mechanistic validation. Future efforts should establish standardized research paradigms, quantitatively delineate the relationship between CE intensity/duration and neural injury, and explore precision intervention targets based on the neuroendocrine-immune-metabolic interaction network, thereby providing theoretical foundations and practical approaches for brain health protection in extreme environments.
Cryopreservation of gametes is an integral component of assisted reproductive technologies and fertility preservation; however, exposure to ultra-low temperatures may be associated with structural, genetic, and epigenetic alterations relevant to reproductive outcomes and offspring health. Although this review focuses on human spermatozoa and oocytes, it also incorporates mechanistic and translational evidence from mammalian animal models, including mouse, bovine, buffalo, caprine, and porcine studies, where direct human evidence remains limited. This distinction is essential, as several mechanisms of cryodamage have been defined experimentally in animal models, necessitating careful consideration of their applicability to human gametes. Spermatozoa and oocytes differ substantially in structural organization, metabolic activity, and regulatory mechanisms, resulting in distinct patterns of cryosensitivity. In spermatozoa, limited cytoplasmic volume, high membrane polyunsaturated fatty acid content, and compacted chromatin increase susceptibility to osmotic stress, oxidative damage, and membrane destabilization during freezing-thawing. Reactive oxygen species are central mediators of sperm cryodamage, contributing to DNA fragmentation, chromatin decondensation, altered protamine-histone balance, and epigenetic changes involving DNA methylation, histone modifications, and RNA profiles. Oocytes are particularly sensitive to temperature fluctuations, cryoprotectant exposure, and spindle disruption because of their complex cytoskeletal organization, mitochondrial activity, and dynamic epigenetic regulation. Cryopreservation-associated damage to the meiotic apparatus, mitochondrial function, and chromatin architecture may promote aneuploidy, DNA strand breaks, and transcriptomic alterations. Overall, this review summarizes current evidence on genetic and epigenetic alterations associated with gamete cryopreservation, highlights mechanisms of cryodamage, and discusses their translational and clinical relevance.
We investigated the optimal thawing solution for one-step warming and assessed the efficacy of the one-step warming method for blastocysts subjected to biopsy. Vit Kit-Warm NX Thawing Solution (TS; Irvine Scientific, Santa Ana, CA, USA; osmolarity: 1641 mOsm), Vit Kit-Warm NX Dilution Solution (DS; Irvine Scientific, Santa Ana, CA, USA; osmolarity: 896 mOsm), and Modified HTF Medium-HEPES (m-HTF; Irvine Scientific, Santa Ana, CA, USA; osmolarity: 280 mOsm) were used to perform one-step warming. Survival, recovery, blebbing rate, and recovery time were compared. In blastocysts that were not subjected to biopsy, the DS group showed a lower rate of blebbing than the TS and m-HTF groups (at least P < 0.05). The DS group also showed a shorter recovery time than the TS and m-HTF groups (P < 0.01). In blastocysts subjected to biopsy, the DS group showed a lower rate of blebbing than the m-HTF group (P = 0.0288). The DS group also showed a significantly shorter recovery time than the TS group (P = 0.0319). The results demonstrate that one-step warming achieved favorable post-warming outcomes under various osmotic conditions, regardless of biopsy status. The DS group exhibited the lowest blebbing rate and the shortest recovery time among the three osmolarity conditions evaluated. These findings suggest that, under our vitrification protocol, DS containing 0.5 M trehalose provided the most favorable post-warming morphological outcomes among the three solutions tested.
The present work aims to determine whether supplementation of cysteine and ascorbic acid in rabbit semen extender can maintain post-thawing sperm physiochemical quality and farm fertility under ambient heat stress conditions, and to clarify the molecular mechanisms involved. Mature bucks (n = 15) were used to collect semen samples, which were pooled and divided into six portions, including the control (C0A0) that extended with Tris-Citric-Glucose (TCG) without any additives. The other extenders were prepared by supplementing TCG with 5 mM cysteine (C5A0); 10 mM cysteine (C10A0); 5 mM cysteine and 5 mM ascorbic (C5A5); 10 mM cysteine and 5 mM ascorbic (C10A5) and 5 mM ascorbic (C0A5). Physiochemical characteristics and transcript abundance of sperm viability and redox status genes were measured in post-thawed semen samples. Results revealed that antioxidant supplementation, especially C5A5 and C10A5, significantly improved post-thaw sperm quality, redox status, and transcript abundance compared to control. This enhanced biochemical profile reduced oxidative stress, subsequently increasing conception rates (ranging from 44.0% to 56.0% in treated groups vs. 40.0% in control) and litter sizes (5.58 - 5.92 in combined treatment groups vs. 4.44 in control). At the molecular regulation level, antioxidant transcripts (SOD1, CAT, PRDX6, TXN, and NRF2) were upregulated while endoplasmic reticulum stress genes (CHOP, XBP1 and ATF6) were downregulated in C5A5 and C10A5 compared with the control. Conclusively, supplementing rabbit semen extenders with cysteine (5 or 10 mM) combined with ascorbic acid (5 mM) synergistically enhance post-thaw sperm quality and in-vivo fertility under ambient heat stress conditions by maintaining sperm DNA and acrosome intactness through modulation of redox enzymes and gene networks regulating cellular death and defense against stress.
