Over the past decades, cryobiology has become increasingly integrated across a variety of domains. With broad intersection, this discipline has recently also emerged as a cornerstone for advancing and clinically translating tissue-engineered constructs (TECs). This review provides a comprehensive overview of recent advances at the intersection of cryobiology and tissue engineering. Vitrification of both scaffold-based and scaffold-free TECs as well as approaches of its upscaling including benefits of polymers and technical devices are comprehensibly discussed. The development of biologically inspired nanoscale materials is outlined as an integral part of this research. Success in vitrification of organoids is also discussed. Developments in controlled-rate slow-cooling/freezing protocols are then examined, with particular attention to xeno-free and Me2SO-free cryoprotective systems that enhance cell viability and biocompatibility. Preclinical studies utilizing cryopreserved TECs in animal models are further outlined as key milestones toward clinical translation. Furthermore, this review introduces emerging synergistic approaches that make TECs more adaptable to cryopreservation by incorporating cryoprotective agents (CPAs), nanoparticles, cold-responsive polymers and ice recrystallization/devitrification inhibitors at the scaffold design stage. Finally, cryobioprinting as an emerging approach that unites cryobiology and tissue engineering, offering new opportunities for the fabrication, storage, and on-demand deployment of viable tissue constructs is reviewed. Overall, the overviewed experimental evidence underscores the transformative role of cryobiology in driving recent advances in the field of tissue engineering and fostering innovative and forward-looking strategies in this field. Ultimately, a closer convergence of cryobiology, tissue engineering, and transplantation science will be essential to advance TECs toward scalable, off-the-shelf availability.
暂无摘要（点击查看详情）
Cryopreservation of marine species is a key tool for biodiversity conservation, population management, and the advance of aquaculture and biological research. Currently exist significant progress in the cryopreservation of gametes, embryos, larvae and even juveniles of several genus of marine invertebrates. However, cnidarians, and particularly jellyfish, remain largely underexplored. In this study, we report on the first successful cryopreservation of Aurelia aurita ephyrae, the first larval stage of this jellyfish species. This species is characterized by extremely high-water content (96.3 %), which represents a unique challenge in the method of cryopreservation. In this study we done toxicity tests using different concentrations ranging from 0.5 to 6 M of dimethyl sulfoxide (Me2SO). On the other hand, we designed two cocktails of cryoprotectants and evaluated the efficiency in a protocol of cryopreservation. The first one is based on 1 M Me2SO + 0.5 M dimethylformamide (DMF) and the second used 1.5 M Me2SO with 0.04 M trehalose (TRE) and 1 % BSA as a post-thaw supplement. The first protocol achieved 100 % survival immediately after thawing and 33 % survival at 48 h, while the second maintained a consistent 33 % survival across both time points. The damage and viability of the larvae was assessed using live/dead fluorescence dye and allowed confirming the integrity of surviving individuals. These results mark the first demonstration of successful cryopreservation protocol for jellyfish larvae and represent a significant advance for the ex-situ conservation of gelatinous zooplankton. This work lays the foundation for further research into cryopreservation protocols for marine species, understand most better water flow for organisms with high water content and opens new possibilities for marine biodiversity conservation and cnidarian model systems.
Sperm cryopreservation is pivotal for conserving fish germplasm, yet cryodamage-induced quality decline limits its application. This study focused on Sichuan bream (Sinibrama taeniatus), an endemic and economically important fish species in the upper Yangtze River. Based on an established cryopreservation protocol, we evaluated sperm quality using computer-assisted sperm analysis (CASA) and fertility assays, followed by a systematic assessment of structural and functional damage via flow cytometry (membrane integrity, mitochondrial potential, reactive oxygen species, and DNA fragmentation), enzymatic assays (energy metabolism and antioxidant enzymes), Western blotting, and ultrastructural observation. Finally, integrated proteomic and metabolomic analyses were employed to elucidate the underlying physiological mechanisms. The results demonstrated that freeze-thawing significantly impaired sperm motility, fertility, and ultrastructure, concurrently disrupting energy metabolism and the antioxidant system. Crucially, multi-omics revealed that these functional declines were linked to dysregulation in key pathways involving cytoskeleton organization, lipid metabolism, energy homeostasis, and oxidative stress, forming a coherent network from initial molecular perturbation to phenotypic dysfunction. This study provides a comprehensive characterization of sperm cryodamage in Sichuan bream, advancing the understanding of fish sperm cryobiology and informing targeted cryoprotection strategy development.
Vitrification is enabling successful cryopreservation of progressively larger organs. Heating the whole volume of an organ simultaneously instead of only the surface during recovery from vitrification has been vital to this success. Volumetric warming enables faster and more uniform warming. Faster warming reduces ice crystal growth, ice recrystallization, and toxic effects of cryoprotectants by reducing ice growth time and cryoprotectant exposure time during warming. Nanowarming of intravascular magnetic nanoparticles by alternating magnetic fields, and direct dielectric warming by alternating electric fields, have both been used successfully for volumetric warming of vitrified organs. Dielectric warming predates vitrification, having been studied intermittently in cryobiology since the 1950s. Most early research was empirical, using microwaves at 915 MHz or 2.45 GHz because of wide availability of magnetron generators and microwave ovens. With greater theoretical understanding, interest later grew in frequencies near 400 MHz and lower. Larger energy absorption and smaller wavelengths inside tissue warming from vitrification instead of thawing from freezing made optimum frequencies for vitrification even lower, below 100 MHz for organs more than 10 cm in diameter. Recent dielectric warming research has used 27, 40, and 55 MHz, achieving heating rates of 200 - 700 °C/min. The advantages of dielectric warming not being dependent upon exogenous particles or vascular volume, high energy efficiency, and ability to monitor temperature by impedance behavior during warming, are counterbalanced by the need for organ immersion before vitrification and pre-warming to a uniform starting temperature above the glass transition temperature. With measurements of two electrical properties of a vitrification solution, permittivity and loss factor as function of temperature, detailed theoretical analysis and modeling of dielectric warming is possible. Comparatively little research has been done on dielectric warming at frequencies optimal for vitrification. This review covers the history, theory, equipment types, practical aspects, and future directions of dielectric warming of cryopreserved tissue.
Microfluidic devices offer precise control over solution mixing and gradient generation, essential for cell-based assays in cryobiology and biomedical research. However, traditional fabrication methods are time-consuming, costly, and require specialized expertise, which limits accessibility. To address these challenges, we developed a cost-effective, reliable, and fully 3D-printed microfluidic device workflow to facilitate rapid and inexpensive prototyping using a consumer-grade printer and biocompatible plastic resins. Here, we demonstrate this workflow with a fluidic mixing device capable of generating programmable concentration gradients and solution combinations. Commercial mixing devices cost more than $300 each and cannot be customized. By utilizing affordable resin materials and an innovative open-channel design sealed with transparent adhesive tape, we overcame common fabrication issues such as channel clogging, enabling rapid and reproducible fabrication of complex microfluidic architectures, all at a materials cost of less than $5. Here we demonstrate this workflow, integrating dual-syringe pumps to create linear osmotic gradients, ranging from iso-osmotic (~300 mOsm/kg) to hyperosmotic (~9,000 mOsm/kg) conditions, followed by a return to isotonicity over defined intervals. To ensure automation and reproducibility, we developed an open-source Python-based software tool that precisely regulates syringe pump activation, flow rates, and gradient timing. The device's performance was validated through continuous osmometric measurements, which confirmed both the linearity and accuracy of gradient generation, and colorimetric measurements to confirm mixing efficacy. This accessible and cost-effective microfluidic platform significantly improves the reproducibility of osmotic exposure studies and shows potential for various biomedical applications, including drug screening and precise chemical modulation.