Temperature critically regulates early development in fish, yet the cellular and molecular mechanisms underlying cold-induced developmental disruption in fish remain poorly defined. Here, we investigated the effects of graded low-temperature exposure on zebrafish embryonic and larval development using integrated organismal, cellular, and molecular analyses. We generated EGFP-transgenic zebrafish to enable direct in vivo fluorescence visualization and evaluation of multiple organs. Wild-type and EGFP-labeled embryos were reared at 28 °C (control) or reduced temperatures (22 °C, 16 °C). Fluorescence imaging showed 22 °C caused developmental delay, reduced growth, impaired eye and cardiac development, and abnormal caudal vein morphology, while 16 °C induced severe developmental arrest and early lethality. At the molecular level, low temperature suppressed growth and cardiac-related genes (igf1, nkx2.5, gata4, tbx5) and upregulated inflammation-, proapoptosis- and antioxidant-related genes (tnfa, il1b, bax, sod1), with concurrent reduced mitochondrial membrane potential indicating impaired mitochondrial function. Collectively, low temperature disrupts zebrafish embryogenesis via coordinated effects on growth signaling, stress responses, and mitochondrial function. These findings link environmental temperature stress to altered gene expression and cellular phenotypes during early zebrafish development, providing insights into mechanisms of cold-induced developmental disruption in this model.
Semen cryopreservation is a key tool for assisted reproduction and genetic conservation, but its efficiency remains limited in donkeys, compromising post-thaw sperm quality. This study aimed to characterize the proteomic profile of Pêga donkey spermatozoa and to investigate molecular mechanisms associated with cryopreservation-induced impairment of sperm function. Semen samples were collected from Pêga jacks and evaluated for sperm motility and vigor before and after cryopreservation. Quantitative proteomic analysis was performed by LC-MS/MS, followed by bioinformatic characterization of differentially abundant proteins. Cryopreservation markedly reduced sperm motility in all animals, whereas sperm vigor showed only a non-significant tendency toward reduction, suggesting sublethal cryoinjury primarily affecting flagellar efficiency. Proteomic profiling identified 554 proteins, of which 98 were differentially abundant between in natura and cryopreserved spermatozoa. Functional enrichment analyses showed that these proteins were mainly associated with energy metabolism, mitochondrial oxidative phosphorylation, glycolysis, cytoskeletal organization, signal transduction, proteostasis, and oxidative stress response. Notably, proteins involved in ATP production, mitochondrial function, and axonemal organization were significantly altered, supporting a mechanistic link between metabolic dysfunction, flagellar structural disorganization, and reduced post-thaw motility. Overall, cryopreservation induced coordinated and compartment-specific remodeling of the donkey sperm proteome, particularly affecting pathways essential for motility and functional competence. These findings provide new molecular insights into the cryobiological vulnerability of donkey spermatozoa and establish a mechanistic basis for the development of more effective, biology-driven cryopreservation strategies for this species.
Sucrose was first included in plant vitrification solutions (PVS) in 1989 and has since remained a primary component of most formulations. Although sucrose is known to stabilize cell membranes, prevent protein denaturation, and promote cellular dehydration, its specific role in successful plant cryopreservation remains unclear. With the growing need to preserve plant germplasm, less toxic yet effective PVS are being developed. However, empirical knowledge on the permeation of sucrose is insufficient for the optimization of PVS and cryopreservation methods. The study reported here evaluates sucrose permeation into rice callus cells for each step of a model cryopreservation cycle. By deuterating sucrose, its C-D bond can be tracked using coherent anti-Stokes Raman scattering microscopy, a highly sensitive, non-invasive vibrational microscopy technique. Results show that sucrose permeates cells within 6 s of initial exposure and takes only 20-30 s to reach steady-state concentration. Sucrose is a permeating cryoprotecting agent regardless of pre-existing intracellular sucrose from preculture and loading steps. Beyond the permeation of sucrose as a CPA component, the results presented here suggest that the method we used could measure permeation by other molecules interacting with live plant material, such as the permeation of herbicides, showing kinetics and sequestration into plant organelles.