Hibernation is a specialized adaptive energy-saving survival strategy evolved by animals to withstand winter cold stress and food scarcity. Its core feature lies in profound metabolic suppression, characterized by a drastic reduction in metabolic rate during hibernation, accompanied by the coordinated downregulation of multiple physiological functions such as body temperature, heart rate, and respiratory rate. The establishment and maintenance of this deep metabolic suppression state essentially rely on the systemic reprogramming of energy metabolism, which serves as the core driving force of hibernation adaptation. During this reprogramming process, lipid metabolism acts as a key executive link: fats stored in adipose tissue not only function as the primary energy reserve pool during hibernation but also undergo precise regulatory remodeling in terms of their compositional characteristics, mobilization efficiency, and catabolic processes, thereby synchronously adapting to the demands of energy supply and environmental adaptation goals. Importantly, metabolic suppression often precedes cooling and can exceed Q10 predictions, indicating active regulatory control rather than passive thermal effects. Reliance on lipid oxidation and cyclic torpor-arousal transitions should heighten oxidative stress risk: electron leakage from mitochondrial complexes I/III during deep torpor, relative hypoxia from reduced perfusion, and rapid "metabolic restart" upon arousal may resemble ischemia-reperfusion. Yet hibernators show minimal oxidative damage, implying robust antioxidant and repair programs. This review summarizes recent advances in the metabolic remodeling of lipids, substrate conversion, and oxidative stress adaptation in hibernating animals. It reveals the evolutionary mechanisms underlying energy metabolism adaptation and provides potential insights for applications in metabolic diseases, cryobiology, and related fields.
Cryopreservation relies on refrigerants for achieving and sustaining ultra-low (>-150 °C) or cryogenic temperatures (<-150 °C) for cooling, transport and storage of biological materials. Liquid nitrogen (LN2) remains the standard refrigerant in cryobiology due to its low cryogenic boiling point, cost effectiveness and availability. However, its limitations in slower cooling rates and increased fracture probability at larger human-scales motivate the search for alternative refrigerants with tailored phase change temperatures to enable faster cooling, reduce thermal stress while offering eco-friendly sustainable options for transport and storage. This study evaluates current cooling, transport and storage technologies for cryopreservation, categorizing them by refrigerant type and delivery method to identify performance gaps that hinder scale-up to tissues and organs. We introduce a refrigerant-selection framework that systematically screens 100+ potential candidates based on thermal performance, environmental impact, and health-safety considerations. Application of this framework highlights hydrofluoroolefins (HFOs), hydrofluoroethers (HFEs), perfluorocarbons (PFCs), and engineered heat-transfer fluids as promising options capable of enabling higher cooling rates, improved thermal annealing near the glass-transition temperature, and reduced reliance on LN2. This work offers practical guidance for selecting refrigerants in next-generation cryopreservation devices to support application-specific performance and offers foundation for innovation in thermally scalable and environmentally responsible cryopreservation technologies.
Haematopoietic stem cell transplantation (HSCT) offers curative potential for several malignant and non-malignant hematologic disorders. Despite its proven efficacy, access to HSCT in sub-Saharan Africa remains limited, especially in francophone countries, due to the lack of infrastructure, cryobiology facilities and trained personnel. Senegal recently launched a national initiative to establish its first HSCT program. We report the first autologous HSCT performed in Senegal in February 2025 at Dalal Jamm University Hospital, in a 51-year-old man diagnosed with high-risk IgA-lambda multiple myeloma (ISS stage III, del17p). Mobilisation was achieved using filgrastim (10 µg/kg/day for 7 days). A total of 2.8 × 10⁶ CD34⁺ cells/kg were collected by apheresis and stored at 4°C for 24 hours without cryopreservation. Conditioning consisted of high-dose intravenous melphalan (200 mg/m2) followed by reinfusion of the graft on day 0. Hematologic recovery occurred by day +10, with transient grade 3 anemia and grade 4 thrombocytopenia requiring transfusion support. The main complications were manageable febrile neutropenia and mild gastrointestinal and renal toxicities. The patient was discharged on day +17, remained infection-free and achieved complete hematologic and biochemical remission at five months post-transplant. Consolidation therapy with bortezomib-thalidomide-dexamethasone and lenalidomide maintenance was subsequently administered. This first non-cryopreserved autologous HSCT in Senegal demonstrates the feasibility, safety and cost-effectiveness of transplantation under resource-limited conditions. Establishing local cryopreservation and molecular diagnostic capabilities will be essential to enable tandem and allogeneic HSCT, ensuring sustainability and regional self-sufficiency in advanced hematologic care.
Partial freezing has been proposed in various forms since the 1990s as a high sub-0°C technique for prolonging the viable preservation window of organs relative to conventional hypothermic storage. However, despite the fundamental dependence of this technique on equilibrium thermodynamic behaviors, solution and protocol design have thus far remained largely empirical. The multisolute osmotic virial equation (MSOVE) has previously been used for quantitative protocol design in cryobiology, including the optimization of cryoprotectant addition and removal procedures and the prediction of osmotic responses in multi-solute systems. Here, we apply the MSOVE to model the freezing process of partial freezing solutions utilized in recent rat liver preservation protocols, computing melting points, liquidus curves, equilibrium ice fractions, and solute concentration trajectories down to -20 °C. The calculations provide simple mechanistic insights into observed differences in preservation quality between different pairings of composition and storage temperature, and suggest practical routes to optimize electrolyte content, polymer concentration, and cryoprotectant choice when rationally designing future partial freezing protocols.
Ice growth inhibition is crucial in cryotechnology, as uncontrolled recrystallization during the frozen state and freeze-thaw cycles causes irreversible damage to biological samples. Nanoscale materials that mimic antifreeze proteins and exhibit ice recrystallization inhibition (IRI) activity have been explored as cryoprotectants; however, the structural features that govern potent IRI activity remain unclear. This study investigated the effects of nanoparticle size and functionality on the IRI activity. Polystyrene nanoparticles (PSNPs, 30-1000 nm) were used as inert nanoscale models, and amino acid derivatives with phenyl groups with or without hydroxyl functionality, including L-phenylalanine monomers, pentamers of L-phenylalanine (Phe-5), L-tyrosine, and 3,4-dihydroxy-L-phenylalanine, were examined. Among these, we found that tyrosine monomer nanocrystals (TMNs) display exceptionally potent IRI activity under both extracellular and intracellular conditions, which is attributed to nanoscale structure formation, hydroxyl functionality, and high colloidal stability. TMNs enhance cell survival during cryopreservation, even at low dimethyl sulfoxide concentrations, whereas Phe-5 and other analogs show limited activity owing to aggregation or lack of hydroxyl groups. These results elucidate the key factors influencing IRI activity, including nanoscale assembly with high colloidal stability and the presence of a hydroxy functional group. Therefore, considering the biocompatibility of L-tyrosine, our study shows that TMNs are promising supplementary materials for cryobiology.
Obesity is a global public health problem, and new non-surgical or pharmacological treatments are being explored. Among these, cryostimulation has emerged as a new beneficial non-invasive method. Several studies have reported a greater decrease in skin temperature following cryostimulation in individuals with higher BMI or body fat percentage. The aim of this study was to evaluate the effect of a single 3-min whole-body cryostimulation (WBC) session on skin temperature and cold perception in male and female obese subjects. Skin temperature changes before and after the 3-min cryostimulation exposure were measured in twenty-one participants. Our results showed that the average skin temperature decreased by 12.9 °C without reaching dangerous levels. Individual characteristics such as sex and age should nevertheless be carefully considered to ensure the safety and efficacy of the treatment.
The Yesso scallop is a commercially important species in the northern coastal regions of East Asia; however, its aquaculture production has recently become increasingly unstable due to global climate change. Cryopreservation of germ cells and surrogate broodstock technologies would be fundamental approaches for safeguarding the genetic resources of local populations of commercial bivalve species. Successful germ cell transplantation in bivalves requires the proper preparation of donor spermatogonia. In the present study, we established and optimized protocols for the enzymatic dissociation and cryopreservation of testicular tissue. Dissociation using 0.025% Liberase TM at 25 °C for 2 h yielded the highest number of viable cells among the conditions tested. Evaluation of cryoprotectants and their concentrations showed that 5-10% dimethyl sulfoxide (DMSO) was the most effective among the cryoprotectants tested. Further evaluation of cooling rates and thawing temperatures at these concentrations demonstrated that rapid freezing followed by thawing at 25 °C using 10% DMSO resulted in the highest cell viability. Immunohistochemical and immunocytochemical analyses confirmed that the resulting cell suspensions contained spermatogonia, indicating that the established methods are suitable for preparing testicular cell suspensions for germ cell transplantation.