Cryopreserved genetic resources from threatened species face substantial logistical challenges, particularly in field settings where laboratory infrastructure is limited. This study evaluated three simplified cryopreservation protocols for wildlife biobanking using threatened Neotropical cervids as experimental models. Ultra-rapid freezing (UF) with cryoprotectants (CPA) using short (s) and long (l) exposure times (30 s and 5 min, corresponding to sCPA-UF and lCPA-UF, respectively), and CPA-free UF (CPAf-UF), were compared with no cryopreservation (fresh tissue, FT). In sCPA-UF, tissues were frozen immersed in the freezing medium, whereas in lCPA-UF, excess medium was removed before ultrarapid freezing. Post-thaw assessment included cell viability analysis, histomorphological evaluation, DNA damage quantification, mitotic index determination, and functional characterization through cell culture, migration, and proliferation assays. All cryopreservation methods yielded viable and proliferating fibroblasts. lCPA-UF demonstrated the highest cell viability (often comparable to FT), while CPAf-UF and sCPA-UF protocols achieved significantly lower but functionally adequate viability (>50%). Histomorphological analysis revealed minor, non-significant structural alterations across all cryopreserved groups, with slightly increased apoptotic nuclei in CPAf-UF and sCPA-UF treatments. Despite reduced proliferation rates compared to lCPA-UF, all protocols maintained cellular adhesion, migration capacity, and proliferative potential. Mitotic indices were largely preserved. This study demonstrates that, although cryoprotectant-based ultrarapid freezing provides superior cell survival, cryoprotectant-free ultrarapid freezing represents a functionally viable alternative for wildlife tissue preservation, offering practical logistical advantages for field applications.
Conventional multi-step warming protocols (MWP) for vitrified blastocysts are labor-intensive and susceptible to handling variability. This study evaluated whether a shortened warming protocol (SWP) could support post-warming recovery and developmental competence comparable to those of conventional MWP. A retrospective propensity score-matched cohort study was conducted using 546 single vitrified-warmed blastocyst transfer cycles (271 SWP and 275 MWP). Post-warming developmental behavior was assessed using time-lapse imaging, including the start of re-expansion (tSRE), completion of re-expansion (tCRE), and hatching start point (tHSP). Morphological recovery, blastocyst quality score (BQS), and inner cell mass (ICM) and trophectoderm (TE) grades were also evaluated. Blastocysts warmed using SWP demonstrated significantly faster post-warming recovery, with shorter tSRE (0.148 vs. 0.179 h, P = 0.009), tCRE (2.16 vs. 2.91 h, P < 0.001), and tHSP (3.08 vs. 5.24 h, P < 0.001). The SWP group showed a significantly higher proportion of blastocysts with improved post-warming morphology and grade A ICM and TE than the MWP group (P < 0.05). The distribution of hatching stages and clinical pregnancy outcomes did not differ significantly between the groups. These findings suggest that SWP provides efficient and biologically stable post-warming recovery without compromising embryonic quality or clinical outcomes.
Cell spheroids and organoids possess three-dimensional cell-cell interactions that more closely recapitulate in vivo tissue architecture than compared to conventional monolayer culture. Pluripotent stem cell-derived organoids demonstrate highly differentiated organ-specific functions and are valuable in applications that range from research and development to clinical therapeutics. Current lack of effective preservation methods is a major bottleneck preventing the broad distribution of spheroids/organoids and restricts their widespread use. Isochoric supercooling is a technology that enables stable, sub-zero, non-freezing preservation of biological material. The isochoric feature significantly reduces the probability of ice nucleation upon impact and vibrational perturbation, an important consideration for the transport and delivery of precious biologic cargo. In this report, we investigated the preservation of self-assembled HepG2 hepatic cell line spheroids in isochoric -6°C supercooling conditions compared to 4°C cold storage. We found that isochoric -6°C supercooling was superior to 4°C cold storage in preserving high viability of hepatic spheroids in University of Wisconsin solution for 3 days. To extend preservation duration to 5 days, it was necessary to use HypoThermosol-FRS as the preservation solution and to implement a gradual cooling and warming protocol. Importantly, isochoric -6°C supercooling for 5 days with gradual cooling/warming preserved both high cell viability and intact spheroid structure, outcomes not achieved with 4°C storage or fast cooling/warming. Our findings indicate that isochoric supercooling may be a critical technology for preserving cellular spheroids and organoids within the timeframe and logistical requirements for facilitating their distribution and utilization.
Cryopreservation serves as a vital technique for long-term germ preservation of aquatic species, and post-thaw sperm viability is essential to successful germplasm conservation. Here, we optimized the ultra-low temperature cryopreservation protocol for sperm of the Manila clam Ruditapes philippinarum. Based on preliminary viability assessment, sterile natural seawater was confirmed as the optimal diluent, and 0.2% (v/v) ammonia seawater was chosen for sperm activation. A 3×3 orthogonal experimental design was applied to screen the optimal combination of cryoprotectant type, concentration (10%, 15%, 20%) and cooling procedure for sperm cryopreservation of R. philippinarum. The optimized cryopreservation protocol was determined as follows. Sperm samples were collected via dissection of clams under low temperature. The obtained sperm were initially diluted with sterile natural seawater at a volume ratio of 1:1, and then blended with 10% dimethyl sulfoxide (DMSO) cryoprotectant at a volume ratio of 1:2. The diluted sperm were first equilibrated 15 cm above liquid nitrogen (LN2) for 5 min, followed by another 10-min equilibration at 5 cm above LN2, and finally preserved by complete immersion in LN2. After 24 h of cryogenic storage, frozen sperm were thawed in a 38 °C water bath and activated with 0.2% ammonia seawater. The average fertilization rate reached 90.99 ± 1.14%, and the hatched larvae developed normally. This study lays a theoretical and technical foundation for germplasm conservation and selective breeding of high-quality Manila clam varieties.