Type 1 Diabetes (T1D) is a devastating disease in which the immune system attacks insulin- producing beta cells in the pancreas, disrupting the normal blood glucose regulation mechanism and resulting in the significant burden of ongoing blood glucose monitoring and management and longer-term damage to organs and tissues. An emerging therapy for T1D includes transplanting insulin-producing stem cell (SC)-derived aggregates into patients, restoring normal regulation of blood glucose and eliminating the need for insulin injections. To enable stable storage, distribution, and clinical administration of this therapeutic, reliable cryopreservation methods are required. However, current cryopreservation protocols result in low cell viability post thaw and have challenges in scalability. This review provides background on stem cell therapy for T1D and the production and storage pipeline of these SC-derived aggregates, with a focus on the challenges of cryopreservation. We review the fundamental physics involved in cryopreservation, including cryoprotective agents (CPAs), CPA loading and unloading, the importance of cooling and rewarming rate selection, and why the cell aggregate microstructure of islets presents a particularly difficult challenge for cryopreservation. Finally, we highlight important developments in SC-derived aggregate cryopreservation and the state of the art.
The objective of this study was to investigate the effects of trehalose and different doses of melatonin and lipid mixtures on the quality parameters of post-thaw bull sperm during cryopreservation. The ejaculates of three mature bulls were pooled and divided into ten equal aliquots. These aliquots were diluted with a Tris-based extender, which was supplemented with either 5% glycerol (G5) or 3% glycerol combined with 60 mM trehalose (G3T), alongside different doses of melatonin and lipid mixtures. Ten experimental groups were established as follows: G5, G5+0.25 mM melatonin (G5M0.25), G5+0.75 mM melatonin (G5M0.75), G5+ 1.25 μl/ml lipid mixtures (G5L1.25), G5+3.75 μl/ml lipid mixtures (G5L3.75), G3T, G3T+0.25 mM melatonin (G3TM0.25), G3T+0.75 mM melatonin (G3TM0.75), G3T+1.25 μl/ml lipid mixtures (G3TL1.25), and G3T+3.75 μl/ml lipid mixtures (G3TL3.75). No significant interaction was detected between any of the groups containing the G5 and G3T extenders, and all groups were found to be similar for sperm motility and flow cytometry analysis results (p > 0.05). The G5M0.25 and G5L1.25 groups had a higher recovery of post-thaw motility compared to the other groups containing 5% glycerol (p = 0.0004). In terms of motility rates, groups G3TM0.25 and G3TL1.25 displayed a higher level of protection compared to the other groups, and this protection was significantly greater than that determined in groups G3TM0.75 and G3TL3.75 (p = 0.001). The expression of the GFPT1, PFKP, FBF2, HK1, and ALDH2 genes was found to be significantly increased in the G5 and G3T groups containing both doses of lipid mixtures (L1.25 and L3.75) compared to the groups without additives (G5 and G3T) (p < 0.001-0.0001). However, the expression of the SORD gene was found to be increased only in the G3TL1.25 and G3TL3.75 groups compared to group G3T (p < 0.01-0.001). GFPT1 and FBF2 gene expressions were significantly increased in the G5M0.75 group compared to group G5 (p < 0.05). The G3T groups containing both doses of melatonin were found to display increased GFPT1 (p < 0.01) and PFKP (p < 0.05-0.01) expression levels compared to group G3T. It was determined that the addition of lipid mixtures at both doses to both G5 and G3T resulted in a significant transcriptional increase in all of the genes studied (except for SORD gene expression in the G5L1.25 and G5L3.75 groups), compared to the lipid mixtures-free groups (p < 0.05-0.001). Based on the evaluation of all results, G3T can be used as a substitute for G5 to reduce glycerol toxicity.
Accurate normalization using stable reference genes (RGs) is essential for RT-qPCR, particularly in single bovine blastocysts with limited RNA. To date, RG validation under vitrification and warming conditions remains limited. This study compared fresh control and vitrified-warmed in vitro produced bovine blastocysts to identify appropriate RGs for normalizing target gene expression in vitrification-related studies of bovine embryos. In the treatment group, embryos were vitrified at the morula stage, warmed, and cultured to the blastocyst before analysis, and blastocysts from both groups were analyzed at the single-embryo level. Cycle threshold values of candidate RGs (ACTB, GAPDH, H2A, HMBS, PPIA, SDHA, YWHAZ, and 18S) were analyzed for stability ranking using the comparative ΔCt method, geNorm, BestKeeper, and NormFinder, followed by comprehensive analysis with RefFinder. ACTB emerged as the most stable RG across both groups, whereas H2A exhibited the lowest stability. To validate the identified RGs, apoptosis-related genes (FOXO3a, BAX, and CASP3) were normalized using ACTB, H2A, and the traditionally used RG (GAPDH). When normalized with ACTB, CASP3 expression, a marker of the downstream apoptotic pathway, was significantly higher in the vitrified-warmed group than the control group, consistent with the increased proportion of TUNEL-positive apoptotic bodies observed in vitrified-warmed blastocysts. However, normalization with GAPDH or H2A abolished these differences or produced opposite expression patterns between the two groups. These findings reveal that appropriate RG selection is essential for reliable RT-qPCR analysis of vitrified bovine embryos and provide a framework for future studies to improve embryo cryopreservation efficiency.
The present study investigated the differential mRNA expression of energy metabolism and stress-response genes (ADCY10, AK1, ATP5F1B, and HSP70) in fresh and frozen-thawed Murrah buffalo spermatozoa subjected to uniform 2-h in vitro capacitation. Semen samples (n = 32 per group) from eight bulls were divided into four groups: fresh non-capacitated (G1, control), fresh capacitated (G2), frozen non-capacitated (G3), and frozen capacitated (G4). Relative expression was analyzed by RT-qPCR using GLUT5 as the reference gene. Results revealed that ADCY10 was significantly upregulated in G2, G3, and G4 compared to G1, with the highest expression in G3 (frozen non-capacitated), indicating cryopreservation-induced premature activation stronger than bicarbonate-driven capacitation. AK1 was exclusively and significantly upregulated only in fresh capacitated spermatozoa (G2), suggesting it as a specific marker of successful physiological capacitation. ATP5F1B showed significant upregulation in both G2 and G3, reflecting high energy demand in true capacitation and metabolic surge post-thawing, but declined in G4. HSP70 was significantly downregulated in all treated groups compared to G1, confirming cryoinjury-related transcript consumption. These findings demonstrate that cryopreservation triggers partial cryo-capacitation with dysregulated energy gene expression, uncoupling true capacitation pathways and contributing to reduced post-thaw fertility in buffaloes.
Cryopreservation of epididymal spermatozoa, recovered post-mortem, enables genetic preservation in dromedary camels; however, freeze-thaw procedures induce oxidative stress that hinders spermatozoal function. This study evaluated the effects of supplementing a commercial semen extender (INRA 96) with various concentrations of Coenzyme Q10 (CoQ10; 0, 0.25, 1.00, and 2.00 μM) on post-thaw quality and fertilizing capacity of dromedary camel epididymal spermatozoa. Post-thaw assessments included motility, vitality, morphology, plasma membrane functionality, and acrosome integrity. Oxidative stress markers [total antioxidant capacity (TAC) and malondialdehyde (MDA)] were measured in the extender post-thaw. Cleavage and blastocyst rates after in vitro embryo production (IVEP) were also recorded. Supplementation of the extender with 0.25 μM CoQ10 significantly improved post-thaw motility (48.33% ± 1.67 vs. control 38.33% ± 1.67), vitality (53.33% ± 2.73 vs. control 41.00% ± 2.08), membrane functionality (50.33% ± 4.10 vs. control 47.33% ± 4.26), and acrosome integrity (63.33% ± 2.73 vs. control 46.67% ± 2.40). Additionally, CoQ10 at 0.25 μM enhanced cleavage (30.39% ± 1.86) and blastocyst formation rates (20.59% ± 2.03) compared to the control groups (23.73% ± 1.20 and 15.25% ± 1.45, respectively). Biochemically, 0.25 μM CoQ10 elevated TAC (0.76 ± 0.02 mM/L) and reduced MDA (6.88 ± 0.38 nmol/mL) compared to controls (0.40 ± 0.04 mM/L and 8.99 ± 0.31 nmol/mL, respectively). In contrast, higher CoQ10 concentrations (1.00 and 2.00 μM) impaired both sperm quality and embryo development potential. In conclusion, low-dose CoQ10 supplementation (0.25 μM) showed beneficial outcomes, while concentrations exceeding 1.00 μM were deleterious. Future research should investigate the molecular mechanisms by which CoQ10 protects spermatozoa, including its effects on mitochondrial function and specific antioxidant pathway activation during the cryopreservation process.
This study assessed, in sheep, the effects of antifreeze protein (AFP) type I on redox balance at different embryonic stages by measuring mitochondrial activity (MITO), reactive oxygen species (ROS), and glutathione (GSH) levels in the same embryos immediately before cryopreservation and after warming. A total of 45 GI-GII embryos were divided into morulae (MO; n = 18) and blastocysts (BL; n = 27), and then, each group was subjected to slow freezing in a solution containing 0.1 μg/mL AFP I or not. In the MO-CONT group, cryopreservation reduced (P < 0.05) GSH levels and MITO, whereas ROS levels remained unchanged. In contrast, no differences (P > 0.05) were observed between pre- and post-cryopreservation in the MO-AFP group. Interestingly, the MO-AFP exhibited higher (P < 0.05) MITO than the MO-CONT group. In blastocysts, cryopreservation led to reductions (P < 0.05) in ROS, GSH levels, and MITO. In conclusion, AFP I supplementation preserved MITO in sheep morula embryos following cryopreservation, suggesting a stage-dependent protective effect.
Despite extreme conditions including freezing temperatures, low water activity, and few nutrients, active microorganisms are thought to inhabit glacial ice, yet little is known about their identities and methods of survival. We used flow cytometry, cultivation, metagenomics, and metatranscriptomics to characterize viable and active microbial communities from near-surface englacial ice from White Glacier in the Canadian High Arctic and Johnsons Glacier on Livingston Island, Antarctica. The ice, though low in microbial biomass (104 cells/ml), harbors communities capable of growth at subzero temperatures (-5°C), high salinity (12% NaCl), and low pH (pH 3). The communities of both poles were different, with metagenome-assembled genomes (MAGs) from White Glacier belonging to Cyanobacteriota and novel phyla and MAGs from Johnsons Glacier belonging to Pseudomonadota and Actinomycetota. Despite this, both glacial communities shared key metabolic functions, including aerobic respiration, aerobic carbon monoxide oxidation, sulfide oxidation, and denitrification. Metatranscriptomics from White Glacier revealed dominant Cyanobacteriota, performing oxygenic photosynthesis and carbon fixation and accompanied by active lithoautotrophs performing metabolisms such as carbon fixation via the 3-hydroxyproprionate cycle, anoxygenic photosynthesis, sulfide oxidation, and nitrate reduction/denitrification. These metabolisms appear to support an active heterotrophic community performing aerobic respiration and aerobic carbon monoxide oxidation. This study highlights the distinct but functionally similar microbial communities in Arctic and Antarctic glaciers, hinting that there may be a core set of metabolisms required for surviving in englacial ice and suggesting that similar communities could persist in glacial ice on Mars or the icy outer moons, Europa and Enceladus.